Differential stimulation of hepatic and brain metallothioneins by ethanol

Administration of ethanol induces the synthesis of hepatic metallothionein and metallothionein mRNA in the liver but not in the brain. Furthermore, ethyl alcohol, methyl alcohol and isopropyl alcohol enhance the synthesis of metallothionein in Chang cells but not in neuroblastoma IMR-32 cells in culture. The results of this study are interpreted to suggest that the mechanisms of synthesis of metallothionein and the utilization of essential metal nutrients in the brain and peripheral tissues are not identical.     

The effects of 6-hydroxydopamine and oxidative stress on the level of brain metallothionein

Oxidative stress, resulting either from excess generation or reduced scavenging of free radicals, has been proposed to play a role in damaging striatal neurons in Parkinson's disease. Since metallothionein is able to regulate the intracellular redox potential, we have undertaken a group of experiments to see whether or not 6-hydroxydopamine, which generates free radicals and is toxic to dopaminergic neurons, could alter the level of zinc and metallothionein. 6-Hydroxydopamine (8 μg in 4 μl 0.02% ascorbic acid) reduced the level of zinc and metallothionein in the striatum but not other brain regions tested. Dopamine plus selegiline increased the synthesis of metallothionein in Chang cells as judged by enhanced incorporation of [³⁵S]cysteine into metallothionein. The effect of dopamine was selective, in that dopamine could not stimulate the synthesis of metallothionein in neuroblastoma IMR-32 cells, which are devoid of dopaminergic receptors. The effect of dopamine in stimulating the synthesis of metallothionein was similar to that of zinc, known to generate the synthesis of metallothionein, and to that of H₂O₂ and FeS0₄, known to generate free radicals. The results of these experiments provide additional evidence that zinc or zinc metallothionein are altered in conditions where oxidative stress has taken place.     

Induction of metallothionein synthesis in Menkes' and normal lymphoblastoid cells is controlled by the level of intracellular copper

A study was carried out on the uptake of copper, zinc, or cadmium ions and their induction of metallothionein synthesis in Menkes' and normal lymphoblastoid cells. The main difference between Menkes' and normal cells in the uptake of these metal ions was an increased uptake of copper ions in Menkes' cells at a low concentration of CuCl2 (2.1 microM). The CuCl2 concentration necessary to induce metallothionein synthesis in Menkes' cells was 50 microM, whereas that in normal cells was about 200 microM. The levels of zinc or cadmium ions needed to induce metallothionein in Menkes' cells were similar to those in normal cells. At least four isomers of metallothionein were induced by copper, zinc, and cadmium ions in both types of cells. Metallothionein synthesis in Menkes' and normal cells was induced when the amounts of intracellular copper reached a threshold level of approximately 0.2 nmol/10(6) cells, and the rate of metallothionein synthesis in these cells was increased as a function of the amounts of intracellular copper (0.2-1.7 nmol/10(6) cells). These results indicate that the induction of metallothionein synthesis in lymphoblastoid cells is controlled by the level of intracellular copper, suggesting that the major defect in Menkes' cells is not due to the abnormal regulation of metallothionein synthesis but to an alteration of the copper metabolism in cells by which the levels of intracellular copper become larger than those in normal cells and just lower than the threshold level for induction of metallothionein synthesis.     

Antagonism by 8-hydroxy-2(di-n-propylamino)tetraline and other serotonin agonists of muscarinic M1-type receptors coupled to inositol phospholipid breakdown in human IMR-32 and SK-N-MC neuroblastoma cells.

