Metal binding compounds in hepatopancreas and haemolymph of porcellio scaber (isopoda) from contaminated and reference areas (original) (raw)
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Biological Trace Element Research, 2005
Metallothioneins (MTs) are ubiquitous low-molecular-weight metalbinding proteins, with a variety of functions in metal metabolism ascribed to them. Among terrestrial invertebrates, MTs have been studied in nematodes, insects, snails, and earthworms. The aim of this study was the characterization of MT-like proteins in the terrestrial isopod crustacean Porcellio scaber in order to analyze their probable role in the metabolism of copper (Cu) and zinc (Zn). Dietary Zn supplementation (793 µg Zn/g dry food, 6 d) was applied to stimulate MT synthesis. After separation of the hindgut postmicrosomic supernatant (cytosol) of Zn-exposed animals by gel filtration on a Sephadex G-75 column, a Cu-and Zn-containing peak was detected in the position of V e /V o~2 , where MTs are expected to elute. Rechromatography of these fractions by size-exclusion chromatography-high-performance liquid chromatography revealed that the 215-nm absorbance peak coincided with the absorbance peak of the rabbit MT II standard. These low-molecular-weight Cu-and Zn-binding compounds, detected in the cytosol of the hindgut cells in Zn-exposed P. scaber, are considered to be Cu,Zn-MT-like proteins. To our knowledge, this is the first report on the characterization of MT-like proteins in isopod crustaceans.
Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology, 1996
Bioassays were conducted with the polychaete Eqthoe complanata to study the patterns of accumulation and depuration of copper and zinc in relation to the levels of metal-binding proteins during sublethal exposure. Acute toxicity tests indicated 96-hr LCSO of 1.3 mg Cu+*.l-' and 139.6 mg Zn+'.l-', from which sublethal levels of 0.2 mg.l-' of copper or 10 mg.l-' of zinc were established for a 24-day exposure. At those sublethal levels, tissue (carcass) concentrations of copper and zinc in E. complanata reached maximal levels of 29.6 and 319 pg.g-' dry mass, respectively, at the end of the exposure period, whereas unexposed worm levels (carcass) were estimated in 5.6 and 35.9 pg.g-' dry mass of copper and zinc, respectively. After 22 days of depuration, tissue levels were reduced to a minimum of 13.7 and 108 /.!g.g-' dry mass, respectively. Exposure to a mixture of metals resulted in lower tissue levels of copper and zinc as compared with those found for each individual metal analysis. During uptake and depuration phases, Sephadex G-75 chromatography revealed metallothionein-like proteins of molecular weights between 10 and 20 kDa with a higher affinity for zinc than for copper, as well as heavier metalloproteins (>60 kDa), displaying a higher affinity for copper. These data suggest different biochemical mechanisms underlying the control of zinc and copper metabolism through the activity of low-and high-molecular-weight metal-binding proteins. COMP BIOCHEM PHYSIOL 114C;3:179-184, 1996.
Comparative Biochemistry and Physiology Part C: Comparative Pharmacology, 1989
After feeding with metal-contaminated substrate from a lead-mining area, high concentrations of cadmium and zinc were measured in the hepatopancreas of the terrestrial slug Arion lusitunicus. 2. Centrifugation of hepatopancreas homogenates from both control and metal-fed animals revealed that most of the cadmium was associated with the supernatant, whereas a large proportion of zinc was bound to the pellet. 3. In both control and metal-fed groups cadmium-binding components have been found by ion exchange and gel chromatography. 4. All the cadmium from control slugs was bound to components with a mol. wt higher than 20,000. 5. In metal-fed animals, however, all cadmium was associated with an inducible protein with an apparent mol. wt of 10,000. 6. This component showed an elevated absorption at 254 nm and reduced absorption at 280 nm. 7. Amino acid analysis revealed high amounts of glycine (18%), cysteine (lo%), aspartate (12%), serine (13%), and glutamate (14%). 8. The component belongs to the family of metallothionein-like proteins and is thought to play a role in the detoxification of cadmium.
