Liver Metallothionein Research Articles

Cadmium downregulates ZnT‐4 and induces mammary gland involution in lactating mice

Numerous studies in various species have shown that cadmium (Cd) is retained in the lactating mammary gland (LMG) and is only poorly transferred to milk suggesting that the LMG may act as a barrier to protect the suckling offspring from exposure to Cd. The mechanism for the Cd retention is unknown but may involve interaction with metallothionein (MT) or members of the family of zinc transporters (ZnTs). The aim of the present study was to examine the morphology of the LMG and protein expressions of MT, ZnT‐2, ZnT‐4 or ZnT‐7 in Cd treated mice. A total of 12 primiparous NMRI dams with normalized litter sizes (n=8) were given 17.8 umol Cd/kg/day or vehicle on postnatal days (PND) 8, 9 and 10. Left inguinal mammary glands (4 and 5) were dissected on PND 11 for morphological examination, Zn‐analyses and immunoblotting. Livers were taken from the dams for MT westerns. The histopathology demonstrated increased fat content in the LMG of the Cd treated dams and a less active feature of the alveolar epithelium as compared to the controls, indicating a Cd induced involution. Total Zn‐levels in the LMG were unaffected by Cd. ZnT‐4 protein was significantly downregulated as a result of the Cd treatment whereas MT, ZnT‐2 and ZnT‐7expressions were unaffected. Liver MT was upregulated in the Cd treated dams. Our results indicate that Cd induces involution of the LMG and that ZnT‐4 is involved in this process.

The plausibility of maternal nutritional status being a contributing factor to the risk for fetal alcohol spectrum disorders: The potential influence of zinc status as an example

There is increasing evidence that human pregnancy outcome can be significantly compromised by suboptimal maternal nutritional status. Poor diet results in a maternal-fetal environment in which the teratogenicity of other insults such as alcohol might be amplified. As an example, there is evidence that zinc (Zn) can interact with maternal alcohol exposure to influence the risk for fetal alcohol spectrum disorders (FASD). Studies with experimental animals have shown that the teratogenicity of alcohol is increased under conditions of Zn deficiency, whereas its teratogenicity is lessened when animals are given Zn-supplemented diets or Zn injections before the alcohol exposure. Alcohol can precipitate an acute-phase response, resulting in a subsequent increase in maternal liver metallothionein, which can sequester Zn and lead to decreased Zn transfer to the fetus. Importantly, the teratogenicity of acute alcohol exposure is reduced in metallothionein knockout mice, which can have improved Zn transfer to the conceptus relative to wild-type mice. Consistent with the above, Zn status has been reported to be low in alcoholic women at delivery. Preliminary data from two basic science and clinical nutritional studies that are ongoing as part of the international Collaborative Initiative on Fetal Alcohol Spectrum Disorders support the potential role of Zn, among other nutritional factors, relative to risk for FASD. Importantly, the nutrient levels being examined in these studies are relevant to general clinical populations and represent suboptimal levels rather than severe deficiencies. These data suggest that moderate deficiencies in single nutrients can act as permissive factors for FASD, and that adequate nutritional status or intervention through supplementation may provide protection from some of the adverse effects of prenatal alcohol exposure.

Metal and metallothionein content in bullfrogs: Study of a whole watershed impacted by agricultural activities

Among the various anthropogenic activities potentially affecting amphibian populations, agriculture has often been evoked. Fertilizers used in agriculture are documented sources of metals that may contaminate nearby rivers. Bullfrogs ( Rana catesbeiana) were collected within the Yamaska River basin (Quebec, Canada) along a gradient of agricultural land use intensity. Hepatic, renal and intestinal metal content, along with hepatic metallothionein content, were determined. In general, dissolved metal concentrations and accumulated metal concentrations in bullfrogs varied little among sites. Hepatic Cu and Zn concentrations were significantly different among subwatersheds and were in accordance with the gradient in agricultural activities in at least one of the three years studied. Liver metallothionein content did not vary significantly among the subwatersheds, but they were correlated with Cu content within two sites and Cd content at one site. We conclude that R. catesbeiana could be used as a biomonitor for Cu contamination and potentially for other metals.

