Regional distribution of metallothionein, zinc, and copper in the brain of different strains of rats

The regional brain distribution of metallothionein (MT), zinc, and copper in the brain was determined in nine anatomical regions (olfactory bulb, cortex, corpus striatum, hippocampus, thalamus plus hypothalamus, pons plus medulla oblongata, cerebellum, midbrain, and white matter) and was compared between two different strains of rat (Sprague-Dawley [SD] and Lewis). No significant difference was observed in the whole-brain MT level between the two strains (17.8 ± 3.4 μg/g in SD rats and 20.3 ± 2.3 μg/g in Lewis rats). In SD rats, however, MT was more highly expressed in the white matter than in the other regions studied. In contrast, MT concentration was highest in the cortex and lowest in the olfactory bulb in Lewis rats. The MT levels in the cortex, corpus striatum, hippocampus, and thalamus plus hypothalamus were significantly lower in SD rats than in Lewis rats. In both strains, the olfactory bulb contained markedly higher levels of both zinc and copper than the other regions (27.9 ±6.8 μg/g zinc in SD rats and 27.6 ± 6.9 μg/g zinc in Lewis rats, and 5.2 ± 1.5 μg/g copper in SD rats and 11.1 ± 4.8 μg/g copper in Lewis rats). The next high-est zinc levels were seen in the hippocampus, whereas the next highest copper levels were in the corpus striatum in both SD and Lewis rats. The high levels of zinc and copper in the olfactory bulb were not accompanied by concomitant high MT concentrations. These results indicate that the strain of rat as well as the anatomical brain region should be taken into account in MT and metal distribution studies. However, the highest concentrations of zinc and copper in olfactory bulb were common to both SD and Lewis rats. The discrepancy between MT and the metal levels in olfactory bulb suggests a role for other proteins in addition to MT in the homeostatic control of zinc and copper.     

Genetic differences in zinc and copper induction of liver metallothionein in inbred strains of the mouse

Differences in Zn-induced levels of hepatic metallothionein (MT) in inbred strains of the mouse are described. Three low-producing strains, C57BL/6, C57BL/10, and NIH, are identified, while C3H and CBA display the highest levels of hepatic MT following Zn treatment. These interstrain differences affect not only the level of MT protein, but also the amount of MT-bound Zn and the total hepatic Zn concentration. Both MT isoforms are equally affected. A similar interstrain difference following Cu treatment is present in C3H and C57BL/6. The origin of these interstrain differences is discussed.     

Effects of 17 beta-estradiol on levels and distribution of metallothionein and zinc in squirrelfish

Females of the squirrelfish family (Holocentridae) accumulate higher levels of zinc in the liver than any other known animal. This zinc accumulation is made possible by high expression of the zinc-binding protein, metallothionein (MT). In the present study, the squirrelfish (Holocentrus ascensionis) MT cDNA was cloned and sequenced. The deduced amino acid sequence was very similar to other teleost MT. The role of estrogens on zinc metabolism was investigated by injecting male and immature female squirrelfish with 17 beta-estradiol (E(2)). E(2) treatment triggered transient increases in plasma zinc and vitellogenin (VTG) levels, and both of these variables showed very similar time courses. These results suggest that E(2) is responsible for the large hepatoovarian translocation of zinc observed in female squirrelfish and that VTG might be a vehicle for zinc. E(2) did not directly alter the levels of zinc or MT mRNA in the liver. However, the hepatic MT protein concentration increased differentially in the nuclear fraction. Thus E(2) is probably responsible for the association of MT with the nuclear fraction previously observed in untreated mature female squirrelfish.     

