Oxygen free radicals and metallothionein.

It is generally accepted that the principal roles of metallothionein lie in the detoxification of heavy metals and regulation of the metabolism of essential trace metals. However, there is increasing evidence that it can act as a free radical scavenger. This article reviews the evidence supporting such a physiological role and describes induction of metallothionein synthesis by oxidative stress, possible mediators for this induction, and the radical scavenging capability of metallothionein in tissues and cells. The relationship between metallothionein and other antioxidant defense systems and the medical implications of the free radical scavenging properties of metallothionein are also discussed.

Manifestations of copper excess.

Although copper is an essential micronutrient normally subject to effective homeostatic control, excess dietary intakes can in some circumstances be toxic. Susceptibility to copper toxicosis depends, however, on many factors, including species, genetics, age, and diet. This appears to reflect not only variations in the efficiency of the absorption and excretion of copper but also differences in the intake of other hepatotoxic or protective factors, differences in the cellular distribution of copper, and differences in the expression of specific copper transport and storage proteins. Many of the toxic effects of copper, such as increased lipid peroxidation in cell membranes and DNA damage, are related to its role in the generation of oxygen free radicals.

Homeostatic regulation of zinc absorption and endogenous losses in zinc-deprived men.

The mechanisms involved in the homeostatic regulation of zinc were studied in five male subjects by using stable 70Zn as a marker. When dietary zinc was reduced from 85 to 12 mumol/d, adaptation was achieved by a mean (+/- SEM) reduction in urine zinc of 48 +/- 7% and in fecal zinc of 46 +/- 12% over 25 d in four subjects. The latter was caused by an increase in the efficiency of zinc absorption from 38 +/- 3% to 93 +/- 1% after 15 d of zinic deprivation and by a reduction in intestinal endogenous losses of zinc. In a fifth subject, who had some evidence of a resolving alcohol-induced hepatitis, urine and fecal zinc were reduced by 64% and 41%, respectively, in 15 d and zinc absorption increased from 46% to 93%. More information on adaptive responses is needed to enable current dietary recommendations to be reconsidered.

Effect of copper and zinc status on susceptibility to cadmium intoxication.

The effects of dietary cadmium on copper and zinc metabolism in animals are described. Emphasis is given to situations involving chronic exposure to low levels of cadmium, to the identification of population groups most at risk, and to the protective effect of dietary supplementation with copper and zinc. The mechanism of the interaction between the metals and the involvement of metallothionein are discussed.

Intravenous iron-dextran: studies on unsaturated iron-binding capacity.

A method is described for measuring the plasma unsaturated iron-binding capacity in the presence of very high concentrations of iron as iron-dextran. The procedure utilizes (59)Fe to label the apotransferrin with subsequent separation of ionic iron from transferrin-bound iron on an ion exchange or Sephadex G.25 column. The unsaturated iron-binding capacity has been measured in rabbits and dogs after intravenous injection of iron-dextran and in human subjects after total dose infusion of iron-dextran. No evidence of saturation of the unsaturated iron-binding capacity was found even when the plasma iron values were greater than 40,000 mug Fe/100 ml.

Symptomatic zinc deficiency in experimental zinc deprivation.

An evaluation of indices of poor zinc status was undertaken in five male subjects in whom dietary zinc intake was reduced from 85 mumol d-1 in an initial phase of the study to 14 mumol d-1. One of the subjects developed features consistent with zinc deficiency after receiving the low zinc diet for 12 days. These features included retroauricular acneform macullo-papular lesions on the face, neck, and shoulders and reductions in plasma zinc, red blood cell zinc, neutrophil zinc and plasma alkaline phosphatase activity. Alcohol induced hepatitis, which was suspected in this subject, may have caused a predisposition to altered zinc metabolism and possible zinc deficiency which was exacerbated by subsequent zinc deprivation. The report supports the value of neutrophil zinc concentration as an indicator of poor zinc status.

Urinary excretion of metallothionein-I and its degradation product in rats treated with cadmium, copper, zinc or mercury.

The metallothionein-I (MT-I) content of urine following administration of cadmium (Cd), copper (Cu), mercury (Hg) or zinc (Zn) to rats was determined by radioimmunoassay. Urinary excretion of MT-I was increased significantly after injection of each of these metals. Fractionation of urine from Cd-treated rats on Sephadex G-50 showed a single immunoreactive component corresponding to native MT-I, whereas in urine from Cu, Zn or Hg-treated rats 2 immunoreactive components corresponding to MT-I and a possible degradation production were observed. Since a comparable low molecular weight component corresponding to this degradation product was not detected to the same extent on fractionation of plasma from Cu-exposed rat, it seemed to be derived from degradation of MT in the kidney.

Metallothionein immunoreactivity in the liver and kidney of copper injected rats.

