Identification of metallothionein isoforms on capillary zone electrophoresis by adding anti-metallothionein antibody

The aim of this study was to identify metallothionein (MT) isoforms in mouse liver by using capillary zone electrophoresis (CZE). Purified MT-1 and MT-2 isoforms were completely separated by CZE using a polyacrylamide-coated tube at physiologic pH. There were two peaks in the cytosol fraction prepared from zinc-injected mouse liver, in which the migration times corresponded with those of purified MT-1 and MT-2 isoforms. When anti-MT monoclonal antibody was added with the purified MT-1 or MT-2 solution, the peaks decreased. Furthermore, the two peaks in the cytosol prepared from Zn-injected mouse liver decreased in a time-dependent manner from the electropherogram after the addition of the antibody. Therefore, those peaks were identified as MT-1 and MT-2 isoforms, respectively. In conclusion, the addition of anti-MT monoclonal antibody to the cytosol fraction of tissues is an effective method for identification of MT isoforms after separation using CZE.     

Identification of metallothionein isoforms with capillary zone electrophoresis using a polyacrylamide-coated tube

Metallothionein (MT) isoforms from various liver tissues were separated with capillary zone electrophoresis (CZE) using a polyacrylamide-coated tube at neutral pH. The electrophoresis was performed on MT-1 and MT-2 purified from mouse, rat, rabbit and human livers. The retention times of mouse and rat MT-1 coincided, while the retention times of rabbit and human MT-1 were longer. The retention times of MT-2 purified from the four sources were the same. MT-1 and MT-2 separated more definitely with N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)-Tris buffer (25 mM, pH 7.4) than with N-tris(hydroxymethyl)methyl-3-aminopropane sulfonic acid (TAPS)-Tris buffer (25 mM, pH 7.7) or with N-(2-acetamido)iminodiacetic acid (ADA)-Tris buffer (25 mM, pH 7.4). In addition, liver MT isoforms prepared from Zn- or Cd-administered mice could be separated.     

The amino acid composition of the zinc-induced metallothionein isoforms in rat brain

Recent investigation from this laboratory has identified in the rat brain a zinc-inducible and actinomycin D-inhibited metallothionein with an elution volume (Ve/Vo) of 2.08 and a molecular weight of smaller than 10,000 daltons. Furthermore, purification of the zinc-induced metallothionein by ion exchange chromatography on DEAE-Sephadex A-25 columns produced two isoforms, eluting, respectively, at 68 and 130 mM of Tris-acetate buffer, pH 7.5. In this paper, we report that zinc-induced metallothionein produces also two distinct isoforms on reverse phase high performance liquid chromatography that exhibit retention times of 17.23 and 18.53 minutes, respectively. Brain metallothionein was characterized further by studies showing that the zinc-induced metallothionein incorporated a large quantity of [³⁵S]cysteine and that isoforms I and II contain 17 and 18 cysteine residues, respectively, while being devoid of any arginine, histidine, leucine, phenylalanine or tyrosine. The precise functions of the brain metallothionein isoforms, which may be related to the transport and homeostasis of essential elements such as zinc and copper, remain to be elucidated.     

Different patterns in the induction of metallothionein mRNA synthesis among isoforms after acute ethanol administration

The induction of metallothionein (MT) isoform synthesis was investigated in mouse cerebral cortex 18 h after oral ethanol administration. The expression of MT-I isoform mRNA increased in a dose-dependent manner after ethanol loading at doses between 2 g/kg (ethanol/body weight) and 8 g/kg. Lipid peroxide formation, measured as the amount of malondialdehyde-reactive substances, remained at the control level after all of the administered ethanol doses. The expression of MT-III isoform mRNA remained at the control level up until an ethanol loading dose of 4 g/kg and then finally increased to a significant level at a dose of 8 g/kg, which is almost the LD₅₀ for oral ethanol in mice. The different patterns of MT synthesis induction among MT isoforms suggests that the MT-I isoform, which is ubiquitous in mammalian tissues, plays a significant role as an antioxidant. On the other hand, the MT-III isoform, which has a limited tissue distribution, especially in the central nervous system, seems to be implicated in tissue repair and/or protection against critical tissue injury.     

