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Metallothionein
Terms modified by Metallothionein Selected AbstractsProtein-based electrochemical biosensor for detection of silver(I) ions,ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2010Sona Krizkova Abstract Silver(I) ions are extremely toxic to aquatic animals. Hence, monitoring of these ions in the environment is needed. The aim of the present study was to suggest a simple biosensor for silver(I) ions detection. The suggested biosensor is based on the modification of a hanging mercury drop electrode (HMDE) by the heavy metal binding protein metallothionein (MT) for silver(I) ions detection. Metallothionein accumulated for 120 s onto the HMDE surface. After rinsing the electrode, the biosensor (MT modified HMDE) was prepared prior to detection of silver(I) ions. The biosensor was immersed in a solution containing silver(I) ions. These ions were bound to the MT structure. Furthermore, the electrode was rinsed and transferred to a pure supporting electrolyte solution, in which no interference was present. Under these experimental conditions, other signals relating to heavy metals naturally occurring in MT were not detected. This phenomenon confirms the strong affinity of silver(I) ions for MT. The suggested biosensor responded well to higher silver(I) ion concentrations. The relative standard deviation for measurements of concentrations higher than 50,µM was approximately 2% (n,=,8). In the case of concentrations lower than 10,µM, the relative standard deviation increased to 10% (n,=,8). The detection limit (3,signal/noise) for silver(I) ions was estimated as 500,nM. Environ. Toxicol. Chem. 2010;29:492,496. © 2009 SETAC [source] Variation of metallothionein and metal concentrations in the digestive gland of the clam Ruditapes decussatus: Sex and seasonal effectsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2001Maria A. Serafim Abstract Metallothionein (MT) and metal (Cd, Cu, and Zn) concentrations were determined in the digestive gland of male and female Ruditapes decussatus. Clams were collected monthly during the period of sexual development, from June to September, at two different sites of a coastal lagoon, the Ria Formosa Lagoon, on the south coast of Portugal. The MT concentrations were determined in the heat-treated cytosolic fraction of the digestive gland of both male and female clams. Total metal (Cd, Cu, and Zn) concentrations in the heat-treated cytosol of the digestive gland were also determined in both sexes. The MT and metal concentrations in the digestive gland were not sex dependent in this species. Therefore, these results suggest that random samples can be used to determine MT and metal concentrations in the digestive gland of R. decussatus. Seasonal and site-specific dependency, however, were detected for MT, Cd, and Cu concentrations. The maximum concentration levels of MT, Cd, and Cu were detected in July and August for both sexes in the same area. Zinc concentrations, however, did not show marked seasonal variations, indicating that this species is able to regulate zinc concentrations. The highest MT concentrations were significantly related to the highest metal concentrations. [source] Ethanol-Mediated Fetal Dysmorphology and its Relationship to the Ontogeny of Maternal Liver MetallothioneinALCOHOLISM, Issue 6 2009Peter Coyle Background:, Fetal zinc (Zn) deficiency arising from ethanol-induction of the Zn-binding protein metallothionein (MT) in the mother's liver has been proposed as a mechanism of teratogenicity. Here, we determine the ontogeny of MT and Zn homeostasis in rats and mice and then examine the effect of acute ethanol exposure in early embryonic development on this relationship. The protective effect of Zn against ethanol-mediated fetal dysmorphology is also examined. Methods:, Study 1: Maternal liver MT and Zn homeostasis was determined in Sprague,Dawley rats and C57BL/6J mice throughout gestation. Study 2: Rats were administered ethanol (25% in saline, intraperitoneal 0.015 ml/g) or vehicle alone on gestational day (GD) 9. Maternal liver MT and Zn, and plasma Zn was determined over the ensuing 24 hours. Study 3: Pregnant rats were treated with ethanol and Zn (s.c. 2.5 ,g Zn/g) on GD9 and fetal dysmorphology was assessed on GD 19. Results:, Study 1: Maternal liver MT began to rise around GD 9 peaking on GD 15 before falling to nonpregnant levels around term. The pregnancy-related increase in MT was associated with a fall in plasma Zn which was significantly lower on GD 15 thereafter returning to nonpregnant levels by parturition. Study 2: Ethanol administered to pregnant rats on GD 9 resulted in a 10-fold induction of MT in the maternal liver and was associated with a 33% rise in liver Zn and a 30% fall in plasma Zn, 16 hours after treatment. Study 3: Ethanol treatment on GD 9 resulted in a significant increase in craniofacial malformations which were prevented by concurrent Zn treatment. Conclusions:, The findings indicate that maternal liver MT levels are lowest in early gestation (before GD 10) making this a sensitive period where ethanol-induction of MT can affect fetal Zn homeostasis and cause fetal dysmorphology. The study further provides evidence of a protective role for Zn against ethanol-mediated teratogenicity. [source] Acquisition of apoptotic resistance in cadmium-induced malignant transformation: Specific perturbation of JNK signal transduction pathway and associated metallothionein overexpression,MOLECULAR CARCINOGENESIS, Issue 8 2006Wei Qu Abstract Prior work has shown that chronic cadmium exposed rat liver epithelial cells (CCE-LE) become malignantly transformed after protracted low level cadmium exposure. Acquisition of apoptotic resistance is common in oncogenesis and the present work explores this possibility in CCE-LE cells. CCE-LE cells were resistant to apoptosis induced by etoposide or an acute high concentration of cadmium as assessed by flow cytometry with annexin/FITC. Three key mitogen-activated protein kinases (MAPKs), namely