Metallothionein inhibits peroxynitrite-induced DNA and lipoprotein damage

Previous studies have demonstrated that metallothionein functions as an antioxidant that protects against oxidative DNA, protein, and lipid damage induced by superoxide anion, hydrogen peroxide, hydroxyl radical, and nitric oxide. The present study was undertaken to test the hypothesis that metallothionein also protects from DNA and lipoprotein damage induced by peroxynitrite, an important reactive nitrogen species that causes a diversity of pathological processes. A cell-free system was used. DNA damage was detected by the mobility of plasmid DNA in electrophoresis. Oxidation of low density lipoprotein was measured by a thiobarbituric acid-reactive substance, which was confirmed by lipid hydroperoxide assay. Plasmid DNA damage and low density lipoprotein oxidation were induced by 3-morpholinosydnomine, which produces peroxynitrite through the reaction between nitric oxide and superoxide anion or by synthesized peroxynitrite directly. DNA damage by 3-morpholinosydnomine was prevented by both metallothionein and superoxide dismutase, whereas the damage caused by peroxynitrite was prevented by metallothionein only. The oxidation of low density lipoprotein by 3-morpholinosydnomine and peroxynitrite was also significantly inhibited by metallothionein. This study thus demonstrates that metallothionein may react directly with peroxynitrite to prevent DNA and lipoprotein damage induced by this pathological reactive nitrogen species.     

Kidney and liver metallothioneins in rats after administration of an organic compound

Intubation of rats with alpha-mercapto-beta-(2-furyl)-acrylic acid (MFA) for 5 days at 50 mg/kg caused a 7-fold increase in kidney copper concentration, a 2-fold increase in kidney zinc concentration, and a 20% increase in liver zinc concentration. The proteins which bound the increased metals were purified and identified as metallothioneins by their amino acid compositions. Two isoforms were isolated from each organ. Renal thioneins appeared identical to counterpart hepatic apoproteins, but the former bound Cu and Zn in a 2:1 mole ratio and the latter bound only Zn. Kidney contained over 10 times more metallothionein per g of tissue than did liver. In rats previously administered MFA, injection of cadmium sulfate resulted in rapid displacement of liver metallothionein-bound Zn by Cd under conditions where minimal metallothionein was found in Cd-dosed animals not administered MFA. We conclude that MFA induces metallothionein biosynthesis in kidney and liver of normal rats; this is a novel effect for an organic compound.     

Induction of metallothionein mRNA in rat liver and kidney after copper chloride injection.

The kinetics of the increase of metallothionein mRNA in rat liver and kidney after CuCl2 injection was determined by cell-free translation and dot-blot hybridization of total RNA isolated at various times after the injection. Both assay procedures gave essentially the same result: a 16-fold increase in hepatic metallothionein mRNA was observed 7h after CuCl2 injection, with a decline to basal values by 15 h. The response in the kidney was less dramatic, with a 6-fold increase in metallothionein mRNA 5 h after injection, and basal values were attained by 12h. The rise in Cu2+ concentration in both organs was closely correlated with the increase in metallothionein mRNA; hepatic Cu2+ was increased 5.9-fold by 5h after injection and renal Cu2+ was increased 4.3-fold 5h after injection. The Zn2+ concentration in the liver had not risen significantly within 5h of Cu2+ injection. Renal Zn2+ concentrations did not alter appreciably in the Cu2+-treated animals. These results support the conclusion that Cu2+ is acting as a primary inducer of metallothionein mRNA in the rat.     

Apparent deficiency of metallothionein in the liver of the Antarctic icefish Chionodraco hamatus. Identification and isolation of a zinc-containing protein unlike metallothionein.

1. A zinc-binding protein has been isolated and purified from the liver of the icefish Chionodraco hamatus. 2. The icefish Zn-protein has characteristics distinct from those of metallothionein. 3. The amino acid composition shows a low content of cysteine and a high content of glutamate and aspartate. 4. No metallothionein has been detected in the extracts from icefish liver.     