Interferonbeta-induced changes in metallothionein expression and subcellular distribution of zinc in HepG2 cells

We evaluated the changes of metallothionein induction and cellular zinc distribution in HepG2 cells by interferonbeta treatment. Immunohistochemical staining of metallothionein was observed in the cytoplasm and nuclei of hepatocytes; which was observed predominantly in the cells treated with interferon and zinc compared to those with zinc alone, interferon alone or the no-treated control. The cellular zinc level was higher in order of the interferon- and zinc-treated cells, the zinc-alone-treated cells, and the interferon-alone-treated cells. Flow cytometry showed that S-phase population increased in interferon-alone-treated cells and interferon- and zinc-treated cells, but not in zinc-alone-treated ones. Cellular elemental distribution was analyzed using in-air micro-particle induced X-ray emission. In zinc-alone-treated sample, X-ray spectra showed good consistency between the enhanced cellular zinc distribution and the phosphorous map. Localizations of bromine followed by interferon treatment were found accompanying a spatial correlation with the phosphorous map. The samples treated with interferon and zinc showed the marked accumulation of zinc and bromine. Discrete bromine accumulation sites were clearly visible with a strong spatial correlation followed by zinc accumulation. These findings suggest that interferonbeta in combination with zinc predominantly induces metallothionein expression in HepG2 cells. In addition, interferonbeta may promote the translocation of metallothionein-bound zinc from cytoplasm to S-phase nuclei.     

Regional distribution of glucose in mouse brain

After rapid inactivation of the enzymes responsible for glucose metabolism by microwave irradiation, concentrations of glucose in 20 regions of the mouse brain were estimated with combined gas chromatography-mass spectrometry (GC-MS). The highest concentrations of glucose were found in the periventricular nuclei of the hypothalamus and nucleus preopticus (P<0.05). The septum and nucleus amygdaloideus showed significantly higher glucose concentration compared with the cerebral neocortex, olfactory bulb, corpus striatum, cingulum, fornix, colliculus inferior, cerebellar cortex, corpus geniculatum laterale, substantia nigra, and nucleus ruber (P<0.05). The glucose concentration in the substantia nigra and nucleus ruber was significantly lower than in the other regions (P<0.01).     

Tumor-host zinc metabolism the central role of metallothionein

Features of tumor and host zinc metabolism are described. Emphasis is placed on tumor-host interactions. Using the model of the Ehrlich ascites tumor in mice, one clear site of modulation of cellular zinc by the amount of nutrient zinc available in the host is a zinc-binding protein with the properties of metallothionein. The selective depletion of zinc from this protein is correlated with the loss of cell proliferation by tumors injected into zinc-deficient animals. The properties of isolated metallothionein are consistent with a role for it as a reactive pool of intracellular zinc which can be donated to apozinc proteins and other structures. The presence of the Ehrlich tumor in mice also perturbs their distribution of zinc: zinc leaves the plasma and is accumulated by liver in the form of newly synthesized zinc metallothionein. During host zinc deficiency, this redistribution is not observed. This may be caused not only by a lack of mobile plasma zinc, but also by an inhibition of the initiation of this host response at the site of the tumor in the peritoneum.     

Dysregulation of metallothionein and zinc aggravates periodontal diseases

BackgroundPeriodontitis (PD) is a multifaceted inflammatory disease connected to bacterial infection that results in the destruction of tooth supporting structures and eventually tooth loss. Given their involvement in infection and inflammation, both metallothionein (MT) and zinc (Zn) might play vital roles in the development and progression of PD. More specifically, both MT and Zn are heavily involved in regulating immune functions, controlling bacterial infection, balancing inflammatory responses, and reducing oxidative stress, all of which are associated with the pathogenesis of PD.ObjectiveThis review paper will explore the physiological functions of MT and Zn and hypothesise how dysregulation could negatively affect periodontal health, leading to PD.FindingsBacterial lipopolysaccharide (LPS) derived from periodontal pathogens, namely P. gingivalis initiates the acute phase response, thus upregulating the expression of MT which leads to the subsequent deficiency of Zn, a hallmark of periodontal disease. This deficiency leads to ineffective NETosis, increases the permeability of the gingival epithelium, and disrupts the humoral immune response, collectively contributing to PD. In addition, the presence of LPS in Zn deficient conditions favours M1 macrophage polarisation and maturation of dendritic cells, and also inhibits the anti-inflammatory activity of regulatory T cells. Collectively, these observations could theoretically give rise to the chronic inflammation seen in PD.ConclusionA disrupted MT and Zn homeostasis is expected to exert an adverse impact on periodontal health and contribute to the development and progression of PD.     