Trace Elements and Metallothionein in Liver and Kidney of Felis catus

Trace metals such as Zn, Cu, and Fe are essential for life; differently, no biochemical function is known for Cd. Changes in dietary metal concentrations can cause deficiency or toxicity. Studies on trace elements in cat are lacking. This paper aimed to analyze Zn, Cu, Fe and Cd concentrations in liver and kidney of pathological domestic cat and to isolate metallothionein (MT) in these tissues. It was not possible to explore a possible correlation between metal concentrations and pathologies because the incidence for each of them was too low. Fe was the most abundant metal; in particular, the liver accumulates average Fe concentrations one order of magnitude higher than Zn and Cu, ranging from 66.75 and 1,444.23 microg/g. Significantly, higher levels of Fe were found in the liver of elder animals. Zn concentrations varied between 26.31 and 84.78 microg/g in the liver whereas in the kidney, ranged between 7.69 and 71.15 microg/g. Cu concentrations were between 2.37 and 112.91 microg/g in liver and between 2.12 and 9.85 microg/g in kidney. Cd was the least abundant metal with the exception of the kidney of the oldest cats where it reached a maximum of 13.71 microg/g. Gel-filtration metal distribution profiles from cytosolic extracts revealed the presence of Cd, Cu, Zn thioneins either in the liver or in the kidney. Because tissue samples were taken from pathological cats from different breed and age, care must be taken to use these data as a baseline profile of trace elements in healthy animals. Our results are indicative that for some specimens the feed levels of Fe and Cu could be higher than the optimal dietary intake and in few cats, there was also an exposure to Cd that was counteracted by MT biosynthesis.

Alignment of Gold Clusters on DNA via a DNA-Recognizing Zinc Finger-Metallothionein Fusion Protein

The complementary recognition of base pairs (bp) is the major strategy in the "DNA lithography" of gold (Au)clusters or nanoparticles, where single-stranded DNAs sulfurized at their termini are generally used to bind Au clusters or nanoparticles. In this report, we discuss a new material that can be used to locate Au clusters on the desired positions of DNA. For this purpose, we combined a two-domain zinc finger (ZF) and the analogue of R domain of rat's liver metallothionein (MT) to utilize the DNA-recognizing ability of ZF motifs and the heavy metal binding ability of MTs, and prepared an artificial fusion protein, ZFZF-MTalpha (1). Titration experiments monitored by absorption and circular dichroism spectroscopies, as well as an electrophoretic mobility shift assay and quantification using 5,5'-dithiobis(2-nitrobenzoic acid), clarified that (1) the ZF domain traps two divalent metal ions to fold in a ZF structure with M(Cys)2(His)2 (M = Co, Zn, and Cd) coordination units, (2) the MT domain traps metal ions to form clusters (Cd2+ particularly forms a Cd4(Cys)9 cluster) without interfering with the folding of the ZF domain, and (3) 1 recognizes the 5'-GGGGGG-3' (G6) bp sequence in the presence of Zn2+ based on the amino acid sequence encoded in the ZF domain. The titration of Au11(PPh3)8Cl3 (Au11) into the solution of 1 in the presence of Zn2+ revealed that the MT domain strongly binds the Au11 cluster with a 1:1 ratio, and a Au11-containing conjugate, ZF(Zn)ZF(Zn)-MTalpha(Au11) was obtained. Transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy showed that this conjugate maintains an Au11 core in the form of Au11(PPh3)4(S-Cys)6 without disturbing the folding nature of the ZF domain. ZF(Zn)ZF(Zn)-MTR(Au11) recognizes the bp sequence G6 with K(d1) = 450 nM, while simultaneously forming a dimer on the DNA with K(d2) = 200 nM. TEM experiments showed that the conjugates form parallelograms or triangles (defective parallelograms) on a double crossover (dx) DNA, according to the positions of G6 encoded in the dx DNA.