Low-molecular weight metalloproteins in tissues of the narwhal (Monodon monoceros)

Narwhal (Monodon monoceros) liver and kidney cytosol were fractionated by gel chromatography, anion-exchange chromatography and electrophoresis. Cadmium was associated largely with low molecular weight proteins, while mercury was associated also with high molecular weight proteins, but apparently not because of saturation of the metallothionein mechanism. Eight different electrophoretic bands, four of which were metalloproteins, were found under the "metallothionein" peak. Anion-exchange chromatography yielded five metal peaks while further fractionation on G-50 gave two peaks, one containing almost pure metallothionein (Mt-1) and the other a metalloprotein having twice the molecular weight of metallothionein. Mt-2 was observed, at a much lower concentration than Mt-1, in liver but not kidney.     

The levels of liver metallothionein and zinc in plaice, Pleuronectes platessa L., during the breeding season, and the effect of oestradiol injection

Wild plaice from two locations were examined for liver metallothionein and liver and serum zinc content, before, during and after the breeding season. During the early stages a number of females had very high liver metallothionein and zinc levels. Egg formation and ripening were accompanied by a reduction in serum zinc. In males, the metallothionein levels did not reach such high values and were not correlated with gonad development.
There was a correlation between the zinc concentration of the liver and the metallothionein concentration of the liver. Above the threshold value for metallothionein formation, approximately half the additional zinc was found in the metallothionein and half in non-metallothionein pool(s).
Intramuscular injection of oestradiol 3-benzoate into immature females caused a significant increase in liver weight but a depression in metallothionein concentration relative to the controls.     

Prevention by copper of cadmium sequestration by metallothionein in liver.

Following chronic CdCl2 administration to rats, more than 98% of the metal in liver supernatant is bound to the low molecular weight binding protein, metallothionein. Simultaneous administration of high doses of Cd and copper salts result in an increase in toxicity which is accompanied by a failure of Cd sequestration by metallothionein in vivo. This may be due to an aggregation of metallothionein which has been observed in the presence of copper in vitro.     

A comparison of the effects of penicillamine, trientine, and trithiomolybdate on [35S]-labeled metallothionein in vitro; implications for Wilson's disease therapy

The synthesis of radiolabeled metallothionein was induced in rats in vivo by the injection of CuSO4 and [35S]-cysteine. Treatment of "cold" rat liver cytosol "spiked" with purified [35S] metallothionein with Penicillamine and Trientine showed that even at relatively high concentrations (up to 50 mg/g liver, wet weight), these compounds had no effect on the copper peak or the position of the [35S] label in the cytosol eluate after Sephadex G-75 gel filtration. By contrast, incubation of the "spiked" liver cytosol with Trithiomolybdate, even at relatively low concentrations (0.5 mg/g liver, wet weight), resulted in a transfer of metallothionein copper to high molecular weight protein fractions; the position of the [35S] apoprotein was unaffected. This copper "stripping" effect on metallothionein supports clinical and other evidence that thiomolybdates have a genuine decoppering effect in vivo whereas Penicillamine and Trientine have another mode of action and indicates that thiomolybdates might provide a more rational alternate therapy for Wilson's disease patients.     

Using MT(-/-) mice to study metallothionein and oxidative stress

Mice with null mutations for metallothionein genes MT-1 and MT-2 were used to study the role that metallothionein plays in protecting cellular targets in vivo from oxidative stress. Wild-type (MT(+/+)) and MT-null (MT(-/-)) mice were treated with either saline or zinc and exposed to two types of oxidative stress: gamma-irradiation or 2-nitropropane. There was no alteration in the antioxidant defense system (superoxide dismutase, catalase, or glutathione peroxidase and glutathione levels) to compensate for the lack of the metallothionein in the MT(-/-) mice. The amount of oxidative damage to liver DNA, lipids, and proteins were similar for the MT(-/-) and MT(+/+) mice even though the levels of metallothionein in the livers of the saline- or zinc-pretreated MT(+/+) mice were 5- to 100-fold greater than found in the MT(-/-) mice. To determine if metallothionein can protect mice from the lethal effects of ionizing radiation, the mean survivals of MT(-/-) and MT(+/+) mice exposed to whole body gamma-irradiation were measured and found to be similar. However, the mean survival increased significantly after zinc pretreatment for both the MT(-/-) and MT(+/+) mice. These results demonstrate that tissue levels of metallothionein do not protect mice in vivo against oxidative stress.     