Effect of zinc on the Stokes' radius of metallothionein

Apothionein was prepared from rat liver metallothionein-II. To this, zinc, as zinc acetate, was re-added in a stepwise fashion to yield a series of complexes with ratios of metal to protein varying from 1:1 to 7:1. The Stokes' radius of the apoprotein and each zinc-metallothionein complex was then determined by gel filtration. A plot of atoms Zn/mol metallothionein versus Stokes' radius yielded a sigmoid curve, with values for Stokes' radius ranging from 20.8 A for the apoprotein 15.4 A for the fully saturated zinc complex.     

Oxidant-induced mobilization of zinc from metallothionein.

Neutrophils which accumulate at sites of inflammation secrete a number of injurious oxidants which are highly reactive with protein sulfhydryls. The present study examined the possibility that this reactivity with thiols may cause protein damage by mobilizing zinc from cellular metalloproteins in which the metal is bound to cysteine. The ability of the three principal neutrophil oxidants, hypochlorous acid (HOCl), superoxide (.O2-), and hydrogen peroxide (H2O2), to cleave thiolate bonds and mobilize complexed zinc was compared using two model compounds (2,3-dimercaptopropanol and metallothionein peptide fragment 56-61), as well as metallothionein. With all compounds, 50 microM HOCl caused high rates of Zn2+ mobilization as measured spectrophotometrically with the metallochromic indicator 4-(2-pyridylazo)resorcinol. Xanthine (500 microM) plus xanthine oxidase (30 mU), which produced a similar concentration of .O2-, also effected a rapid rate of Zn2+ mobilization which was inhibited by superoxide dismutase but not catalase, indicating that .O2- is also highly reactive with thiolate bonds. In contrast, H2O2 alone was much less reactive at comparable concentrations. These data suggest that HOCl and .O2- can cause damage to cellular metalloproteins through the mobilization of complexed zinc. In view of the essential role played by zinc in numerous cellular processes, Zn2+ mobilization by neutrophil oxidants may cause significant cellular injury at sites of inflammation.     

Parenteral zinc and tissue metallothionein in normal and diabetic rats

The effect of parenteral zinc on tissue metallothionein (MT) was studied in normal and streptozotocin-induced diabetic rats. The accumulation of Zn-MT in liver and pancreas of normal and diabetic rats following the administration (ip) of various amounts of zinc was not different. Renal Zn-MT was higher in the diabetic group, and this was not changed by zinc injection. Although diabetic rats, relative to normal, possessed a markedly higher concentration of Cu-MT in kidney initially, this difference decreased considerably after zinc injection. The ratio of Cu-MT to cytosolic Cu in kidney was not affected by parenteral zinc and was highest in diabetic rats. Zinc injection markedly reduced food intake, water consumption, and urine output in both normal and diabetic rats. Blood glucose of diabetic rats also decreased 24 h after zinc administration. Our results indicate that relative to normal, MT and zinc metabolism are different in kidney, and to some extent liver, but not different in the pancreas of the chemically induced diabetic rat.     

Expression of mouse metallothionein in the cyanobacteriumSynechococcus PCC7942

A cDNA encoding mouse metallothionein was cloned into the shuttle vector pUC303, creating a translational fusion with the bacterial chloramphenicol acetyltransferase gene. The resulting fusion protein has been expressed in the cyanobacteriumSynechococcus PCC7942. Cyanobacterial transformants expressed mouse metallothionein-specific mRNA species as detected by RNA slot blots. In addition, the transformants expressed a unique cadmium ionbinding protein corresponding to the predicted size of the mouse metallothionein fusion protein. Expression of this fusion protein conferred a two-to five-fold increase in cadmium ion tolerance and accumulation onSynechococcus PCC7942.     