ERK1/2, JNK1/2, and p38, were phosphorylated in CCE-LE cells after acute cadmium exposure. However, the levels of phosphorylated JNK1/2 were markedly decreased in CCE-LE cells compared to control. JNK kinase activity was also suppressed in CCE-LE cells exposed to cadmium. Epidermal growth factor (EGF), used as a positive control for stimulating JNK phosphorylation, was much less effective in CCE-LE cells than control cells. Ro318220 (Ro), a strong activator of JNK, increased phosphorylated JNK1/2 to levels similar to the cadmium-treated control cells and also enhanced apoptosis in response to cadmium in CCE-LE cells. Metallothionein (MT), which is thought to potentially inhibit apoptosis, was strongly overexpressed in CCE-LE cells. Further, in MT knockout (MT,/,) fibroblasts, JNK1/2 phosphorylation was markedly increased after cadmium exposure compared with similarly treated wild-type (MT+/+) cells. These results indicate cadmium-transformed cells acquired apoptotic resistance, which may be linked to the specific suppression of the JNK pathway and is associated with MT overexpression, which, in turn, may impact this signal transduction pathway. The acquisition of apoptotic resistance may play an important role in cadmium carcinogenesis by contributing to both tumor initiation and malignant progression. Published 2006 Wiley-Liss, Inc. [source] Metallothionein as a biomarker for mercury in tissues of rat fed orally with cinnabarAPPLIED ORGANOMETALLIC CHEMISTRY, Issue 6 2004Zhi-Yong Huang Abstract Cinnabar, as one of the most widely used mineral drugs in traditional Chinese medicines, has been proven to have prominent curative effects in clinical use for more than 2000 years. But the safety and toxicity of the drug has been under constant debate in clinic usage. Metallothionein (MT) contains about 30% of cysteine in the molecule, and plays an important detoxification role against heavy metals. In this study, it was used as a biomarker to assess mercurial accumulation in rats fed orally with cinnabar. After feeding rats with cinnabar by gastric gavage at different dosages and at different times, the distribution of heavy metals (including mercury, copper and zinc) and MT was investigated among rat tissues, including liver, kidney, heart, brain, testis and blood. Metals and MT determinations were carried out using inductively coupled plasma mass spectrometry (ICP-MS) and a modified mercury saturation assay technique respectively. The results indicated that mercury was easily accumulated in the tissues of rats exposed to cinnabar, especially in kidney. For example: at a feeding dosage of 5 g kg,1 (bw) for 4 weeks, the mercury concentrations in kidney were 13, 8.7, 21.6 and 26 times those in liver, testis, brain and heart respectively; and at 2.5 g kg,1 (bw) for 2 weeks, the mercury concentrations in kidney were 21, 2.1, 3 and 21 times those in liver, testis, brain and heart respectively. In addition, mercury in kidney and liver of all cinnabar groups was significantly higher than that of the control group (P < 0.01). A high positive correlation observed between MT concentrations and mercury levels in both liver and kidney (R2 = 0.9299, P < 0.02 for liver; R2 = 0.9923, P < 0.0008 for kidney) indicated that MT could be used as a biomarker for mercury in tissues. Copyright © 2004 John Wiley & Sons, Ltd. [source] Induction of Metallothionein in Mesothelial Cells by ZincARTIFICIAL ORGANS, Issue 6 2007Dominik M. Alscher Abstract:, Patients on peritoneal dialysis (PD) are exposed to peritoneal dialysis fluids with unphysiological properties. Local defense systems are of importance. In this respect, metallothionein (MT) might play an important role. Because nothing is known about the achievability of MT induction in peritoneum by zinc, we performed the following study. We investigated human peritoneal mesothelial cells (HPMC) from omentum and a mesothelioma cell (MTC) line after addition of zinc in concentrations from 35 to 350 µM. Measurements of MT-mRNA and protein (by immuncytochemistry [IHC], Western blots, and dot blots) were performed. Zinc caused a clear and highly significant fourfold increase of RNA in MTC and to a lower extent in HPMC (1.6-fold, P < 0.001). IHC demonstrated a clear induction in HPMC and MTC. Western and dot blots confirmed this and showed an increase of MT from 112-mg/g total protein (TP) to 410-mg/g TP. Zinc was able to upregulate MT significantly in HPMC and MTC on the RNA and protein level. Fourfold increases of MT were achievable. [source] Increased Hepatic and Decreased Urinary Metallothionein in Rats after Cessation of Oral Cadmium ExposureBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 4 2010Yihuai Liang Wistar rats of both genders were given CdCl2 in drinking water at daily doses of 0, 2.5, 5.0 or 10.0 mg Cd/kg body-weight for 12 weeks. Half of the animals were then killed; the others were given Cd-free water for the following 16 weeks, i.e. until 28 weeks after start of the experiment (28-week rats). We observed dose-dependent increases in the levels of MT in the tissues of rats 12 weeks after beginning the experiment (12-week rats). After the exposure ceased, levels of MT in the 28-week rats changed in three ways: an increase in the liver, persistence in the kidney cortex and a decrease in the medulla, relative to those levels in their 12-week counterparts. Biomarkers of kidney dysfunction were determined to be urinary MT (UMT) and urinary N -acetyl-,- d -glucosaminidase (UNAG). After 12 weeks, we observed dose-related statistically significant increases in UMT and UNAG in all of the Cd-exposed groups. A statistically significant decrease for UNAG between the 12- and 28-week rats occurred among males at the lowest Cd dose and for UMT in all of the Cd-exposed groups. The unchanged tissue levels of MT in the kidney cortex suggest that decreased UMT is a sign either of (i) decreased transport of Cd-MT from the liver via blood plasma to the renal tubules or (ii) increased tubular reabsorption and recovery of renal tubular