Cadmium induction of metallothioneins in several dogfish organs

Dogfish Scyliorhinus canicula were exposed to 50 mg/l Cd for 4 days for inducing metallothionein synthesis. Spleen, pancreas, kidney and gonads were dissected out and metallothionein presence was checked by means of gel filtration chromatography in Sephadex G-75 and sodium dodecyl sulphate polyacrylamide electrophoresis. In pancreas and kidney, a cadmium-binding protein with spectroscopical and electrophoretic properties similar to those of dogfish liver metallothionein was found. In the other organs, the existence of an analogue protein but at very low concentrations is feasible.     

Developmental variation in copper, zinc and metallothionein mRNA in brindled mutant and nutritionally copper deficient mice.

The concentrations of copper, zinc and metallothionein-I (MT-I) mRNA were determined in the liver, kidney and brain of the brindled mutant mouse from birth until the time of death. Despite accumulation of copper in the kidney of the mutant, MT-I mRNA concentrations were normal. There was no difference between the MT-I mRNA in the brain of mutant and normal in the first 10 days of life, but after day 10 metallothionein mRNA levels were increased in the mutant. The concentration of copper was very low in the liver of the mutant, and on day 6 after birth the metallothionein mRNA was also reduced by about 50%. This reduction was not seen in copper-deficient 6-day-old pups, despite very low hepatic copper levels. This suggests that the lower hepatic MT-I mRNA in the day 6 brindled mouse was not simply due to the reduction in hepatic copper and also that hepatic copper is not regulating metallothionein gene expression the liver of neonatal mice. After day 12 hepatic MT-I mRNA levels were elevated in mutant and in copper deficient mice, both of which die at 14 to 16 days. These increases and the increase in brain MT-I mRNA in older mutant mice are likely to be caused by stress. Overall the results support the conclusions that the brindled mutation does not cause a constitutive activation of the metallothionein genes, and that the differences in metallothionein mRNA between mutant and normal are most probably secondary consequences of the mutation.     

Developmental variation in copper,zinc and metallothionein mRNA in brindled mutant and nutritionally copper deficient mice

The concentrations of copper, zinc and metallothionein-I (MT-I) mRNA were determined in the liver, kidney and brain of the brindled mutant mouse from birth until the time of death. Despite accumulation of copper in the kidney of the mutant, MT-I mRNA concentrations were normal. There was no difference between the MT-I mRNA in the brain of mutant and normal in the first 10 days of life, but after day 10 metallothionein mRNA levels were increased in the mutant. The concentration of copper was very low in the liver of the mutant, and on day 6 after birth the metallothionein mRNA was also reduced by about 50%. This reduction was not seen in copper-deficient 6-day-old pups, despite very low hepatic copper levels. This suggests that the lower hepatic MT-I mRNA in the day 6 brindled mouse was not simply due to the reduction in hepatic copper and also that hepatic copper is not regulating metallothionein gene expression the liver of neonatal mice. After day 12 hepatic MT-I mRNA levels were elevated in mutant and in copper deficient mice, both of which die at 14 to 16 days. These increases and the increase in brain MT-I mRNA in older mutant mice are likely to be caused by stress. Overall the results support the conclusions that the brindled mutation does not cause a constitutive activation of the metallothionein genes, and that the differences in metallothionein mRNA between mutant and normal are most probably secondary consequences of the mutation.     

A single form of metallothionein is Present in both heavy metal induced and neonatal chicken liver

MultiPlicity of metallothionein and their genes in higher animals are documented extensively in recent literature. In contrast, chicken liver Produced aPParently a single form of metallothionein uPon heavy metal exPosure. This Protein was Purified by gel filtration and ion exchange chromatograPhy and another technique based on heat treatment and acetone fractionation, followed by ion exchange chromatograPhy. In adult uninduced chicken liver the Presence of metallothionein was below the detection limit. But, like mammalian system, chicken liver was found to contain high amount of metallothionein at neonatal stage. This naturally occurring neonatal chicken hePatic metallothionein was Purified and comPared with the heavy metal induced adult hePatic metallothionein. The biochemical and immunobiological comParative analysis of adult and neonatal hePatic metallothionein showed identical characteristics. The neonatal metaltothionein exPressed naturally was a zinc metallothionein and unlike few other mammalian neonatal metallothionein did not contain any coPPer. Metallothionein was undectable in unfertilized eggs, in early embryos, and in Postnatal chicken, from 4 weeks after birth. The highest level of this naturally occurring neonatal metallothionein was found in 1–4 day old neonatal liver, which was about 1.5% of the total cytosolic Protein. This is the first rePorted evidence for the Presence of ontogenically modulated exPression of metallothionein in avian system. Possible biological role of neonatal metallothionein and their cellular interactions has been discussed.     