IMR-32 and SK-N-MC cells were found to contain [3H]quinuclidinyl benzilate specific binding sites inhibited by pirenzepine in a manner suggesting the presence of both M1-type and M2-type muscarinic receptor recognition sites. Neither cell had detectable [3H]8-OH-DPAT binding sites. Carbachol stimulated the rate of inositol phospholipid breakdown in IMR-32 and SK-N-MC human neuroblastoma cells with an EC50 value of about 50 microM in both cases. Pirenzepine inhibited the carbachol (100 microM)-stimulated inositol phospholipid breakdown in both cells with Hill slopes of unity and IC50 values of 15 nM (IMR-32) and 12 nM (SK-N-MC). The 5-HT1A receptor agonist 8-OH-DPAT competitively inhibited carbachol-stimulated inositol phospholipid breakdown with pA2 values of 5.78 (IMR-32) and 5.61 (SK-N-MC). These values are consistent with the inhibitory potency of 8-OH-DPAT towards [3H]quinuclidinyl benzilate binding in these cells. The 5-HT agonists 5-MeODMT and buspirone at micromolar concentrations inhibited carbachol-stimulated breakdown in IMR-32 cells. The inhibition by 8-OH-DPAT and 5-MeODMT was not affected by preincubation with (-)alprenolol. 5-HT (10-100 microM) was without effect on either basal or carbachol-stimulated breakdown. It is concluded that IMR-32 and SK-N-MC neuroblastoma cells express muscarinic M1-type but not serotoninergic receptors coupled to phosphoinositide-specific phospholipase C. 8-OH-DPAT acts as a weak antagonist at these muscarinic receptors.     

Isolation and characterization of metallothionein from the tortoise Clemmys mutica

A relatively high concentration of metallothionein was present in the liver of the control mature tortoise Clemmys mutica. The administrations of cadmium and copper to Clemmys mutica increased the metals in the liver with concomitant decrease of zinc, which was due to substitution of zinc in the native metallothionein with cadmium and copper. The Clemmys metallothionein was a mixture of three isoproteins which were abundant in half-cystine (34.2-34.6 mol%) and lysine (10.8-11.9 mol%), but lacked aromatic amino acids. One of the isoproteins, MT-I, contained one residue of histidine, which was an identical feature with frog and avian metallothioneins.     

In vivo and ex vivo displacement of zinc from metallothionein by cadmium and by mercury

Divalent cadmium and mercury ions are capable in vitro of displacement of zinc from metallothionein. This process has now been studied in vivo and ex vivo, using the isolated perfused rat liver system, in order to determine if this process can occur in the intact cell. Rats with normal and elevated (via preinduction with zinc) levels of hepatic zinc thionein were studied. Cd(II) completely displaces zinc from normal levels of metallothionein and on a one-to-one basis from elevated levels of metallothionein, both in vivo and ex vivo. Hg(II) displaces zinc from metallothionein (normal or elevated) rather poorly, as compared with Cd(II), in vivo, probably due to the kidneys preference for absorbing this metal. Ex vivo Hg(II) displaces zinc from metallothionein (normal or elevated) on a one-to-one basis, with considerably more mercury being incorporated into the protein than in vivo. The results of double-label ex vivo experiments using metal and [35S]cysteine (+/- cycloheximide) were consistent with the above experiments, indicating that de novo thionein synthesis was not required for short term incorporation of cadmium and mercury into metallothionein. These data are supportive of the hypothesis that cadmium and mercury incorporation into rat hepatic metallothionein during the first few hours after exposure to these metals can occur primarily by displacement of zinc from preexisting zinc thionein by a process which does not require new protein synthesis.     

Cadmium induces a novel metallothionein and phytochelatin 2 in an aquatic fungus

Cadmium stress response was measured at the thiol peptide level in an aquatic hyphomycete (Heliscus lugdunensis). In liquid culture, 0.1 mM cadmium increased the glutathione (GSH) content and induced the synthesis of additional thiol peptides. HPLC, electrospray ionization mass spectrometry, and Edman degradation confirmed that a novel small metallothionein as well as phytochelatin (PC2) were synthesized. The metallothionein has a high homology to family 8 metallothioneins (http://www.expasy.ch/cgi-bin/lists?metallo.txt). The bonding of at least two cadmium ions to the metallothionein was demonstrated by mass spectrometry (MALDI MS). This is the first time that simultaneous induction of metallothionein and phytochelatin accompanied by an increase in GSH level has been shown in a fungus under cadmium stress, indicating a potential function of these complexing agents for in vivo heavy metal detoxification. The method presented here should be applicable as biomarker tool.     