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.     

Basal and copper-induced expression of metallothionein isoform 1,2 and 3 genes in epithelial cancer cells: The role of tumor suppressor p53

Metallothioneins (MTs) are a ubiquitous low-molecular weight, cysteine rich proteins with a high affinity for metal ions. The expression and induction of MTs have been associated with protection against DNA damage, oxidative stress, and apoptosis. Our past research had shown that p53 is an important factor in metal regulation of MTs. The present study was undertaken to explore further the interrelationship between p53 and MTs. We investigated whether silencing of p53 could affect expression pattern of basal and copper induced metallothioneins. The silencing of wild-type p53 (wt-p53) in epithelial breast cancer MCF7 cells affected the basal level of MT-2A RNA, whereas the levels of MT-1A and MT-1X RNA remained largely unchanged. The expression of MT-3 was undetectable in MCF7 with either functional or silenced p53. MCF7 cells with silenced wt-p53 failed to upregulate MT-2A in response to copper and showed a reduced sensitivity toward copper induced cell apoptotic death. Similarly in MCF7-E6 and MDA-MB-231 cells, the presence of inactive/mutated p53 halted MT-1A and MT-2A gene expression in response to copper. Constitutive expression of MT-3 RNA was detectable in the presence of mutated p53 (mtp53). Transient transfection of MDA-MB-231 cells with wt-p53 enabled copper induced upregulation of both MT-1A and MT-2A but not basal level of MT-2A, MT-1E, MT-1X and MT-3. Inactivation of p53 in HepG2 cells amplified the basal expression of studied MT isoforms, including MT-3, as well as copper-induced mRNA expression of MTs except MT-1H and MT-3. Presented data demonstrate a direct relation between p53 and MT-1A and MT-2A and they also indicate that wt-p53 might be a negative regulator of MT-3 in epithelial cancer cells.     

Challenging the model for induction of metallothionein gene expression

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich metal-binding proteins found in a wide variety of organisms including bacteria, fungi and all eukaryotic plant and animal species. MTs bind essential and non-essential heavy metals. In mammalian cells MT genes are highly inducible by many heavy metals including Zn, Cd, Hg, and Cu. Aquatic systems are contaminated by different pollutants, including metals, as a result of man's activities. Bivalve molluscs are known to accumulate high concentrations of heavy metals in their tissue and are widely used as bioindicators for pollution in marine and freshwater environments, with MTs frequently used as a valuable marker of metal contamination. We here describe the MT isoform gene expression patterns of marine and freshwater molluscs and fish species after Cd or Zn contamination. Contamination was carried out at a river site polluted by a zinc ore extraction plant or in the laboratory at low, environmentally relevant metal concentrations. A comparison for each species based on the accumulated MT protein levels often shows discrepancies between gene expression and protein level. In addition, several differences observed in the pattern of MT gene expression between mollusc and mammalian species enable us to discuss and challenge a model for the induction of MT gene expression.     

Radical scavenging abilities of fish MT-A and mussel MT-10 metallothionein isoforms: An ESR study

Metallothioneins (MTs) are cysteine-rich proteins involved in homeostasis of essential metals, detoxification of toxic metals and scavenging of free radicals. Scavenging of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was measured by means of ESR spectroscopy for two recombinant MTs from aquatic species: MT-10 from the sea mussel Mytilus galloprovincialis, and MT-A from the fish Oncorhyncus mykiss. Both the zinc- and the cadmium-loaded forms (Zn(7)-MTs and Cd(7)-MTs) were analysed, using the commercial MT-II (Zn(7)-MT-II and Cd(7)-MT-II, respectively) from rabbit liver as a reference. A decrease in the scavenging ability was observed for all the three MTs passing from the Zn- to the Cd-loaded forms, because of the higher stability of the Cd-mercapto complex. The Zn(7)-MTs from aquatic species were more effective in scavenging DPPH signal than the rabbit Zn(7)-MT-II (2.8 and 4-folds, respectively). Similar results were obtained also for the Cd(7)-MTs, thus confirming the stronger antioxidant power of MTs from aquatic organisms compared with the rabbit MT-II. Moreover, mussel MT-10 was more active in DPPH scavenging than fish MT-A. When the complete release of metals from MTs was obtained by lowering the pH to 3 or, alternatively, by adding the chelating agent diethylenetriaminepentaacetic acid (DTPA), an increase in the scavenging ability of MTs was observed.     