Levels of metallothionein messenger RNA in foetal, neonatal and maternal rat liver

We have used translation in vitro of hepatic polyadenylated RNA to characterize the levels of metallothionein mRNA in foetal, neonatal, pregnant and nulliparous rats. The translation products of foetal hepatic metallothionein mRNA increased relative to other mRNA translation products from day 18 of gestation to birth and attained a maximum, maintained throughout suckling, which is tenfold above 17-day foetal hepatic levels and fourfold above adult levels. Maternal liver metallothionein mRNA decreased fivefold between 17 days and 20 days of gestation, rose sharply immediately before birth, and was low throughout lactation.     

On the sensitivity of metallothioneins to oxidation during isolation.

It is demonstrated that the distribution of metals among the Sephadex G-75 fractions of rat liver and horse kidney supernatant is altered by exposure to oxidizing conditions. In particular, the metals bound to metallothionein are displaced into high-molecular-weight fractions and, to a lesser extent, into the low-molecular-weight forms, under aerobic conditions. In this process, metallothionein zinc is much more labile than cadmium. An appreciable proportion of the thionein is also found in the high-molecular-weight fractions and can be recovered along with the metals by treatment with mercaptoethanol. This result shows that the distributions obtained aerobically with large cadmium content in the high-molecular-weight fractions are an artefact due to metallothionein oxidation and suggests that 'spillage' of metals such as cadmium may be due in large part to oxidative processes rather than saturation effects. Evidence is presented that disulphide-bond formation occurs as thionein becomes bound in the high-molecular-weight region and that chemical reduction is necessary to restore its normal elution behaviour. Mercaptoethanol added to the homogenates maintains the reducing conditions normally found in the cellular milieu and prevents the oxidation of the metallothionein redistribution of the metals during isolation. Under these conditions the rat liver metallothionein isolated from animals exposed to chronic low concentrations of cadmium in drinking water contains appreciable quantities of copper as well as zinc and contains much of the zinc that is present in horse kidney supernatants. Metallothionein can also be extracted from a 40 000g pellet after sonication of the pellet. Thus careful analytical studies of the sites of cadmium deposition in rat liver indicate that greater than 95% is bound to metallothionein.     

Age-dependent variation for inducibility of metallothionein genes in mouse liver by cadmium

Cadmium is a toxic metal that induces the expression of metallothionein genes in many tissues and that binds avidly to metallothionein, a soluble transition metal binding protein. The present study examined the temporal pattern and magnitude of accumulation of metallothionein mRNA in liver of C57BL/6J mice of various ages treated with cadmium. In adult female mice, accumulation was dependent on the dosage level of cadmium and related to the concentration of this metal in liver. The accumulation of metallothionein mRNA in liver depended on age at exposure to cadmium. Intraperitoneal administration of 2 mg of cadmium per kg provoked small increases (two- to threefold) in levels of metallothionein mRNA in livers of 7- and 14-day-old mice. In contrast, cadmium treatment of 28- and 56-day-old mice resulted in 12- to 19-fold increases in levels of metallothionein mRNA in liver with maximum increases occurring 3 to 4 hr after treatment. Because similar patterns for the accumulation of cadmium of liver were found in 7-, 28-, and 56-day-old mice, observed age-dependent differences in induction of metallothionein mRNA in liver were probably not due to differences in the accumulation of cadmium in this organ. Taken together, these data suggest that tissue-specific factors controlling the expression of metallothionein genes may account for developmental variation in the inducibility of these genes by cadmium. Ontogenic variation in accumulation of metallothionein mRNA after cadmium treatment may be a factor in developmental variation in the acute lethality of cadmium in C57BL/6J mice.