Reaction of the zinc sensor FluoZin-3 with Zn7-metallothionein: Inquiry into the existence of a proposed weak binding site

It has been reported that Zn(7)-metallothionein (MT), contains one weak binding site for Zn(2+). To test this conclusion, rabbit liver MT isolated at pH 7 was reacted with chelating agents of modest affinity for Zn(2+). Contrary to the previous study, no evidence was found for Zn(2+) stoichiometrically bound to the protein with an apparent stability constant of about 10(8). Indeed, stability constant measurements based upon competition between Zn(7)-MT and ligands of known stability with Zn(2+) showed that all of the protein bound Zn(2+) displayed the same stability constant at pH 7.4 and 25 degrees C of (1.7+/-0.6)x10(11). Brief reaction of Zn(7)-MT with strong acid converted it into MT(*) and upon reneutralization into Zn(7)-MT(*), which demonstrated reactivity of about 1 Zn(2+)/mol MT with competing ligands. Acid titration of Zn(7)-MT to pH 2 or below rapidly resulted in the formation of Zn(7)-MT(*) that displayed biphasic titration with base, revealing the rebinding of lower affinity Zn(2+) between pH 5 and 7. Since MT is commonly acidified during preparation, care must be taken to document which form of the protein is present in subsequent experiments at pH 7.

Mechanism of protection by metallothionein against acetaminophen hepatotoxicity

Acetaminophen (APAP) overdose is the most frequent cause of drug-induced liver failure in the US. Metallothionein (MT) expression attenuates APAP-induced liver injury. However, the mechanism of this protection remains incompletely understood. To address this issue, C57BL/6 mice were treated with 100 μmol/kg ZnCl 2 for 3 days to induce MT. Twenty-four hours after the last dose of zinc, the animals received 300 mg/kg APAP. Liver injury (plasma ALT activities, area of necrosis), DNA fragmentation, peroxynitrite formation (nitrotyrosine staining), MT expression, hepatic glutathione (GSH), and glutathione disulfide (GSSG) levels were determined after 6 h. APAP alone caused severe liver injury with oxidant stress (increased GSSG levels), peroxynitrite formation, and DNA fragmentation, all of which were attenuated by zinc-induced MT expression. In contrast, MT knockout mice were not protected by zinc. Hydrogen peroxide-induced cell injury in primary hepatocytes was dependent only on the intracellular GSH levels but not on MT expression. Thus, the protective effect of MT in vivo was not due to the direct scavenging of reactive oxygen species. Zinc treatment had no effect on the early GSH depletion kinetics after APAP administration, which is an indicator of the metabolic activation of APAP to its reactive metabolite N-acetyl- p-benzoquinone imine (NAPQI). However, MT was able to effectively trap NAPQI by covalent binding. We conclude that MT scavenges some of the excess NAPQI after GSH depletion and prevents covalent binding to cellular proteins, which is the trigger for the propagation of the cell injury mechanisms through mitochondrial dysfunction and nuclear DNA damage.

Behavioural effects of cadmium (Cd) in free-living willow ptarmigan (Lagopus lagopus)