Separation and quantitation of metallothionein isoforms from liver of untreated rats by ion-exchange high-performance liquid chromatography and atomic absorption spectrometry

A sensitive method for determination of metallothionein (MT) isoform levels in rat liver by ion-exchange high-performance liquid chromatography and atomic absorption spectrometry was developed. Critical steps in sample preparation, like MT extraction, MT saturation with Cd and protein separation, were optimized. This method is capable of measuring levels of 2.0 μg/g liver for metallothionein-1 (MT-1) and 1.3 μg/g liver for metallothionein-2 (MT-2), respectively, with a high recovery of 103% on average. The method described, thus, proved suitable for analyzing metallothionein isoform concentrations even in untreated animals. The ratio of MT-1 to MT-2 was found to be 1:1 on average. MT decomposition during storage was very high in whole livers, but could be reduced by about 80% when extracted liver samples were used.     

The rice metallothionein gene promoter does not direct foreign gene expression in seed endosperm

We generated transgenic tobacco and rice plants harboring a chimeric gene consisting of the 5-upstream sequence of the rice metallothionein gene (ricMT) fused to the -glucuronidase (GUS) gene. The activity and tissue-specific expression of the ricMT promoter were demonstrated in these transgenic plants. In the transgenic rice plants, despite substantial levels of GUS activity in the shoot and root, almost no GUS signal was detected in the endosperm. Thus, the ricMT promoter could be useful in avoiding accumulation of undesired proteins in the seed endosperm.     

Serum-induced hypha formation in the dimorphic yeast Yarrowia lipolytica

The dimorphic yeast Yarrowia lipolytica forms true hyphae in a medium containing N-acetylglucosamine. We made a new finding that serum is a very effective inducer of hypha formation of Y. lipolytica: serum induced its hyphal growth very quickly compared to N-acetylglucosamine (4 h vs. 10 h). Osmotic and oxidative stresses (0.2 M NaCl and 20 mM H2O2) inhibited the hypha formation induced by N-acetylglucosamine, but did not suppress the hypha formation triggered by serum. Serum-specific morphological mutants, which formed hyphae in the N-acetylglucosamine medium but not in serum medium, could be isolated. These results suggest that the signal triggered by serum may be transduced through a different pathway, at least in part, from that used for the N-acetylglucosamine signal in Y. lipolytica.     

Alterations in metal toxicity and metal-induced metallothionein gene expression elicited by growth medium calcium concentration

The calcium content of the growth medium has been shown to influence the growth and differentiation of primary epithelial cells in culture. The goal of the present study was to determine if growth medium calcium concentration could influence the susceptibility to metal toxicity and metallothionein gene expression of an immortalized human prostate-derived epithelial cell line (RWPE-1). The RWPE-1 cell line was grown in medium containing either 0.1 or 1.4 mM calcium. Confluent cells were exposed to either Zn⁺² (50, 100, or 150 μM) or Cd⁺² (3, 6, or 12 μM) for 13 days, and cell toxicity and MT gene expression were determined along the time course of exposure. It was demonstrated that the calcium content of the growth medium had a marked influence on Zn⁺² toxicity and a lesser but significant effect on Cd⁺² toxicity to the RWPE-1 cells. Calcium concentration of the growth medium was also shown to alter the accumulation of MT-1/2 protein and MT-1E, MT-1X, and MT-2A mRNAs. It was shown that MT-1/2 protein was markedly increased for metal-exposed cells grown in medium containing 0.1 mM calcium; however, the increased expression did not cause an increase in the resistance of the cells to Zn⁺² or Cd⁺² exposure. These observations show that growth medium calcium concentration can influence metal toxicity and the pattern of expression of the MT mRNAs and protein for RWPE-1 cells. The results suggest that caution should be exercised when comparing toxicological responses between cell lines that may be grown in growth formulations differing in calcium concentration.     