Metallothioneins and resistance to cadmium poisoning in Drosophila cells

Toxicity of cadmium on Drosophila cell lines has been studied. Maximal tolerance for cadmium chloride is 10 microM. Metallothioneins are induced in Drosophila cells following cadmium addition. A stable cadmium resistant cell line (Cd R200) has been selected starting from the haploid D clone. The Cd R200 cells are diploid and display metallothionein levels 22 times higher than cells of the original line fully induced with cadmium. The 200 microM CdCl2 tolerance upper limit in Cd R200 line is overcome if L-cysteine is supplemented to the medium. It is thus possible, in the presence of 5 mM L-cysteine, to select cells able to resist 800 microM CdCl2. These cells produce 4 times more metallothioneins than Cd R200 cells.     

Metabolism of parenterally administered zinc and cadmium in livers of newborn rats

The interaction of injected zinc and cadmium with metallothionein was investigated in newborn rats. Tissues of 5-day-old rats were removed 24 h after a single injection (Sc) of saline or zinc (20 mg/kg, body wt.) or cadmium (1 mg/kg, body wt.) with 2.5 μCi of ⁶⁵Zn or ¹⁰⁹Cd or 5 μCi of [³⁵S]cysteine. Injection of zinc resulted in a 75% increase in the hepatic zinc concentration with a concomitant elevation of metallothionein (P < 0.001), zinc in metallothionein increased by 45% (P < 0.05); [³⁵S]cysteine incorporation indicated the induced synthesis of metallothionein. Injection of cadmium did not alter either metallothionein or zinc levels in liver, but cadmium in cytosol was preferentially bound to metallothionein. Neither treatment altered hepatic copper metabolism and copper in metallothionein, nor renal zinc and metallothionein levels. These data indicate that zinc injection can elevate hepatic zinc levels and induce metallothionein synthesis in newborn rats despite high basal levels; cadmium injection does not induce metallothionein synthesis, though cadmium is avidly sequestered by pre-existing metallothionein. The differences in the induction of metallothionein by these divalent cations can be explained by the differences in their binding affinities for thiol groups in intracellular metallothionein.     

Cadmium, copper and zinc in tissues of deceased copper smelter workers

Workers at a copper and lead smelter in northern Sweden have a multifactorial exposure to a number of heavy metals. The concentrations of cadmium, copper and zinc in liver, lung, kidney and brain tissues have been determined by atomic absorption spectrometry in 32 deceased long-term exposed male lead smelter workers, and compared with those of 10 male controls. Furthermore, copper and zinc levels in hair and nails were determined by energy-dispersive X-ray fluorescence.

The highest cadmium concentrations among both workers and controls were observed in kidney, followed in order by liver, lung and brain. The levels in kidney, liver and lung were all significantly higher in the workers than in the controls (p < 0.03). Among the workers relatively strong positive correlations (p < 0.03) were observed between cadmium concentrations in liver and lung, liver and kidney, liver and brain, and lung and brain. In the exposed workers a positive correlation was observed between cadmium and zinc concentrations in the kidney (r_s = 0.38; p = 0.034). This is probably mainly due to the protein metallothionein, which is stored in the kidney, binding equimolar amounts of these two metals.

The highest concentrations of copper were found in hair and nails among both workers and controls, followed in order by liver, brain, kidney and lung. The tissue concentrations of copper in brain, lung and kidney were all significantly higher among the smelter workers than in the controls (p ≤ 0.036). Copper levels in lung and age at time of death were positively correlated among the exposed workers (r_s = 0.39; p = 0.029). In the same group, positive correlations between copper and zinc concentrations in kidney (r_s = 0.45; p = 0.009) and nails (r_s = 0.68; p < 0.001) were also observed, reflecting possible biological interactions between these two metals.