The objective of this study was to examine effects of sublethal cadmium (Cd) contamination on parental behaviour and chick survival in willow ptarmigan (Lagopus lagopus) in an area that does not have high environmental levels of Cd due to geology but have high environmental levels of Cd due to anthropogenic deposition and acidification. One group of willow ptarmigan hens were exposed to Cd (83.2 mg Cd/L) after egg-laying using osmotic minipumps; whereas, another group given saline served as a control. No statistically significant difference in Cd concentration between the two groups was found. There was a significant correlation between the Cd concentrations in the liver and kidney of the birds and between Cd and metallothionein (MT) concentration in the kidney. No significant differences were found between the control hens and the Cd-contaminated hens with respect to any of the behaviour parameters. Age seemed to be more important than Cd treatment as adult hens showed more distraction display, had lower flushing distance, and higher brood attendance than juveniles. There were no difference in chick survival between Cd-contaminated and control hens, and there was no correlation between Cd concentrations and chick survival. The non-significant effects of Cd concentrations on parental behaviour may be due high environmental levels of Cd caused by anthropogenic deposition and acidification. We suggest that willow ptarmigan in the area have gradually evolved abilities to cope with high environmental levels of Cd, for instance, by binding Cd to MT in kidney and liver. This may suggest that Cd exerts a strong selection pressure on birds causing a relatively rapid evolution of protection mechanisms.

Ethanol‐Mediated Fetal Dysmorphology and its Relationship to the Ontogeny of Maternal Liver Metallothionein

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. 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 microg Zn/g) on GD9 and fetal dysmorphology was assessed on GD 19. 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. 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.

Conjugation of Au11 cluster with Cys-rich peptides containing the α-domain of metallothionein

The stoichiometrically controlled complexation of abiological inorganic clusters with proteins is a challenging task that enables us to combine the functions of inorganic clusters with those of biological systems on the basis of chemical characterisation. In this study, we synthesised a 1:1 conjugate between Au11(PPh3)8Cl3 (Au11) and CGMTIIalpha, where Au11 is the smallest cluster, which is situated on the boundary of molecules and nanoparticles, having discrete electronic states and exhibiting Coulomb blockade; on the other hand, CGMTIIalpha is a twelve-cysteine (Cys)-containing peptide consisting of the alpha-domain of rat liver metallothionein II (MTIIalpha)--a heavy metal-binding protein--and an addendum, cysteine-glycine (CG). The Cys residue of the N-terminus is used as a site for elongation by the native chemical ligation (NCL) method. Titration experiments monitored by circular dichroism spectroscopy indicate that CGMTIIalpha reacts with Au11 in a ratio of 1:1. The product exhibits a negative Cotton effect at 222 nm, indicating the formation of a short a-helix. The composition of this hybrid module is quantified as Au11(PPh3)4(S-Cys)6 by transmission electron microscopy (core size observation), energy-dispersive X-ray spectroscopy (elemental analysis), Ellman test (uncoordinated Cys residues), and high-performance liquid chromatography (quantification of eliminated PPh3). These results, as well as those obtained from the simulations of Au 4f X-ray photoelectron spectra, suggest that the Au cluster of this product is Au11(PPh3)4(S-Cys)6. We also prepared F3F3-CGMTIIalpha, where F3F3 is a zinc finger peptide with two domains, in order to demonstrate the applicability of the NCL method to the elongation of CGMTIIalpha. F3F3-CGMTIIalpha also reacts with Au11 to yield a 1:1 conjugate without interfering in the folding of the zinc finger.

Metallothionein and metal levels in liver, gills and kidney of Sparus aurata exposed to sublethal doses of cadmium and copper

The levels of metallothionein (MT), a biomarker of metal exposure, and of Cd and Cu, known as MT inducers, were investigated in Sparus aurata intraperitoneally injected with 500 microg/kg of Cu and Cd for 2 days. MT and metal concentrations (Cd and Cu) were determined in liver, gills and kidney. MT levels were significantly increased in all investigated tissues, with the highest value in liver of Cu as Cd-treated fishes (3.56-fold and 3.3- fold, respectively). Metal concentrations were statistically different between all tissues. Highest metal concentrations were in the liver. The higher metal concentrations and MT induction levels support the main role of MT in metal homeostasis and detoxification.