Multielement analysis of metal-binding proteins in cytosol fractions
Science of The Total Environment, 1983
The distribution of 24 elements among the cytosol protein tractions was aetermined for specimens ot the bivalve mollusc Macoma balthica obtained from three estuarine locations subject to varying levels ot metal pollution and on specimens ot Rangia cuneata from three areas subject to varying degrees of thermal pollution. Ot the 24 elements examined 15 occurred at levels above aetection limits and in association with one or more ot tour distinct protein tractions. Levels ot Ag and Cu associated with high molecular weight proteins and with "metallothionein-like" proteins permitted iaentification ot those Macoma balthica specimens exposed to the greatest degree of metal stress. INTRODUCIl ON Recent research efforts dealing with toxicity and bioaccumulation of trace elements in marine organisms have tocusea on metal binding proteins, particularly the so-called metallothioneins or metallothionein-like proteins. Roesijaai [1,2i provides an extensive review of this literature.
Aquatic Toxicology, 2010
Metallothioneins (MT) are essential metal binding proteins involved in metal homeostasis and detoxification in living organisms. Numerous studies have focused on MT response to metal exposure and showed an important variability according to species, metal, concentration and time of exposure. In this study, the expression of one isoform of MT gene (Cemt1) and associated MT protein synthesis were determined after 1, 3, 9, 24, 72 and 168 h of cadmium (Cd) or mercury (Hg) exposures in gills of the cockle Cerastoderma edule. This experiment, carried out in laboratory conditions, revealed that in Cd-exposed cockles, induction of Cemt1 is time-dependent following a "pulse-scheme" with significant upregulation at 24 h and 168 h intersected by time point (72 h) with significant downregulation. MT protein concentration increases with time in gills of exposed cockles in relation with the progressive accumulation of Cd in soluble fraction. On contrary, Hg exposure does not lead to any induction of Cemt1 mRNA expression or MT protein synthesis compared to control, despite a higher accumulation of this metal in gills of cockles compared to Cd. The localization of Hg (85-90%) is in insoluble fraction, whereas MT was located in the cytoplasm of cells. This gives us a first clue to understand the inability of Hg to activate MT synthesis. However, other biochemical processes probably occur in gills of C. edule since the remaining soluble fraction of Hg exceeds MT sequestration ability. Finally, since one of the first main targets of metal toxicity in cells was the mitochondria, some genes involved in mitochondria metabolism were also analyzed in order to assess potential differences in cellular damages between two metal exposures. Indeed, until T 168 , no impact on mitochondrial genes was shown following Hg exposure, despite the complete lack of MT response. This result indicated the presence of other effective cellular ligands which sequester the cytosolic fraction of this metal and consequently inhibit metal reactivity. Such competition mechanisms with other cytosolic ligands more sensitive to Hg were particularly argued in the discussion.
Association of selected essential (Co, Cu, Fe, Mn, Mo, Se, and Zn) and nonessential (Cd, Pb) trace elements with cytosolic proteins of different molecular masses was described for the liver of European chub (Squalius cephalus) from weakly contaminated Sutla River in Croatia. The principal aim was to establish basic trace element distributions among protein fractions characteristic for the fish living in the conditions of low metal exposure in the water. The fractionation of chub hepatic cytosols was carried out by size exclusion high performance liquid chromatography (SE-HPLC; Superdex™ 200 10/300 GL column), and measurements were performed by high resolution inductively coupled plasma mass spectrometry (HR ICP-MS). Elution profiles of essential elements were mostly characterized by broad peaks covering wide range of molecular masses, as a sign of incorporation of essential elements in various proteins within hepatic cytosol. Exceptions were Cu and Fe, with elution profiles characterized by sharp, narrow peaks indicating their probable association with specific proteins, metallothionein (MT), and ferritin, respectively. The main feature of the elution profile of nonessential metal Cd was also single sharp, narrowpeak, coinciding with MT elution time, and indicating almost complete Cd detoxification by MT under the conditions of weak metal exposure in the water (dissolved Cd concentration ≤0.3 μg L−1). Contrary, nonessential metal Pb was observed to bind to wide spectrum of proteins, mostly of medium molecular masses (30– 100 kDa), after exposure to dissolved Pb concentration of ~1 μg L−1. The obtained information within this study presents the starting point for identification and characterization of specific metal/metalloid-binding proteins in chub hepatic cytosol, which could be further used as markers of metal/ metalloid exposure or effect on fish.