function. [source] X-Ray Absorption Spectroscopy of Cuprous-Thiolate Clusters in Saccharomyces cerevisiae MetallothioneinCHEMISTRY & BIODIVERSITY, Issue 10 2008Limei Zhang Abstract Copper (Cu) metallothioneins are cuprous-thiolate proteins that contain multimetallic clusters, and are thought to have dual functions of Cu storage and Cu detoxification. We have used a combination of X-ray absorption spectroscopy (XAS) and density-functional theory (DFT) to investigate the nature of Cu binding to Saccharomyces cerevisiae metallothionein. We found that the XAS of metallothionein prepared, containing a full complement of Cu, was quantitatively consistent with the crystal structure, and that reconstitution of the apo-metallothionein with stoichiometric Cu results in the formation of a tetracopper cluster, indicating cooperative binding of the Cu ions by the metallothionein. [source] Electrochemical Investigation of Strontium,Metallothionein Interactions , Analysis of Serum and Urine of Patients with OsteoporosisELECTROANALYSIS, Issue 3-5 2009Ivo Fabrik Abstract The main aim of this paper is to study interaction between strontium and metallothionein (MT), and to determine changes in strontium and thiols (MT, reduced glutathione, cysteine, and homocysteine) level in plasma, serum, and urine samples of patients treated with strontium ranelate (SrR). To investigate the interactions between MT and strontium(II) ions, adsorptive transfer stripping technique coupled with differential pulse voltammetry (DPV) the Brdicka reaction was employed. Besides standard Brdicka signals (Co, RS2Co, Cat1, Cat2, Cat3), we observed new signal related to Sr-MT interaction. Further we investigated the effect of various time of interaction, concentration of strontium(II) ions and temperature of supporting electrolyte on Brdicka signals. Optimal time of interaction was 240,s. Under temperature of supporting electrolyte 20,°C, we measured linear dependence of Cat3 signal height on strontium(II) ions concentration. After that we have investigated the possibility of strontium-MT interactions, we were interested in strontium, MT and low molecular mass thiols levels in serum and urine of patients treated with strontium(II) ions to cure osteoporosis. Strontium concentration determined by atomic absorption spectrometry was 55±5,,g/L before and 10,500±1,400,,g/L at the 30th day of SrR administration. Levels of metallothionein in serum ranged from 0.1 to 6.4,,M. Correlation between serum strontium concentration and MT level was determined and correlation coefficient was R=0.93. [source] Cover Picture: Electrophoresis 20'2008ELECTROPHORESIS, Issue 20 2008Article first published online: 7 NOV 200 Issue 20/08 has an emphasis on "Bioanalysis" since it comprises 9 research articles on this topic including the human ABO genotyping, proteins markers of dysfunctioning pancreatic beta cells, alpha amylase depletion from human saliva, analysis of high molecular mass proteins by 2-DE, analysis of the oxidation products of metallothionein, determination of pathogenic bacteria by CE, boronate affinity saccharide electrophoresis for carbohydrate analysis, fluorophore-assisted carbohydrate electrophoresis, and glycan analysis by CGE. In addition, this issue includes a "Fast Track" article on the sequence variation in part of the 60 kDa glycoprotein gene within Cryptosporidium hominis and Cryptosporidium parvum isolates from citizens of the UK, which have been inferred to have been infected while traveling abroad or in the UK. [source] Partial oxidation and oxidative polymerization of metallothioneinELECTROPHORESIS, Issue 20 2008Hajo Haase Abstract One mechanism for regulation of metal binding to metallothionein (MT) involves the non-enzymatic or enzymatic oxidation of its thiols to disulfides. Formation and speciation of oxidized MT have not been investigated in detail despite the biological significance of this redox biochemistry. While metal ion-bound thiols in MT are rather resistant towards oxidation, free thiols are readily oxidized. MT can be partially oxidized to a state in which some of its thiols remain reduced and bound to metal ions. Analysis of the oxidation products with SDS-PAGE and a thiol-specific labeling technique, employing eosin-5-iodoacetamide, demonstrates higher-order aggregates of MT with intermolecular disulfide linkages. The polymerization follows either non-enzymatic or enzymatic oxidation, indicating that it is a general property of oxidized MT. Supramolecular assemblies of MT add new perspectives to the complex redox and metal equilibria of this protein. [source] The resistance of metallothionein to proteolytic digestion: An LC-MS/MS analysisELECTROPHORESIS, Issue 16 2007Rongying Wang Abstract Metallothioneins (MTs) are a family of cysteine-rich metalloproteins which strongly bind to heavy metals, such as Cd(II), Zn(II), and Cu(I). Previous works by other group using gel electrophoresis and fluorescence showed MTs were resistant to proteolytic digestion by a variety of enzymes, raising the difficulties in proteomic identification of MTs. The present work was attempted to analyze the resistance of MTs to trypsin using LC with MS/MS (LC-MS/MS), which was able to determine the sequences of the produced peptides and thus precisely characterize the cleavages. The results showed that metal-saturated MTs were completely resistant to trypsin. This resistance problem could be overcome by the addition of EDTA to MT samples, which rendered MTs readily digested into peptides and identified by MS/MS. Interestingly, the partially metal binding MTs were digested into peptides predominantly with miss cleavages which were well dependent on the amount of heavy metals bound to MTs. An explanation for these observations was proposed. The potential applications of the MT's resistance to trypsin in isolation and identification of MTs in complex mixtures such as cultured cells was demonstrated. The preliminary data also showed the same proteomic approach of proteolytic digestion followed by MS/MS analysis may provide