Increased zinc absorption but not secretion in the small intestine of metallothionein-null mice

Metallothionein (MT) has been assigned a role in intestinal Zn absorption and secretion. The influence of MT was investigated in isolated segments of the small intestine from mice lacking the expression of MT I and II genes (MT−/−). To measure Zn absorption, washed 10- to 12-cm segments of the proximal and distal small intestine of MT−/− and control MT+/+ mice were filled with ⁶⁵Zn as ZnSO₄ (10 μg/mL), and the amount of ⁶⁵Zn appearing in the external buffer was measured over 4 h. To measure Zn secretion, the same procedure was followed using everted gut segments. The ⁶⁵Zn absorption from the small intestine was significantly greater in MT−/− mice, but only in the absence of albumin. In the proximal small intestine, the inclusion of 2% albumin in the external buffer significantly increased Zn absorption from 6.8% (no albumin) to 13.2% (with albumin) for MT−/−, and from 4.9% (no albumin) to 14.2% (with albumin) for MT+/+. In the distal segment, the respective values, with and without albumin respectively were 9.5% and 15.1% for MT−/− mice and 4.3% and 16.1% for MT+/+ mice. Regarding ⁶⁵Zn secretion, there was no difference between MT+/+ and MT−/− in either segment. However, the rate of secretion was higher in the proximal small intestine for both genotypes. Although it can be demonstrated that MT limits Zn absorption under controlled conditions in vitro, the ability of albumin to overcome this effect emphasizes the importance of circulating ligands in Zn transport.     

Dose Effects of Apical versus Basolateral Zinc Supplementation on Epithelial Resistance,Viability, and Metallothionein Expression in Two Intestinal Epithelial Cell Lines

Zinc supplementation is used to reduce diarrhea incidence in piglets and it has been shown in vitro that the antisecretory effects are maximal after basolateral zinc application. To examine whether the application site and dose of zinc also influence passive ion permeability and viability, porcine (IPEC‐J2) and human (Caco‐2) intestinal epithelial cells were treated with increasing zinc concentrations (0–200 μM) at either the apical or basolateral side. Transepithelial electrical resistance and viability decreased and expression of metallothionein and the efflux zinc transporter 1 increased most prominently when zinc was added in high concentrations at the basolateral side of IPEC‐J2 cells. Zinc transporter 4, a zinc importer, was not affected. Heat shock protein 70 mRNA expression increased only after basolateral addition of 200 μM zinc in IPEC‐J2 cells. Thus, zinc can elicit toxic effects especially when added at the basolateral side, with IPEC‐J2 cells being more susceptible than Caco‐2 cells.     

Protective effects of metallothionein against dopamine quinone-induced dopaminergic neurotoxicity

Dopamine (DA) quinone as DA neuron-specific oxidative stress conjugates with cysteine residues in functional proteins to form quinoproteins. Here, we examined the effects of cysteine-rich metal-binding proteins, metallothionein (MT)-1 and -2, on DA quinone-induced neurotoxicity. MT quenched DA semiquinones in vitro. In dopaminergic cells, DA exposure increased quinoproteins and decreased cell viability; these were ameliorated by pretreatment with MT-inducer zinc. Repeated L-DOPA administration markedly elevated striatal quinoprotein levels and reduced the DA nerve terminals specifically on the lesioned side in MT-knockout parkinsonian mice, but not in wild-type mice. Our results suggested that intrinsic MT protects against L-DOPA-induced DA quinone neurotoxicity in parkinsonian mice by its quinone-quenching property.     

Molecular characterization and function analysis of MT-10 and MT-20 metallothionein isoforms from Mytilus galloprovincialis

Structure and function of molluscan metallothioneins (MTs) are still poorly understood. The sea mussel Mytilus galloprovincialis displays two MT isoforms which differ in both primary sequences and physiological functions. MT-10 is the constitutive isoform, whereas MT-20 is mainly induced by cadmium (Cd). Both MTs were produced as recombinant proteins and showed identical Cd content and similar Cd-binding properties. Conversely, circular dichroism disclosed marked differences in the secondary conformations of the two Cd(7)-MTs. The possible relapses of these structural differences on protein stability and function were assessed. MT-10 presented a higher thermal stability and a more compact structure than MT-20, as it was inferred by absorption and emission spectroscopy studies. Moreover, the kinetics of Cd-release clearly indicated that MT-10 is much more sensitive to oxidation than is MT-20. The observed differences between MT-10 and MT-20 are discussed in terms of the different physiological roles exerted by the two isoforms in mussel.     

Cellular stress and intracellular zinc dyshomeostasis

Various stressful conditions like oxidative or nitrosative stress, heavy metal load or thiol-modifying compounds have been shown to disturb the intracellular zinc homeostasis leading to increasing concentrations of free zinc within the cytoplasm or nuclei of cells. However, much less is known about the consequences of a disturbed intracellular Zn2+ homeostasis under these conditions. Current knowledge is reviewed here.     