Tissue Metallothionein Concentrations in Mice and Humans with Hyperglycemia

Besides participating in tissue zinc homeostasis and protecting against heavy metal toxicities, metallothionein (MT) is known as an antioxidant. Increased MT activity can ameliorate diabetic hyperglycemia, and subjects with less MT synthesis are more prone to diabetic complications. However, whether tissue MT status is varied in the subjects with diabetes mellitus remains unclear. This study was undertaken to measure tissue MT levels in laboratory mice (serum, liver, and epididymal adipose tissue) and humans (serum) with hyperglycemia. Tissue MT levels were measured by enzyme-linked immunosorbent assay. The results showed that MT status in serum and adipose tissue did not markedly differ between the subjects with and without hyperglycemia. In addition, streptozotocin- and high-fat-diet-induced hyperglycemic mice had higher while ob/ob mice had lower liver MT levels than that of normal control mice. Furthermore, serum MT levels tended to correlate with glycemia values in mice. The results of this study indicate that serum MT value does not differ in subjects with hyperglycemia and cannot be used as an index to evaluate the susceptibility or progress of diabetes mellitus.

Production of Metallothionein Polyclonal Antibodies Using Chickens as Model

The production of polyclonal antibodies (pAbs) against metallothioneins (MT) has been done in mammals. In this work, we describe a model where pAbs against rat liver MT were produced in chickens. Liver MT-1 and MT-2 isoforms isolated from rats were used as immunogens. MT was purified by exclusion chromatography and MT isoforms isolated by ionic exchange chromatography. Chickens were immunized with each isoform emulsified with Freund adjuvant over 6 weeks. MT-pAbs obtained from egg yolk were purified by ammonium sulfate precipitation followed by thiophilic interaction chromatography. MT-pAbs were characterized by ELISA, SDS-PAGE electrophoresis, and Western blot assays. Results showed significant titers (1:1,000) of MT-1 and MT-2 IgY in the eggs collected 30 days after the first immunization as determined by a direct ELISA assay; results also show a cross-reaction between MT-1 and MT-2 isoforms: however, the Abs obtained did not react with other non-MT proteins in hepatic homogenates. Sensitivity assays showed that MT-pAbs detected MT-1 and MT-2 at nanogram levels. These data suggest that chickens are an alternative model for producing pAbs against mammal high-homology proteins such as MT.

Dose- and Time-Dependent Effects of Luteolin on Liver Metallothioneins and Metals in Carbon Tetrachloride-Induced Hepatotoxicity in Mice

The aim of this study was to investigate the protective effect of luteolin on liver Ca, Mg, Zn, Cu, Fe, and Mn content in mice with carbon tetrachloride (CCl4)-induced hepatotoxicity. Additionally, liver metallothionein (MT) expression was studied. Luteolin was administered intraperitoneally (i.p.) as a single 5- or 50-mg/kg dose or once daily for two consecutive days, respectively. Two hours after the last injection, the mice were treated with CCl4 (20 mg/kg, i.p.). CCl4 injection reduced hepatic level of all metals except Ca, with an intense cytoplasmic staining pattern in hepatocytes located in periportal areas, indicating induction of MTs. Pretreatment with 50 mg/kg of luteolin for 2 days remarkably elevated metal content to control values (Mg and Cu) or even above them (Zn and Fe). Luteolin pretreatment increased pericentral MTs immunopositivity and histological architecture improvement in a time- and dose-dependent manner, being the most prominent in mice pretreated with 50 mg/kg for 2 days. The liver in this group showed pronounced MT expression in almost all hepatocytes throughout the liver parenchyma. In conclusion, these results suggest the protective effect of luteolin on CCl4-induced hepatotoxicity and an enhancement of hepatocyte proliferative capabilities.

Responses of biochemical markers in carp Cyprinus carpio from two field sites in Western Ukraine

The aim of this study was to compare environmental quality in two sites of the river in Western Ukraine, rural (R) and industrial (I) during three seasons via a set of biochemical markers in carp Cyprinus carpio L. Upon comparing the values of the I-site with those of the R-site, we found that Mn- and Cu, Zn-superoxide dismutase (SOD) activities decreased and O(.) production increased; metallothionein (MT) and glutathione levels increased in most cases, and acetylcholinesterase (AChE) activity decreased in summer. This confirms our hypothesis about continuous environmental press at the I-site. The higher activity of catalase and lipid peroxidation (TBARS), as well as the increased levels of vitellogenin-like proteins at the R-site, compare to the I-site in spring reflects the permitting effect of agricultural discharges. According to the results of PCA, the most sensitive biomarkers of pollution are MT, TBARS, and AChE in liver.