information on metal binding status of MTs, along with the identification of MTs in a mixture. [source] Multimarker approach in transplanted mussels for evaluating water quality in Charentes, France, coast areas exposed to different anthropogenic conditionsENVIRONMENTAL TOXICOLOGY, Issue 5 2003M. Roméo Abstract An active biomonitoring experiment was performed using mussels collected at a clean site, Fier d'Ars, and transplanted to two locations, outside the harbor of La Rochelle and in the Baie de L'Aiguillon along the coast of Charentes (French Atlantic coast) beginning in April for several months. Mussels were collected in June and October. The cadmium, copper, and zinc concentrations of all resident and transplanted mussel samples and the polycyclic aromatic hydrocarbon and polychlorinated biphenyl concentrations in some mussel samples and in the sediment samples were determined. Mussel response was evaluated for several biochemical biomarkers: concentrations of metallothionein, activities of glutathione S-transferase and acetylcholinesterase (AChE) and levels of thiobarbituric reactive substance (TBARS). The physiological status of the animals was assessed using the condition index. A principal component analysis performed with the chemical and biochemical results of the evaluations of the resident and transplanted mussels collected in June allowed them to be separated into three groups: resident mussels from la Rochelle with high metal and TBARS levels, resident mussels from Baie de L'Aiguillon with a very high condition index, and resident mussels from Fier d'Ars and transplanted mussels at La Rochelle and Baie de L'Aiguillon with low TBARS and AChE activities. Strong seasonal variation from June to October of all parameters was noted. Mussels transplanted to La Rochelle appeared to be the most "polluted" in their pollutant concentrations and biochemical responses; moreover, the La Rochelle site had the highest concentration of organics in sediments of all the sites. The choice of Fier d'Ars as a reference site may be questionable because some of the biomarker responses of the mussels were higher than expected there, although these pollutants in mussels and sediment were present at the lowest concentrations measured. © 2003 Wiley Periodicals, Inc. Environ Toxicol 18: 295,305, 2003. [source] Uptake kinetics and subcellular compartmentalization of cadmium in acclimated and unacclimated earthworms (Eisenia andrei)ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 7 2010Shuo Yu Abstract Acclimation to cadmium (Cd) levels exceeding background concentrations may influence the ability of earthworms to accumulate Cd with minimum adverse effects. In the present study, earthworms (Eisenia andrei) were acclimated by exposure to 20,mg/kg Cd (dry wt) in Webster soil for 28 d. A 224-d bioaccumulation test was subsequently conducted with both acclimated and unacclimated worms exposed in Webster soils spiked with 20,mg/kg and 100,mg/kg Cd (dry wt). Uptake kinetics and subcellular compartmentalization of Cd were examined. Results suggest that acclimated earthworms accumulated more Cd and required a longer time to reach steady state than unacclimated worms. Most of the Cd was present in the metallothionein (MT) fraction. Cadmium in the MT fraction increased approximately linearly with time and required a relatively longer time to reach steady state than Cd in cell debris and granule fractions, which quickly reached steady state. Cadmium in the cell debris fraction is considered potentially toxic, but low steady state concentrations observed in the present study would not suggest the potential for adverse effects. Future use of earthworms in ecological risk assessment should take into consideration pre-exposure histories of the test organisms. A prolonged test period may be required for a comprehensive understanding of Cd uptake kinetics and compartmentalization. Environ. Toxicol. Chem. 2010;29:1568,1574. © 2010 SETAC [source] Protein-based electrochemical biosensor for detection of silver(I) ions,ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2010Sona Krizkova Abstract Silver(I) ions are extremely toxic to aquatic animals. Hence, monitoring of these ions in the environment is needed. The aim of the present study was to suggest a simple biosensor for silver(I) ions detection. The suggested biosensor is based on the modification of a hanging mercury drop electrode (HMDE) by the heavy metal binding protein metallothionein (MT) for silver(I) ions detection. Metallothionein accumulated for 120 s onto the HMDE surface. After rinsing the electrode, the biosensor (MT modified HMDE) was prepared prior to detection of silver(I) ions. The biosensor was immersed in a solution containing silver(I) ions. These ions were bound to the MT structure. Furthermore, the electrode was rinsed and transferred to a pure supporting electrolyte solution, in which no interference was present. Under these experimental conditions, other signals relating to heavy metals naturally occurring in MT were not detected. This phenomenon confirms the strong affinity of silver(I) ions for MT. The suggested biosensor responded well to higher silver(I) ion concentrations. The relative standard deviation for measurements of concentrations higher than 50,µM was approximately 2% (n,=,8). In the case of concentrations lower than 10,µM, the relative standard deviation increased to 10% (n,=,8). The detection limit (3,signal/noise) for silver(I) ions was estimated as 500,nM. Environ. Toxicol. Chem. 2010;29:492,496. © 2009 SETAC [source] Biokinetics of cadmium and zinc in a marine bacterium: Influences of metal interaction and pre-exposureENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 8 2008Dongshi Chen Abstract The uptake kinetics of Cd and Zn, as influenced by metal interaction and metal pre-exposure, was examined in the gram-positive marine bacterium Bacillus firmus over a wide range of ambient free-Cd and -Zn concentrations. Bacteria were exposed to experimental media with different concentrations