Among both workers and controls, the highest zinc concentrations were found in hair, followed in order by nails, liver, kidney, brain and lung. Significantly higher tissue concentrations among the workers as compared with the reference group were noted in kidney, liver and brain (p ≤ 0.033).

Neither copper nor zinc concentrations in hair and nails seemed to provide a useful measure of the trace element status of the smelter workers.     

Influence of dietary cadmium on the distribution of the essential metals copper,zinc and iron in tissues of the rat

The effect of long-term dietary cadmium treatment upon the distribution of the metals copper, iron and zinc has been compared in various organs of male and female rats. The renal accumulation of cadmium was similar in both sexes without a plateau being reached. In contrast, the hepatic accumulation of cadmium was higher in the female than in the male rat and a plateau was observed after 30–35 weeks of dietary cadmium treatment. Most of the cadmium which accumulated in these organs was recovered in the metallothionein fraction and the concentration of hepatic cadmiumthionein in the female rat was correspondingly higher than in the male rat. Accumulation of cadmium was associated with an increased zinc concentration in the liver and an increased copper concentration in the kidney; these increases were correlated with increases in liver and kidney metallothioneins induced by cadmium. Uptake of cadmium into organs other than liver and kidney occurred to a small extent but was not associated with changes in the concentration of copper and zinc. Cadmium also accumulated in the intestinal mucosa where it could be recovered in a fraction corresponding to metallothionein. A loss of iron from the liver and kidney was also observed following dietary cadmium treatment and involved mainly a loss of iron from ferritin.     

Analysis of hepatic copper, zinc, metallothionein and metallothionein-Ia mRNA in developing sheep

The concentrations of zinc, copper, metallothionein and metallothionein-Ia mRNA in sheep livers during development was determined. It was found that early sheep foetuses (30-40 days gestation) had very high concentrations of hepatic zinc (2305 +/- 814 micrograms/g dry mass), and that these levels declined steadily to 644 +/- 304 micrograms/g near to term. The copper concentrations in the foetal livers were not higher than those in the adult. The concentrations of metallothionein and metallothionein-Ia mRNA were also very high in the foetal livers and declined steadily during gestation from 261 +/- 94 molecules/pg RNA to 71 +/- 18 molecules/pg near to term. Metallothionein-Ia mRNA concentrations were closely correlated with hepatic zinc concentrations but not with copper. Metallothionein concentrations also decreased during gestation: e.g. 3044 micrograms/g (wet mass) in one foetus on day 34 of gestation to 862 micrograms/g on day 125. After birth, however, the concentrations of metallothionein declined to less than 100 micrograms/g and this decline occurred despite the presence of significant quantities of mRNA. The ratio of metallothionein/metallothionein-Ia mRNA decreased from 1.3 to 3.2 x 10(5) molecules metallothionein/molecule of metallothionein-Ia mRNA during gestation to between 0.28-0.64 x 10(5) molecules/molecule in the postnatal animals. We conclude that the major function of metallothioneins in the foetal liver is protection of the liver against the potentially toxic accumulation of zinc. In the postnatal sheep there appears to be a decreased synthesis or increased degradation of metallothionein.     

Zinc resistance impairs sensitivity to oxidative stress in HeLa cells: protection through metallothioneins expression.