Variation in metallothionein gene expression is associated with adaptation to copper in the earthworm Dendrobaena octaedra

Evolution of resistance to heavy metals has been reported for several populations of soil living organisms occurring at metal contaminated sites. Such genetically based and heritable resistance contribute to the persistence of populations in contaminated areas. Here we report on molecular responses to experimental copper in populations of the earthworm, Dendrobaena octaedra, originating from copper contaminated soil near Gusum (Sweden) where heavy metal pollution has been present for several decades. We studied gene expression of six genes potentially involved in resistance to copper toxicity using F2-generations of D. octaedra populations, originating from reference sites and contaminated (High, Medium and Low) sites around Gusum. The main result was different expression patterns of genes encoding for two different isoforms (mt1 and mt2) of metallothionein proteins during experimental exposure to copper contaminated soil. Expression of mt1 showed a fast and significant upregulation in the High population and a slower, albeit significant, upregulation in Medium and Low populations. However, in the three reference populations no upregulation were seen. In comparison, a fast upregulation was also seen for the High population in the isoform mt2, whereas, gene expression of all other populations, including reference populations, showed slower upregulation in response to experimental copper. The results indicate that copper resistance in D. octaedra from contaminated areas is related to an increased expression of metallothioneins.     

Fluconazole downregulates metallothionein expression and increases copper cytotoxicity in Microsporum canis

Azole antifungals are widely used to treat infections with dermatophyte fungi. Whereas it is well established that this class of drugs interferes with fungal ergosterol synthesis, little is known about its potential other biological effects. Here we report the isolation and structural organization of Microsporum canis metallothionein gene and demonstrate that fluconazole is able to downregulate the baseline as well as copper-induced expression of this gene. Since this effect occurred within 30 min after exposure of the fungus to fluconazole, it is unlikely that it is due to impaired ergosterol synthesis. Our additional demonstration that fluconazole enhances copper toxicity for M. canis suggests that inhibition of metallothionein expression by fluconazole is biologically relevant and may represent an important additional mode of the antifungal action of this drug. Therefore our data indicate that antifungal effects of azole derivatives might not only be due to interference with cell wall synthesis but may also affect other biological circuits within the fungal cells.     

Implication of the differential roles of metallothionein 1 and 2 isoforms in the liver of rats as determined by polyacrylamide-coated capillary zone electrophoresis

Metallothioneins (MTs), determined by polyacrylamide-coated capillary zone electrophoresis (CZE), coincided well with those described by enzyme-linked immunosorbent assay. By using CZE, MT isoforms 1 (MT-1) and 2 (MT-2) were well separated and determined in the liver cytosol of LEC rats and Wistar rats administered CdCl(2). The total concentrations of MTs in the liver cytosol of LEC rats increased age-dependently as 1.0, 2.1, and 7.2mg/g wet weight of the liver at the age of 5, 10, and 15 weeks, respectively, and those of Wistar rats that had received daily CdCl(2) also increased with time of CdCl(2) as 0.5 and 1.2mg/g wet weight of the liver for 3 and 6 consecutive administration days, respectively. The MT-1/MT-2 ratio in the liver cytosol of LEC rats decreased age-dependently as 1.75, 1.49, and 0.76 at the age of 5, 10, and 15 weeks, respectively. In contrast, that of Wistar rats increased with time of exposure to the metal ion CdCl(2) as 1.1 and 1.6 for 3 and 6 administration days, respectively. Copper accumulation in the liver of LEC rats has already been reported. The present results indicated that the mechanism of the induction of MT synthesis differs between LEC rats, who lack ATP7B, and Wistar rats, who were given a toxic metal ion. On the basis of these results, we propose that MT-1 is related to the metabolism or detoxification of toxic metals such as Cd, and in contrast, MT-2 is responsible for the homeostasis of essential metals such as Cu.