of Cd and Zn over a short, 15-min period. Zinc was found to be an effective competitive inhibitor of Cd uptake when the Zn2+ concentration ([Zn2+]) was increased to 10,8 M, whereas the Cd concentration (ranging from 10,9 to 10,6 M) did not affect Zn uptake. Inhibition of Cd uptake was dependent on [Zn2+] instead of the [Zn2+] to Cd2+ concentration ratio. Cadmium uptake at different [Zn2+] was significantly inhibited by a sulfur ligand (SH) blocker (N -ethylmaleimide) and a Ca-channel blocker (lanthanum), suggesting that competition between Cd and Zn most likely occurred via binding to the same transport sites. Cadmium efflux also was determined in the presence of different [Zn2+]. A biphasic depuration of Cd was found when [Zn2+] was greater than 10,8 M, whereas the calculated Cd efflux rate was independent of [Zn2+]. We further exposed B. firmus at different Cd or Zn concentrations for 24 h, then determined the metal uptake and efflux kinetics as well as the metallothionein (MT) induction. Both the Cd and Zn cellular concentrations increased with greater exposed metal concentration, but the MT levels and efflux were little affected by the elevated metal concentration. To some extent, however, the Cd uptake was reduced with an elevated intracellular Zn concentration, suggesting that at high Cd concentrations, intracellular Zn can suppress the Cd uptake in B. firmus. These results help to understand the interactions of metals in the marine environments. [source] The effect of food rations on tissue-specific copper accumulation patterns of sublethal waterborne exposure in Cyprinus carpioENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 7 2007Shodja Hashemi Abstract Common carp (Cyprinus carpio) were fed to two different food rations, 0.5% body weight (low ration [LR]) and 5% body weight (high ration [HR]), and were exposed to sublethal (1 ,M) copper levels for 28 d in softened Antwerp (Belgium) city tap water (Ca2+, 79.3 mg/L; Mg2+, 7.4 mg/L; Na+, 27.8 mg/L; pH 7.5,8.0). Copper accumulations in the liver, gills, kidney, anterior intestine, posterior intestine, and muscle were determined. Copper accumulation in the gills, liver, and kidney of LR fish was significantly higher than in HR fish. The only time copper uptake in HR fish was significantly higher than in LR fish was in the posterior intestine after two weeks of exposure. No difference was found between the two rations in the anterior intestine. Copper accumulation in the liver of both feeding treatments occurred in a time-dependent manner and did not reach steady state in any treatment. On the contrary, copper concentration in the gills reached a steady state for both HR and LR fish within the first week of exposure. No copper accumulation was found in muscle tissues of either treatment. Copper concentration dropped to control levels in all tissues, except liver tissue, two weeks after the exposure ended. Our studies indicated that copper uptake was influenced by the food ration in carp. The difference in copper accumulation probably is related to the amount of dietary NaCl and different rates of metallothionein synthesis. Low food availability provides less Na+ influx and leads to increased brachial uptake of Na+ and copper. In addition, it has been shown that starved animals show increased levels of metallothionein, possibly causing higher copper accumulation. [source] Metallothionein gene expression and protein levels in triploid and diploid oysters Crassostrea gigas after exposure to cadmium and zincENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 2 2006Véronique Marie Abstract Quantitative real-time polymerase chain reaction (PCR) was used to compare for the first time the differential expression of metallothionein (MT) isoform genes, together with biosynthesis of the total MT proteins, in the gills of triploid and diploid juvenile Pacific oyster Crassostrea gigas in response to cadmium (Cd) and zinc (Zn) exposure. Oysters were exposed to Cd (0.133 ,M), Zn (15.3 ,M), and Cd+Zn for 14 d. Results showed similar response capacities to metal exposures in the two populations. No significant difference was revealed in terms of MT gene expression, MT protein synthesis, and Cd accumulation. However, triploid oysters bioaccumulated Zn 30% less efficiently than diploid oysters. Among the three MT isoform genes, CgMT2 appeared to be more expressed than CgMT1, whereas CgMT3 appeared to be anecdotal (106 times lower than CgMT2). CgMT2 and CgMT1 gene expression levels were increased sevenfold in the presence of Cd, whereas Zn appeared to have no effect. A twofold increase in MT protein levels occurred in response to Cd exposure. Discrepancies between mRNA and protein levels suggest that in C. gigas MT are regulated at the transcriptional level, as well as at the translational level. [source] Subcellular cadmium distribution, accumulation, and toxicity in a predatory gastropod, Thais clavigera, fed different preyENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 1 2006Ma-Shan Cheung Abstract Bioaccumulation and toxicity of Cd were investigated in a marine predatory whelk, Thais clavigera, after being fed with the rock oyster, Saccostrea cucullata, or the herbivorous snail, Monodonta labio, for up to four weeks. The oysters and snails had different subcellular Cd distributions and concentrations in their bodies given their different metal-handling strategies and were exposed to dissolved Cd for two weeks before being fed to the whelks. After four weeks of dietary exposure, the Cd body concentrations in T. clavigera increased from 3.1 ,g/g to between 22.9 and 41.8 ,g/g and to between 22.7 and 24.1 ,g/g when they were fed with oyster and snail prey, respectively. An increasing proportion of Cd was found to be distributed in the metallothionein (MT)-like proteins and organelle fractions, whereas the relative distribution in the metal-rich granules fraction decreased when the whelks were fed Cd-exposed prey. At the highest Cd dosage, more Cd was distributed in the pool of metal-rich granules when the