Adult male rats were injected intraperitoneally with copper sulphate in physiological saline (3 mg copper/kg body wt). Metallothionein-I (MT-I) levels in liver, kidney, plasma and red blood cells were measured by radioimmunoassay (RIA), prior to the injection and after 7, 16 and 24 h. Copper and zinc levels in liver and kidneys were also monitored. Concentrations of MT-I in liver and kidneys showed a rapid increase and remained elevated for 24 h. Copper concentrations also increased in both tissues but zinc levels remained constant in the kidney and rose only slightly in the liver. MT-I levels increased gradually in plasma but decreased in the red blood cells. Immunochemistry of liver and kidney, using the direct peroxidase technique with antiserum to rat MT-I, revealed an increase in staining in both tissues after copper administration, consistent with the RIA results. The change in distribution of immunoreactive material with time after copper injection indicates a role for MT in the sequestration and excretion of copper in acutely loaded animals.

Effect of acute stress on the absorption and distribution of zinc and on Zn-metallothionein production in the liver of the chick.

A study has been made of the effects of chloroform inhalation, Escherichia coli endotoxin injection and hydrocortisone injection on the absorption of a single intragastric dose of 65Zn by the chick. Injection of hydrocortisone increased the absorption of the 65Zn by 30-55% in both Zn-deficient and Zn-supplemented chicks. The influence of chloroform and endotoxin was less consistent; the former treatment only increased 65Zn absorption and endotoxin was less consistent; the former treatment only increased 65Zn absorption in Zn-supplemented chicks fed ad libitum whereas endotoxin only increased that in Zn-supplemented chicks on a restricted food intake. Injection of endotoxin increased the hepatic uptake of the absorbed 65Zn in both Zn-deficient and Zn-supplemented chicks, whereas hydrocortisone had a similar effect in the Zn-supplemented birds only. Chloroform inhalation increased hepatic 65Zn uptake in Zn-deficient chicks only. The increase in hepatic Zn concentrations in the stressed chicks was mainly associated with a protein in the cytosol identified as metallothionein. Both endotoxin and hydrocortisone decreased total plasma Zn concentrations in Zn-supplemented and Zn-deficient chicks; chloroform decreased plasma 65Zn content only.

Effects of tetrathiotungstate and dithiotungstate on copper metabolism in rats.

The effects of dietary supplementation with ammonium tetrathiotungstate and dithiotungstate on copper metabolism in young rats have been investigated. The addition of WS4(2-) (4-32 mg W/kg diet) decreased growth rates and induced clinical and biochemical signs of Cu deficiency. These were overcome by increasing the dietary content of Cu. The intestinal absorption of 64Cu was impaired and the tissue distribution of absorbed Cu modified by the administration of WS4(2-). No clinical or biochemical defects indicative of Cu deficiency developed in rats receiving WO2S2(2-) in their diet at concentrations up to 32 mg W/kg. In contrast, this oxythioanion enhanced the content of Cu in plasma liver and kidney, especially when dietary Cu was increased. Most of the additional Cu retained by plasma and kidney was associated with albumin and metallothionein, respectively. The effects of these W sources are compared with those caused by their thio- and oxythiomolybdate analogs and are discussed in relation to the mechanisms whereby Mo induces Cy deficiency in ruminants.

Copper metabolism in rats given di- or trithiomolybdates.

As one of a series of studies on the influence of S substitution of MoO4(2-) on the action of Mo as an antagonist of Cu, the effects of MoO2S2(2-) and MoOS3(2-) on the metabolism of Cu by rats have been investigated. Administration of either oxythiomolybdate increased both plasma Cu concentration and the retention of Cu by the albumin fraction of plasma. In contrast to the effects of MoS4(2-), described previously, neither oxythiomolybdate inhibited 64Cu absorption from the digestive tract or induced biochemical or clinical signs of Cu deficiency. Evidence is discussed suggesting that the potency of the thio- and oxythiomolybdates as inhibitors of Cu utilization is directly proportional to the degree of thio substitution within the series series MoO4- chi S chi 2-.

The effects of age, sex, and zinc status on the accumulation of (copper, zinc)- metallothionein in rat kidneys.

A study has been made of the distribution of copper in the kidneys of growing rats. Renal copper concentrations increased steadily with age and were greater in female than in male animals. Most of the copper was present as (copper, zinc)-metallothionein and two forms of this protein were isolated and characterized from the kidneys of mature female rats. That copper metabolism in kidneys is subject to hormonal influence was indicated by a reduction in the concentrations of copper and (copper, zinc)-metallothionein in ovariectomized rats and by an increase in their concentrations after the administration of progesterone. Concentrations of renal (copper, zinc)-metallothionein were less in zinc-deficient than zinc-adequate rats during pregnancy and after progesterone administration.

Intestinal metallothionein and the mutual antagonism between copper and zinc in the rat.

A study has been made of the mechanism of the mutual antagonism between copper and zinc in rats. Dietary zinc concentrations of up to 450 mg/kg had no effect on intestinal 64Cu absorption but 900 mg/kg caused a 40% reduction. This was associated with an increase in the mucosal uptake of 64Cu in the small intestine. This occurred mainly in the form of metallothionein and it appeared that copper displaced zinc from the protein after its synthesis had been induced by zinc. Ths intestinal absorption of 65Zn was decreased by 20% when the dietary copper intake was increased from 3 to 24 mg/kg. Further increases in copper intake to 300 mg/kg did not cause any additional decrease in 65Zn absorption or any change in the association of intestinal 65Zn with metallothionein. Concentrations of this protein in the intestinal mucosa were not influenced by dietary copper intake.