Environmental Toxicology and Chemistry, 2004
Distribution of Cu, Zn, Cd, Ag, Hg, and Se were determined in hepatocytosol of northern fur seals (Callorhinus ursinus), black-footed albatrosses (Diomedea nigripes), and Dall's porpoises (Phocoenoides dalli). Copper, Zn, and Cd were accumulated preferentially in metallothionein (MT) fraction and their contents in MT fraction increased with the amounts in the hepatocytosol. Silver was bound to both high-molecular-weight substances (HMWS) and MT in the hepatocytosol for all three species, whereas the distribution of Ag in the cytosol was different among the three species. In northern fur seals, Ag mainly was bound to MT, whereas it mainly was associated with HMWS in Dall's porpoises. In contrast, Ag was distributed almost equally in both HMWS and MT for black-footed albatrosses. Mercury content in HMWS and Se content in HMWS and low-molecular-weight substances (LMWS) increased with their contents in hepatocytosol for all the three species. A significant positive correlation was found between Se and Hg contents in high-molecular weight (HMW) fraction in cytosol. The molar ratio of Hg and Se was close to unity in HMW fraction of the specimens with high Hg concentration in cytosol, implying that the Hg-Se complex was bound to the HMWS. Analysis of metals in the hepatocytosol by high-performance liquid chromatography/inductively coupled plasma-mass spectrometry (HPLC/ICP-MS) suggests that multiple isoforms of MT are present in hepatocytosol of the three species and that the metal profiles in hepatocytosols are different among the species. To our knowledge, this is the first report on the association of Ag with HMWS and MT in hepatocytosol of marine mammals and seabirds. Also, distribution and interaction of Hg and Se were investigated for the first time in hepatocytosol of the higher trophic marine animals.
Marine Biology, 2006
The decapod crustacean Penaeus indicus accumulated Cd and Zn in diVerent subcellular compartments of hepatopancreas and gill cells. Most of the Cd and part of the Zn accumulates within the soluble fraction of the cells, while the remainder of the Zn is found in insoluble inclusions, associated with P, Ca, Mg and Si in B-, F-and R-cells in the hepatopancreas, and haemocytes, nephrocytes and epithelial cells in the gills. No presence of Cd was observed in metal-rich inclusions in any cell analysed. Metallothionein-like proteins (MTLP), analysed by diVerential pulse polarography, were present in the hepatopancreas (12-18 mg g ¡1 ) and gills (7-8 mg g ¡1 ) of metal-exposed prawns. Binding to MTLP is the detoxiWcation mechanism for cadmium, while the detoxiWcation of zinc involves both binding to MTLP and incorporation into insoluble metal-rich inclusions.
Chemosphere
Zinc, Cu, Cd and Pb concentrations were determined in protein fractions of digestive gland and in the whole digestive gland of Octopus vulgaris collected from two areas of the Portuguese coast. Approximately 95% of Zn, 99% of Cu, 85-96% of Cd and 77-86% of Pb were stored in the cytosol, suggesting the predominance of cytosolic proteins in the trapping these elements. Gel filtration chromatography evidenced the presence of two major groups of proteins, with high molecular weight (HMW, 144 000-130 000 Da) and low molecular weight (LMW, 11 000-6000 Da). The following metal-protein associations were found: Zn was distributed between HMW and LMW; Cu and Cd in LMW proteins with a minor association with HMW; and Pb in HMW proteins. The strong positive correlations between Cd, Zn and Cu and LMW proteins point to the presence of metalloproteins with high affinity to these elements. A shift was registered between the maximum of the ratio 254:280 nm and metal concentrations in the chromatographic profiles. This shift may result from metallothioneins having a small participation in the metal binding or protein purification was insufficient and various LMW proteins may be interfering.