whelks were fed the oysters than when they were fed the snails. Among all the biomarkers measured (MT induction, condition index, lipid peroxidation, and total energy reserve including carbohydrate, lipid, and protein), only MT showed a significant difference from the control treatments, and MT was the most sensitive biomarker for dietary Cd exposure. No toxicity was found in the whelks fed different Cd-exposed prey as revealed by various biomarkers at the different biological levels. Our results imply that metal fractionation in prey can alter the subsequent subcellular metal distribution in predators and that dietary Cd toxicity to the whelks was low, even when the accumulated Cd body concentrations were high. [source] Gene expression in caged fish as a first-tier indicator of contaminant exposure in streamsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 12 2005Aaron P. Roberts Abstract The development of sensitive, biologically based indicators of contaminant exposure (i.e., biomarkers) is an ongoing topic of research. These indicators have been proposed as a first-tier method of identifying contaminant exposure. The primary objective of this research was to implement a biomarker-based method of exposure assessment using caged fish and real-time reverse-transcriptase polymerase chain reaction (rtRT-PCR) measurements of gene expression. Primers were developed for the CYPIA, metallothionein, and vitellogenin genes in rainbow trout (Oncorhynchyus mykiss), cutbow trout (Oncorhynchyus clarkii × mykiss), and Atlantic salmon (Salmo salar). Each of these genes has been shown to respond specifically to planar aromatic compounds, heavy metals, and environmental estrogens, respectively. Juvenile fish were placed in cages and exposed in situ at reference and contaminated sites on the Cache la Poudre River (CO, USA), the Arkansas River (CO, USA), the St. John River (NB, Canada), and two urban creeks near Dayton (OH, USA). Quantitative gene expression was determined using rtRT-PCR. Biomarker expression profiles were obtained that demonstrated differences in CYP1A, metallothionein, and vitellogenin mRNA production unique to each site, indicating that specific types of compounds were bioavailable and present in sufficient concentrations to elicit transcriptional responses in the organism. These findings support the use of a biomarker-based approach to exposure identification and assessment. [source] Association of heavy metals with metallothionein and other proteins in hepatic cytosol of marine mammals and seabirdsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 8 2004Tokutaka Ikemoto Abstract 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. [source] Isoform-specific quantification of metallothionein in the terrestrial gastropod Helix pomatia.ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 4 2004Abstract The two function-specific metallothionein (MT) isoforms characterized from the midgut gland and mantle tissue of Helix pomatia differ substantially in their metal-binding preferences, as well as molecular and biochemical features. These differences make them potential candidates for biomarker studies based on a differential, isoform-specific approach. To prove this hypothesis, induction experiments with two metals (Cd and Cu) that are normally bound by the two isoforms were compared with a range of organic chemicals and physical stressors under laboratory conditions to test the responsiveness of the two isoforms to the stressors applied. In addition, field studies were conducted with Roman snails and substrate samples collected from different metal-contaminated sites in Austria to test the suitability of the two isoforms as biomarkers under field conditions. The results of these combined laboratory and field studies confirmed the validity of the biomarker approach with the two metal- and tissue-specific isoforms. It is demonstrated that the Cd-binding MT specifically and exclusively responds to Cd exposure by increasing concentrations, whereas the Cu-binding MT isoform decreases in its concentration upon exposure to physical stress (X-ray irradiation and cold). This suggests researchers should adopt, under certain preconditions, a dual biomarker approach by combining the simultaneous quantification of Cd-MT concentrations in the midgut gland as a biomarker for Cd pollution and of Cu-MT concentration in the mantle as a biomarker for the impairment of snails by additional physical stressors. [source] Oxidative stress biomarkers in bivalves transplanted to the Guadalquivir estuary after Aznalcóllar spillENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 1 2003Antonio Romero-Ruiz Abstract Biological effects of metals were studied in clams (Scrobicularia plana) transplanted to Guadalquivir estuary (Spain) at several times after the spill of toxic metals from Aznalcóllar pyrite mine (southwest Spain) (April 1998) using biochemical biomarkers responsive to reactive oxygen species. Significant As, Cd, Fe, Mn, Ni, and Pb uptake was found in clams living for seven months at the estuary (from July 1999). Increased activity of antioxidant (catalase, glucose-6-phosphate, and 6-phosphogluconate dehydrogenase) and glutathione-related (glutathione reductase and glyoxalase I and II) enzymes was also found after short exposures; the levels of malondialdehyde and metallothionein increased also, particularly with long exposures. Clams living four weeks at the estuary (from March 2000) but not at a reference site also accumulated metals. The higher malondialdehyde and lower reduced-glutathione levels and the more oxidized glutathione status confirmed the oxidative stress of clams living at the estuary, while no marked increase of antioxidant activities was found this time. Lower metal availability along the second transplant could explain the limited responses in this shorter experiment. Although the status of Guadalquivir estuary has recovered since Aznalcóllar spill, continuous monitoring is needed to confirm its progress and to be alert to possible deterioration