Regional distribution of protein kinases in normal and odor-deprived mouse olfactory bulbs

Unilateral naris closure produced dramatic down-regulation of tyrosine hydroxylase (TH) gene expression in periglomerular dopaminergic neurons in the olfactory bulb. To explore molecular mechanisms of TH gene regulation, the present study investigated the regional distribution of protein kinase A (PKAalpha), protein kinase C (PKCalpha), and CaM kinases II (CaMKIIalpha, beta) and IV (CaMKIV) in the normal olfactory bulb and in response to odor deprivation. Strong PKAalpha immunostaining was found in the glomerular, granule cell, external plexiform and olfactory nerve layers. PKCalpha staining was strong in granule cell and external plexiform layers but weak in the glomerular layer. Whereas CaMKIV was primarily found in granule cells, CaMKII was present in the glomerular, external plexiform, mitral cell and granule cell layers. No change in immunoreactivities of these kinases occurred in the olfactory bulb ipsilateral to naris closure. The expression of PKAalpha, PKCalpha and CaMKII, but not CaMKIV, in periglomerular cells suggests that these three kinases may play a role in TH gene regulation in the olfactory bulb. The lack of change in kinase protein levels after naris closure also suggests that any involvement of these kinases in TH gene expression in the olfactory bulb must be through altered kinase activity and not protein levels.     

An EXAFS study of the zinc sites in sheep liver metallothionein

Measurement and interpretation of the EXAFS associated with the K-absorption edge of zinc atoms in sheep liver metallothionein indicate that the primary coordination shell of each of these metal atoms comprises four sulphur atoms, with the Zn-S distance being 2.29 ± 0.02 Å.     

Cu+ distribution in metallothionein fragments

The differential distribution of Cu+ between separate alpha and beta domains of metallothionein (the isolated peptide fragments) and the rate of transfer of Cu+ between the two domains using copper-thiolate specific emission spectroscopy are reported. Kinetic data show the rate of transfer of Cu+ from the Cu6alpha to the Cd3beta domain is 2 x 10(-1) s(-1) while the transfer from Cu6beta to the Cd4alpha domain is much slower at 8 x 10(-3) s(-1), indicating the greater binding affinity of Cu+ for the MT beta domain. We report that the emission intensity of Cu6beta is 0.45 the emission intensity of Cu6alpha-MT. Lambda(max) is shown to be a probe of the environment of the Cu+. A series of copper-containing domain intermediates to the formation of the filled Cu6S9-beta and Cu6S11-alpha-clusters are identified. A mechanism is proposed for the formation of Cu12(betaalpha)-MT that involves metal exchange reactions of Cu+ ions from the alpha to the beta domain with initial formation of a Cu4beta-cluster.     

Postnatal changes in subcellular distribution of copper,zinc and metallothionein in the liver of bank vole (Clethrionomys glareolus): a possible involvement of metallothionein and copper in cell proliferation

1. Dramatic interdependent changes in the intracellular concentrations of copper (Cu), zinc (Zn) and metallothionein (MT) in the liver of bank voles during the first 30 days of their life were observed.2. The post-mitochondrial Cu, Zn and MT (ZnMT) abruptly decreased between 1 and 3 days following birth but the nuclear MT (CuMT) and Cu increased at the same time, suggesting that Cu displaced Zn already bound to MT in the cytoplasm and subsequently the complex CuMT was translocated to the nuclei.3. The nuclear Cu concentration reached the highest level (62–71% of the total tissue Cu) in the period from day 3 to day 20 post-partum, just prior to and during a rapidly growing liver.4. The data indicate that MT and Cu may be involved in the hepatocyte proliferation.     

Localization of metallothionein in the brain of rat and mouse.

Metallothionein (MT) is a low molecular mass protein inducible by heavy metals such as cadmium (Cd), zinc, and copper, and having high affinity for these metals. In the present study, we investigated the immunohistological localization of MT in the brains of rats and mice. In adult rat brain, almost no MT immunostaining was observed, whereas in adult mouse brain strong MT immunostaining was found in the ependymal cells, some glial cells, arachnoid, and pia mater. No immunostaining was detected in neurons and endothelial cells. In younger rats (1-3 weeks old), strong MT immunostaining was observed in ependymal cells, choroid plexus epithelium, arachnoid, and pia mater. The overall MT concentration in adult mouse brain appeared higher than that of the brains of young and adult rats. When adult rats were administered Cd, MT was induced not only in some glial cells, ependymal cells, arachnoid, and pia mater but also in endothelial cells. Although Cd treatment resulted in an increase in the MT immunostaining in the specific cells described above, the MT induction was not great enough to significantly affect the overall MT level in the brain. The present result suggest a possible link of MT with cell growth of choroid plexus epithelium and ependymal cells, as well as a detoxifying role of MT in the blood-brain barrier and the cerebrospinal fluid-brain barrier.     