Metallothioneins (MTs) and δ-aminolevulinic acid dehydratase (ALAd) as biomarkers of metal pollution in great tits ( Parus major) along a pollution gradient

Biomonitoring allows an integrated evaluation of different aspects of exposure, accumulation and effects to environmental pollution, simultaneously accounting for the natural variety between individuals in an ecosystem. In this study, the effects of increased metal accumulation were evaluated at the biochemical level in terms of two biomarker responses in the great tit ( Parus major), a small insectivorous songbird, along an established metal pollution gradient. Metal concentrations in internal tissues (liver and kidney) and blood indicated that lead and cadmium were the most important metals in the pollution gradient under study. At the biochemical level, induction of metal binding protein metallothionein (MT) in liver and kidney reflected cadmium concentrations in these tissues ( R 2 = 0.42 and R 2 = 0.94 respectively, n = 19), although in kidney, MT induction was not sufficient to complex all cadmium present. Secondly, the activity of the enzyme δ-aminolevulinic acid dehydratase (ALAd) in blood decreased exponentially in response to increased lead accumulation ( R 2 = 0.70, n = 18) and represents therefore a specific effect marker for lead exposure. In the highest polluted area, an ALAd inhibition of 85% was reported. Since a higher metal exposure resulted in an increased metal accumulation and subsequent biomarker responses in a dose-dependent way, this study indicates the applicability of ALAd and MT levels in great tits for biomonitoring responses to heavy metal pollution.

Cadmium-induced oxidative stress and DNA damage in kidney of pregnant female rats

In the present study, we have investigated the influence of sub-acute treatment with cadmium (Cd) on some parameters indicative of oxidative stress and DNA damage in tissues of pregnant female rats. Pregnant female rats ( n = 6 ) were injected subcutaneously, daily with a dose of cadmium chloride of 3 mg/kg body weight (b.w.) from day 6 to day 19 of pregnancy, and they were allowed to deliver normally. MDA level and GPx, CAT and SOD activities were used as markers of oxidative stress in liver and kidney. The 8-oxo-dG level was measured by the HPLC-EC system. Cd treatment increased MDA (+116%, p < 0.01 ) in kidney. Moreover, Cd treatment also decreased CuZn-SOD (−11%, p < 0.05 ) and GSH level (−52%, p < 0.05 ) in kidney. Treated rats displayed an increase of the liver metallothionein (MT) level. Induction of MT in liver was probably implicated in the detoxification of Cd. The high level of Cd (3 mg/kg) used in the present study is partially neutralized by MT in liver, whereas the free fraction could be implicated in the oxidative stress and DNA oxidation observed in kidney. Cd treatment failed to alter 8-oxodGuo, indicating the absence of DNA oxidation in liver; by contrast, the same treatment increased the 8-oxodGuo level (+51%, p < 0.05 ) in the kidney of pregnant female rats, indicating an oxidative stress associated with DNA damage only in kidney. To cite this article: S. Chater et al., C. R. Biologies 331 (2008).