after heavy rains. [source] Variation of metallothionein and metal concentrations in the digestive gland of the clam Ruditapes decussatus: Sex and seasonal effectsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2001Maria A. Serafim Abstract Metallothionein (MT) and metal (Cd, Cu, and Zn) concentrations were determined in the digestive gland of male and female Ruditapes decussatus. Clams were collected monthly during the period of sexual development, from June to September, at two different sites of a coastal lagoon, the Ria Formosa Lagoon, on the south coast of Portugal. The MT concentrations were determined in the heat-treated cytosolic fraction of the digestive gland of both male and female clams. Total metal (Cd, Cu, and Zn) concentrations in the heat-treated cytosol of the digestive gland were also determined in both sexes. The MT and metal concentrations in the digestive gland were not sex dependent in this species. Therefore, these results suggest that random samples can be used to determine MT and metal concentrations in the digestive gland of R. decussatus. Seasonal and site-specific dependency, however, were detected for MT, Cd, and Cu concentrations. The maximum concentration levels of MT, Cd, and Cu were detected in July and August for both sexes in the same area. Zinc concentrations, however, did not show marked seasonal variations, indicating that this species is able to regulate zinc concentrations. The highest MT concentrations were significantly related to the highest metal concentrations. [source] Aldehydes release zinc from proteins.FEBS JOURNAL, Issue 18 2006A pathway from oxidative stress/lipid peroxidation to cellular functions of zinc Oxidative stress, lipid peroxidation, hyperglycemia-induced glycations and environmental exposures increase the cellular concentrations of aldehydes. A novel aspect of the molecular actions of aldehydes, e.g. acetaldehyde and acrolein, is their reaction with the cysteine ligands of zinc sites in proteins and concomitant zinc release. Stoichiometric amounts of acrolein release zinc from zinc,thiolate coordination sites in proteins such as metallothionein and alcohol dehydrogenase. Aldehydes also release zinc intracellularly in cultured human hepatoma (HepG2) cells and interfere with zinc-dependent signaling processes such as gene expression and phosphorylation. Thus both acetaldehyde and acrolein induce the expression of metallothionein and modulate protein tyrosine phosphatase activity in a zinc-dependent way. Since minute changes in the availability of cellular zinc have potent effects, zinc release is a mechanism of amplification that may account for many of the biological effects of aldehydes. The zinc-releasing activity of aldehydes establishes relationships among cellular zinc, the functions of endogenous and xenobiotic aldehydes, and redox stress, with implications for pathobiochemical and toxicologic mechanisms. [source] Comparative metal binding and genomic analysis of the avian (chicken) and mammalian metallothioneinFEBS JOURNAL, Issue 3 2006Laura Villarreal Chicken metallothionein (ckMT) is the paradigm for the study of metallothioneins (MTs) in the Aves class of vertebrates. Available literature data depict ckMT as a one-copy gene, encoding an MT protein highly similar to mammalian MT1. In contrast, the MT system in mammals consists of a four-member family exhibiting functional differentiation. This scenario prompted us to analyse the apparently distinct evolutionary patterns followed by MTs in birds and mammals, at both the functional and structural levels. Thus, in this work, the ckMT metal binding abilities towards Zn(II), Cd(II) and Cu(I) have been thoroughly revisited and then compared with those of the mammalian MT1 and MT4 isoforms, identified as zinc- and copper-thioneins, respectively. Interestingly, a new mechanism of MT dimerization is reported, on the basis of the coordinating capacity of the ckMT C-terminal histidine. Furthermore, an evolutionary study has been performed by means of in silico analyses of avian MT genes and proteins. The joint consideration of the functional and genomic data obtained questions the two features until now defining the avian MT system. Overall, in vivo and in vitro metal-binding results reveal that the Zn(II), Cd(II) and Cu(I) binding abilities of ckMT lay between those of mammalian MT1 and MT4, being closer to those of MT1 for the divalent metal ions but more similar to those of MT4 for Cu(I). This is consistent with a strong functional constraint operating on low-copy number genes that must cope with differentiating functional limitation. Finally, a second MT gene has been identified in silico in the chicken genome, ckMT2, exhibiting all the features to be considered an active coding region. The results presented here allow a new insight into the metal binding abilities of warm blooded vertebrate MTs and their evolutionary relationships. [source] The effects of physiologically important nonmetallic ligands in the reactivity of metallothionein towards 5,5,-dithiobis(2-nitrobenzoic acid)FEBS JOURNAL, Issue 18 2001A new method for the determination of ligand interactions with metallothionein The reaction of Cd5Zn2 -metallothionein (MT) with 5,5,-dithiobis(2-nitrobenzoic acid) (Nbs2) has been studied at different reagent stoichiometries, pH and temperature conditions and in the presence of several ligands. At stoichiometries of Nbs2 to MT from 0.5 to 5, the reaction followed first order kinetics. The first order rate constants obtained were independent from the concentration of Nbs2 but were linearly dependent on the concentration of MT. At higher Nbs2/MT stoichiometries, the reaction deviates from first order kinetics and the observed rate constant increases. The reactivity of MT towards Nbs2 has been probed at 4 µm concentration of both reagents where the reaction is monophasic and is characterized by a linear Arrhenius plot (Ea = 45.8 ± 2.7 kJ·mol,1). It has been demonstrated that metal release at low pH or subtraction from MT by EDTA substantially