The zinc/thiolate redox biochemistry of metallothionein and the control of zinc ion fluctuations in cell signaling

Free zinc ions are potent effectors of proteins. Their tightly controlled fluctuations ("zinc signals") in the picomolar range of concentrations modulate cellular signaling pathways. Sulfur (cysteine) donors generate redox-active coordination environments in proteins for the redox-inert zinc ion and make it possible for redox signals to induce zinc signals. Amplitudes of zinc signals are determined by the cellular zinc buffering capacity, which itself is redox-sensitive. In part by interfering with zinc and redox buffering, reactive species, drugs, toxins, and metal ions can elicit zinc signals that initiate physiological and pathobiochemical changes or lead to cellular injury when free zinc ions are sustained at higher concentrations. These interactions establish redox-inert zinc as an important factor in redox signaling. At the center of zinc/redox signaling are the zinc/thiolate clusters of metallothionein. They can transduce zinc and redox signals and thereby attenuate or amplify these signals.     

Regional distribution of brain-derived neurotrophic factor mRNA in the adult mouse brain.

Brain-derived neurotrophic factor (BDNF) is a protein that allows the survival of specific neuronal populations. This study reports on the distribution of the BDNF mRNA in the adult mouse brain, where the BDNF gene is strongly expressed, using quantitative Northern blot analysis and in situ hybridization. All brain regions examined were found to contain substantial amounts of BDNF mRNA, the highest levels being found in the hippocampus followed by the cerebral cortex. In the hippocampus, which is also the site of highest nerve growth factor (NGF) gene expression in the central nervous system (CNS), there is approximately 50-fold more BDNF mRNA than NGF mRNA. In other brain regions, such as the granule cell layer of the cerebellum, the differences between the levels of BDNF and NGF mRNAs are even more pronounced. The BDNF mRNA was localized by in situ hybridization in hippocampal neurons (pyramidal and granule cells). These data suggest that BDNF may play an important role in the CNS for a wide variety of adult neurons.     

Inheritance and expression of the mouse metallothionein gene in tobacco: impact on cd tolerance and tissue cd distribution in seedlings

Genetically engineered seedlings obtained from self-fertilized transgenic tobacco (Nicotiana tabacum) contained and expressed the mouse metallothionein and kanamycin resistance marker genes and were more tolerant to cadmium stress than untransformed controls. Cadmium accumulation in leaves of transgenic seedlings exposed to a low, field-like Cd concentration (0.02 micromolar) was about 20% lower than that in untransformed controls. Genetic analysis of R1 and R2 progeny showed inheritance of the marker gene to be as a dominant Mendelian trait. These results suggest the possibility of developing transgenic plants with modified tolerance to heavy metal stress and food crops having lower Cd content.     

Intestinal metallothionein in lethal-milk mice with systemic zinc deficiency

Lethal-milk (C57BL/6J-lm) mice over 12 months of age exhibit clinical signs of systemic Zn deficiency. Such lm mice have increased concentrations of metallothionein (MT) in the intestinal mucosa. Various concentrations of Cd or Zn were added to the drinking water. MT was assayed using the Cd-saturation/hemolysate method and for sulfhydryl concentration (MT has 33% cysteine residues) with Ellman's reagent. As assayed by both methods, mucosa from untreated lm mice contained approximately twice as much MT as did the C57BL/6J-(+^lm/+^lm) (B6) controls. Treatment with 150 and 500 ppm Zn removed the genotypic differences observed for the untreated and Cd-treated mice. These results are consistent with the lm mutation affecting Zn metabolism through impaired MT metabolism as measured for the intestinal mucosa. These studies do not eliminate the possibility that the liver may also contribute.