Role of metallothioneins in superoxide radical generation during copper redox cycling: defining the fundamental function of metallothioneins

In order to demonstrate the in vivo antioxidant properties of metallothioneins (MTs), the bacteria Escherichia coli was used as a cell reactor in which we compared the metal binding and antioxidative functions of MTs from different species, with different structures and polypeptide lengths. No protective effects of cytoplasmic MTs from cadmium (Cd) or zinc (Zn) contamination were observed in a wild-type E. coli strain, although these MTs can efficiently bind both Cd and Zn. To test their antioxidant properties, MTs were expressed within the cytoplasm of a sodA sodB deficient mutated strain (QC1726). However, a paradoxical MT toxicity was found when this strain was contaminated with Cd and Zn, suggesting that in a wild-type strain, superoxide dismutase counteracts MT toxicity. The most toxic MT was the one with the strongest Cd and Zn binding capacities. This toxic effect was linked to the generation of superoxide radicals, since a Cd-contaminated QC1726 strain expressing oyster MT isoforms produced 75-85% more O(2)*(-) than the control QC1726 strain. Conversely, under anaerobiosis or in the presence of a copper chelator, MTs protected QC1726 strain from Cd and Zn contamination. A model is proposed to explain the observed MT toxicity.     

Isolation and characterization of a metallothionein-1 protein in Chloris virgata Swartz that enhances stress tolerances to oxidative, salinity and carbonate stress in Saccharomyces cerevisiae

Chloris virgata Swartz (C. virgata) is a gramineous wild plant that is found in alkaline soil areas in northeast China and is highly tolerant to carbonate stress. We constructed a cDNA library from C. virgata seedlings treated with NaHCO₃, and isolated a type1 metallothionein (MT1) gene (ChlMT1: AB294238) from the library. The amino acid sequence of ChlMT1 contained 12 cysteine residues that constituted the Cys-X-Cys (X = amino acid except Cys) motifs in the N- and C-terminal regions. Northern hybridization showed that expression of ChlMT1 was induced by several abiotic stresses, from salts (NaCl and NaHCO₃), a ROS inducer (paraquat), and metals (CuSO₄, ZnSO₄, and CoCl₂). ChlMT1 expression in leaf was induced by 200 mM NaCl and 100 mM NaHCO₃. About 5 μM Paraquat, 500 μM Zn²⁺, and 500 μM Co²⁺ also induced expression of ChlMT1 in leaf after 6 h, and 100 μM Cu²⁺ induced it after 24 h. Saccharomyces cerevisiae when transformed with the ChlMT1 gene had dramatically increased tolerances to salts (NaCl and NaHCO₃) and ROS.     

Metallothionein, zinc, and mercury levels in tissues of young rats exposed to zinc and subsequently to mercury

Several studies have described mercury toxicity and the role of metallothioneins (MT) in the detoxification and regulation of metal homeostasis. However, little data exist on this topic during the specific post-natal developmental phase in young mammals. This developmental phase is particularly important since young animals are more sensitive to toxicants than adults. The objective of this work was to investigate whether MT participates in the mechanism of protection conferred by zinc pre-treatment on the toxic effects induced by mercury in neonate rats. Pups were exposed to ZnCl(2) (5 doses of 27 mg/kg/day, s.c.) and subsequently to HgCl(2) (5 doses of 5 mg/kg/day, s.c.); metal (Zn and Hg) and MT contents were analyzed in the liver, kidney, and blood. MT was induced in the liver and kidney of pups of both Zn-sal and Zn-Hg groups, although the greatest increase was in neonates exposed to Zn only. A direct relationship exists between MT and metals for both hepatic and renal tissues, which indicates that the increase in metal levels occurs in parallel to the increase in MT content. Although the heat-treated cytosolic fraction is rich in MT and metals, higher Zn and Hg contents were detected in the insoluble fraction of all tissues. These results suggest that MT is, at least in part, responsible for preventing Hg accumulation in the liver and blood and decreasing renal toxicity.     

A calcium channel blocker nifedipine distorts the effects of nano-zinc oxide on metal metabolism in the marsh frog Pelophylax ridibundus

Global decline of amphibian populations causes particular concern about their vulnerability to novel environmental pollutants, including engineering nanomaterials and pharmaceutical products. We evaluated the bioavailability of nanoform of zinc oxide (n-ZnO) in frog Pelophylax ridibundus and determined whether co-exposure to a common pharmaceutical, a calcium-channel blocker nifedipine (Nfd) can affect this bioavailability. Male frogs were exposed for 14 days to the tap water (Control) and n-ZnO (3.1 μM), Zn²⁺ (3.1 μM, as a positive control for n-ZnO exposures), Nfd (10 μM), and combination of n-ZnO and Nfd (n-ZnO + Nfd) in environmentally-relevant concentration. Exposure to Zn²⁺ or n-ZnO led to up-regulation of metal-binding proteins, metallothioneins (MTs) in the liver and Zn-carrying vitellogenin-like proteins in the blood plasma. Notably, upregulation of MTs by Zn²⁺ or n-ZnO exposures combined with increased binding of Zn and Cu to MTs. This was associated with the more reducing conditions in the liver tissue indicated by elevated lactate to pyruvate ratio. Nfd suppressed the binding of Zn and Cu to MTs and led to a decrease in Lactate/Pyruvate ratio and elevated protein carbonylation indicating pro-oxidant conditions. Redox status parameters were not directly related to DNA fragmentation, nuclear abnormalities or suppression of cholinesterase activity indicating that factors other than oxidative stress are involved in cytotoxicity of different pollutants and their combinations. Furthermore, activity of Phase I biotransformation enzyme (CYP450 oxidase measured as EROD) was elevated in Nfd-containing exposures and in Zn²⁺ exposed frogs. Tyrosinase-like activity in the frog liver was strongly stimulated by Zn²⁺ but suppressed by n-ZnO, Nfd and n-ZnO + Nfd. These findings show that Nfd modulates homeostasis of essential metals in amphibians and emphasize that physiological consequences of combined n-ZnO and Nfd exposures are difficult to predict based on the mechanisms of single stressors.