increases the reactivity of MT towards Nbs2. At the same time, a number of nonmetallic ligands moderately accelerate the reaction of MT with Nbs2 and hyperbolic dose,response curves were obtained. The data have been interpreted with the binding of ligands to MT and following MT. Ligand binding constants were calculated as follows: ATP, K = 0.31 ± 0.06 mm; ADP, K = 0.26 ± 0.07 mm. Several compounds such as AMP, S -methylglutathione, and phosphate had no effect on the reaction, but Zn2+ ions showed an inhibitory effect at micromolar concentrations. [source] Regulation of gene expression in inflammatory bowel disease and correlation with IBD drugs.INFLAMMATORY BOWEL DISEASES, Issue 1 2004Screening by DNA microarrays Abstract Potential biomarkers for Crohn's disease (CD) and ulcerative colitis (UC) were identified from two sets of full thickness pathologic samples utilizing DermArray® and PharmArray® DNA microarrays relative to uninvolved (Un) colon or normal colon. Seven of the over-expressed genes were verified using quantitative RT-PCR (i.e., TMPT, FABP1, IFI27, LCN2, COL11A2, HXB, and metallothionein). By correlating gene expression profiles between inflammatory bowel disease (IBD) tissue samples and IBD drug-treated cell cultures it might be possible to identify new candidate molecular target genes for IBD therapy and drug discovery. Potential biomarkers for CaCo2 cell cultures, which are routinely used as a GI tract surrogate model for in vitro pharmacokinetic studies, treated with azathioprine, 5-aminosalicylic acid, metronidazole, and prednisone were also identified from another experiment. Metallothionein mRNA expression was found to be down-regulated in azathioprine-treated CaCo2 cells, and was coincidentally up-regulated in the CD sample, thus resulting in an anti-correlation. These results suggest that this new screening methodology is feasible, that metallothioneins might be biomarkers for azathioprine therapy in vivo in CD, and that azathioprine might mechanistically down-regulate metallothionein gene expression. Correlations were also observed between IBD samples and either metronidazole- or 5-aminosalicylic acid-treated CaCo2 cells. Similar comparisons of disease tissue samples in vivo vs drug-treated cell cultures in vitro might reveal new mechanistic insights concerning established or experimental drug therapies. This affordable in vitro methodology is promising for expanded studies of IBD and other diseases. [source] Detoxification and antioxidant effects of curcumin in rats experimentally exposed to mercuryJOURNAL OF APPLIED TOXICOLOGY, Issue 5 2010Rakhi Agarwal Abstract Curcumin, a safe nutritional component and a highly promising natural antioxidant with a wide spectrum of biological functions, has been examined in several metal toxicity studies, but its role in protection against mercury toxicity has not been investigated. Therefore, the detoxification and antioxidant effects of curcumin were examined to determine its prophylactic/therapeutic role in rats experimentally exposed to mercury (in the from of mercuric chloride-HgCl2, 12,µmol,kg,1 b.w. single intraperitoneal injection). Curcumin treatment (80,mg,kg,1 b.w. daily for 3 days, orally) was found to have a protective effect on mercury-induced oxidative stress parameters, namely, lipid peroxidation and glutathione levels and superoxide dismutase, glutathione peroxidase and catalase activities in the liver, kidney and brain. Curcumin treatment was also effective for reversing mercury-induced serum biochemical changes, which are the markers of liver and kidney injury. Mercury concentration in the tissues was also decreased by the pre/post-treatment with curcumin. However, histopathological alterations in the liver and kidney were not reversed by curcumin treatment. Mercury exposure resulted in the induction of metallothionein (MT) mRNA expressions in the liver and kidney. Metallothionein mRNA expression levels were found to decrease after the pre-treatment with curcumin, whereas post-treatment with curcumin further increased MT mRNA expression levels. Our findings suggest that curcumin pretreatment has a protective effect and that curcumin can be used as a therapeutic agent in mercury intoxication. The study indicates that curcumin, an effective antioxidant, may have a protective effect through its routine dietary intake against mercury exposure. [source] The effect of superoxide dismutase deficiency on cadmium stressJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 1 2004Paula D. B. Adamis Abstract Saccharomyces cerevisiae mutant strains deficient in superoxide dismutase (Sod), an antioxidant enzyme, were used to analyze cadmium absorption and the oxidation produced by it. Cells lacking the cytosolic Sod1 removed twice as much cadmium as the control strain, while those deficient in the mitochondrial Sod2 exhibited poor metal absorption. Interestingly, the sod1 mutant did not become more oxidized after exposure to cadmium, as opposed to the control strain. We observed that the deficiency of Sod1 increases the expression of both Cup1 (a metallothionein) and Ycf1 (a vacuolar glutathione S-conjugate pump), proteins involved with protection against cadmium. Furthermore, when sod1 cells were exposed to cadmium, the ratio glutathione oxidized/glutathione reduced did not increase as expected. We propose that a high level of metallothionein expression would relieve glutathione under cadmium stress, while an increased level of Ycf1 expression would favor compartmentalization of this metal into the vacuole. Both conditions would reduce the level of glutathione-cadmium complex in cytosol, contributing to the high capacity of absorbing cadmium by the sod1 strain. Previous results showed that the glutathione-cadmium complex regulates cadmium uptake. These results indicate that, even indirectly, metallothionein also regulates cadmium transport. © 2004 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:12,17, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20000 [source] | |||||||||||||||||||