To analyze the effects of high concentrations of zinc ions on oxidative stress protection, we developed an original model of zinc-resistant HeLa cells (HZR), by using a 200 microM zinc sulfate-supplemented medium. Resistant cells specifically accumulate high zinc levels in intracellular vesicles. These resistant cells also exhibit high expression of metallothioneins (MT), mainly located in the cytoplasm. Exposure of HZR to Zn-depleted medium for 3 or 7 d decreases the intracellular zinc content, but only slightly reduces MT levels of resistant cells. No changes of the intracellular redox status were detected, but zinc resistance enhanced H2O2-mediated cytotoxicity. Conversely, zinc-depleted resistant cells were protected against H2O2-induced cell death. Basal- and oxidant-induced DNA damage was increased in zinc resistant cells. Moreover, measurement of DNA damage on zinc-depleted resistant cells suggests that cytoplasmic metal-free MT ensures an efficient protection against oxidative DNA damage, while Zn-MT does not. This newly developed Zn-resistant HeLa model demonstrates that high intracellular concentrations of zinc enhance oxidative DNA damage and subsequent cell death. Effective protection against oxidative damage is provided by metallothionein under nonsaturating zinc conditions. Thus, induction of MT by zinc may mediate the main cellular protective effect of zinc against oxidative injury.     

Effect of metallothionein on cell viability and its interactions with cadmium and zinc in HEK293 cells

Metallothioneins (MTs) are thought to participate in a wide variety of physiological roles, but the mechanisms involved are still unclear. The study was designed to examine the possible factors related to these mechanisms. Methods, including transfection, MTT assay and flow cytometry, were used to investigate the effect of MTs on cell viability and their interactions with cadmium and zinc in HEK293 cells. The results showed that transient overexpression of human MT1A, MT2 and MT3 genes dynamically affected cell viability, and the effect was influenced by zinc and cadmium ions. Overexpressed MTs with added zinc showed a greater inhibitory effect on cell viability. Overexpressed MTs protected cells against low concentrations of cadmium ions (10 microM), but increased cell death in response to high concentrations (20-50 microM). Out of the three MTs, MT1A was more efficient than MT2 and MT3 in its resistance to cadmium (10 microM), and MT3 together with zinc showed more cell growth inhibition than MT1 and MT2. These results indicate that both of the divalent metal ions that could bind MTs, as well as the individual MT isoforms, affect the role of MTs on cell viability, which may explain in part why the comprehensive effect of MTs on the cells was elusive.     

Inhibition of LPS toxicity for macrophages by metallothionein-inducing agents

Parenteral administration of adrenal corticosteroids or particular transition metal salts are known to protect mice from the lethal effects of bacterial lipopolysaccharides (LPS). To determine if both groups of substances act through similar biologic mechanisms, their capacity to protect macrophages from the direct toxic effects of LPS was examined in vitro. When added simultaneously with LPS at culture initiation, 10 to 100 microM cortisone increased the viability of normal peritoneal macrophages as determined by trypan blue exclusion. Prednisolone and corticosterone protected LPS-treated macrophages at even lower concentrations (0.1 to 1 microM); estradiol and testosterone failed to alter cell viability at any concentration tested. Protection was dependent on de novo synthesis because inclusion of 20 nM actinomycin C1 or 1 microM cycloheximide with 10 microM corticosterone during a 4-hr pretreatment period blocked induction of the protective effect. Murine macrophages were also protected by micromolar concentrations of zinc, cadmium, mercury, and manganese, but not by calcium or lead. As was obtained with corticosteroids, heavy metal-induced protection depended on de novo RNA and protein synthesis. Because all substances that protected against LPS are known inducers of metallothionein in somatic cells, peritoneal macrophages were assayed for the presence of this unique, cytoplasmic protein. Within 2 to 8 hr, 10 microM cadmium caused three to fivefold increases in the incorporation of 35S-cysteine and in the binding of 203Hg into the TCA-soluble fraction of cell lysates that was excluded on centrifugally accelerated Sephadex G-10 columns. These results suggest macrophages may be protected from LPS-mediated cytotoxicity through synthesis of a sulfhydryl-rich, metal-binding protein. Although its mechanism of action remains unknown, it is proposed that metallothionein may function homeostatically by altering intracellular concentrations of zinc or may play a regulatory role by facilitating transfer of heavy metals among metal-requiring apoproteins.