Voltammetric Studies of Cadmium‐ and Zinc‐Containing Metallothioneins at Nafion‐Coated Mercury Thin Film Electrodes

AbstractVoltammetric studies of rabbit liver metallothioneins (MTs, containing both Zn and Cd ions) and Zn7‐MT were carried out at Nafion‐coated mercury film electrodes (NCMFEs). The accumulation of MT molecules into the NCMFEs enhances the voltammetric signals and the electrostatic interaction between the Nafion membrane and MT facilitates facile electron transfer reactions. Two well‐defined redox waves, with reduction potential (Epc) values at −0.740 and −1.173 V, respectively, were observed. The peak at Epc =−0.740 V is attributable to the reduction of the Cd‐MT complex, whereas that at Epc=−1.173 V was assigned to the reduction of the Zn‐MT complex. Zn7‐MT exhibits only one redox wave with Epc=−1.198 V. The NCMFE was found to be more advantageous than thin mercury film electrode (MFE), because the pristine metal ions in MTs (e.g., Cd2+ and/or Zn2+) are not significantly replaced by Hg2+. The NCMFE is also complementary to Nafion‐coated bismuth film electrode in that it has a greater hydrogen overpotential, which allows the reduction of the Zn‐MT complex to be clearly observed. Moreover, intermetallic compound formation between Cd and Zn appears to be less serious at NCMFEs. Consequently, the amounts of Cd and Zn deposited into the electrode upon the reduction reactions can be quantified more accurately.

A field survey of metal binding to metallothionein and other cytosolic ligands in liver of eels using an on-line isotope dilution method in combination with size exclusion (SE) high pressure liquid chromatography (HPLC) coupled to Inductively Coupled Plasma time-of-flight Mass Spectrometry (ICP-TOFMS)

The effect of metal exposure on the accumulation and cytosolic speciation of metals in livers of wild populations of European eel with special emphasis on metallothioneins (MT) was studied. Four sampling sites in Flanders showing different degrees of heavy metal contamination were selected for this purpose. An on-line isotope dilution method in combination with size exclusion (SE) high pressure liquid chromatography (HPLC) coupled to Inductively Coupled Plasma time-of-flight Mass Spectrometry (ICP-TOFMS) was used to study the cytosolic speciation of the metals. The distribution of the metals Cd, Cu, Ni, Pb and Zn among cytosolic fractions displayed strong differences. The cytosolic concentration of Cd, Ni and Pb increased proportionally with the total liver levels. However, the cytosolic concentrations of Cu and Zn only increased above a certain liver tissue threshold level. Cd, Cu and Zn, but not Pb and Ni, were largely associated with the MT pool in correspondence with the environmental exposure and liver tissue concentrations. Most of the Pb and Ni and a considerable fraction of Cu and Zn, but not Cd, were associated to High Molecular Weight (HMW) fractions. The relative importance of the Cu and Zn in the HMW fraction decreased with increasing contamination levels while the MT pool became progressively more important. The close relationship between the cytosolic metal load and the total MT levels or the metals bound on the MT pool indicates that the metals, rather than other stress factors, are the major factor determining MT induction.

Metallothionein levels in Algerian mice (Mus spretus) exposed to elemental pollution: An ecophysiological approach

The potential use of metallothioneins (MTs) as biomarkers of trace metal contamination was evaluated for the first time in the Algerian mouse (Mus spretus). Mice were collected seasonally in an abandoned mining area (Aljustrel) and in a reference area, both located in southern Portugal. MT levels were quantified in liver and kidney by differential pulse polarography and hepatic elemental concentrations (Mn, Fe, Cu, Zn, Se) were determined by particle-induced X-ray emission. Hepatic iron and selenium concentrations were elevated in mice from Aljustrel mine when compared to reference animals. MTs levels were averagely higher in mice from Aljustrel than those originated from the reference area. A season-dependent significant effect was found on the hepatic and renal MT concentrations, characterized by higher levels in winter and lower in autumn. In contaminated mice positive relationship between liver elemental contents (Cu in autumn and Fe in winter) and MTs were found. The seasonal variation of MT suggests that probably physiological and environmental factors could influence hepatic and renal MT induction. Results seem to imply that some environmental disturbance occur in the vicinity of the Aljustrel mine. Therefore, for the management purposes MT levels should be followed in liver of M. spretus, especially in winter. Furthermore, other physiological factors that could influence MT expression and turnover in Algerian mouse should also be monitored.