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 A.

Copper and molybdenum absorption by rats given ammonium tetrathiomolybdate.

Previous studies have shown that the tetrathiomolybdate ion [MoS4(2-)] is a potent antagonist of Cu metabolism. Effects of orally administered MoS4(2-) on the absorption and tissue distribution of 64Cu in rats have now been investigated. Four or 12 mg Mo/kg diet, when given as MoS4(2-), strongly inhibited 64Cu absorption and modified the fate of absorbed Cu, decreasing hepatic and renal uptake but increasing plasma retention of 64Cu. These effects were not induced by equivalent dietary concentrations of Mo as MoO4(2-) or when S2- was given as CaS. Clinical and biochemical effects induced by orally administered MoS4(2-) were abolished by increasing dietary concentrations of Cu. Such treatment also inhibited the absorption and tissue retention of 99Mo derived from 99MoS4(2-). Intraperitoneal administration of Cu ameliorated clinical effects attributable to MoS4(2-) but neither inhibited 99Mo absorption nor the appearance of systemic defects in Cu metabolism. Since the absorption of MoS4(2-) (or its derivatives) from the gastrointestinal tract is inhibited by Cu, it is evident that the site of its action as an antagonist influencing either the absorption or the subsequent metabolic fate of Cu depends upon the ratio Cu/MoS4(2-) in the diet.

Effects of molybdate, sulfide, and tetrathiomolybdate on copper metabolism in rats.

Species differences in the response to dietary MoO4(2)- as a metabolic antagonist of Cu are considered briefly. Suggestions that (i) the potency of MoO4(2)- as a Cu antagonist is enhanced by normally innocuous dietary concentrations of S20 and (ii) that MoS4(2)- may be a more effective antagonist than either MoO4(2)- or S2- were investigated in a series of studies with rats. Diets including MoS4(2)- but not of MoO4(2)- or S2- alone promoted a decline in hepatic Cu and ceruloplasmin activity and induced clinical signs of Cu deficiency. Evidence of concurrent anomalies in the partition of Cu between tissues and in the distribution of Cu between proteins of plasma and kidney cytosol suggested that such effects were partly attributable to the development of systemic defects in Cu metabolism. The relationship of such findings to the suggested involvement of MoS4(2)- or its derivatives in the etiology of Mo-induced Cu deficiency in ruminant animals is considered.

Thiomolybdates in rumen contents and rumen cultures.

Examination of direct and (Cu)-difference spectra of i) the aqueous supernatants of in vitro cultures of bovine rumen contents incubated with MoO42- and potential sources of S2- and ii) samples drawn directly from the rumen of animals receiving high Mo diets yielded evidence of the presence of thiomolybdates. Only MoS42- was detected in the soluble phase of in vitro cultures. Although intense and variable background absorbance precluded full characterization of thiomolybdate species in samples drawn directly from the rumen, both spectral data and the biochemical and clinical responses of animals given high Mo diets were consistent with the conclusion that MoS42- rather than MoOS32- was the predominant thiomolybdate species present in the aqueous phase. Addition of Ca2+ either to rumen cultures before incubation or as a supplement to diets high in MoO42- content inhibited the appearance of MoS42- in the aqueous phase. Evidence of the sequestration of MoS42- and MoOS32- by particulate or microbial fractions of rumen contents is considered in relation to the inhibitory action of Mo upon Cu absorption by ruminants.

Intestinal absorption and losses of copper measured using 65Cu in zinc-deprived men.

The absorption and intestinal losses of endogenous Cu in response to a low Zn diet were studied in five young male subjects using stable 65Cu as an oral tracer. The subjects received a semi-purified formula diet providing 85 mumol (5.6 mg) Zn/d during 15-day baseline and repletion phases and 12 mumol (0.8 mg) Zn/d during an intervening period of 25 days. Thirty-eight mumol (2.4 mg) Cu/d was provided throughout the study. In four of the subjects, the mean +/- SEM luminal disappearance of 65Cu was 37 +/- 4 per cent during the baseline phase and was unaffected by Zn deprivation (32 +/- 7 per cent) or repletion (30 +/- 7 per cent) as were intestinal losses of endogenous Cu [7 +/- 4, 8 +/- 3, 8 +/- 3 mumol/d (0.4 +/- 0.1, 0.5 +/- 0.1, 0.5 +/- 0.1 mg/d) during baseline, Zn deprivation and Zn repletion phases, respectively]. In a fifth subject, who had some evidence of a resolving alcohol-induced hepatitis, the luminal disappearance of 65Cu was 31, 44 and 42 per cent and the intestinal losses of endogenous Cu 11, 2 and 6 mumol/d (0.7, 0.1 and 0.4 mg/d) during the baseline, Zn deprivation and Zn repletion phases respectively. Plasma Cu concentrations, however, fell throughout the study in all the subjects, despite consistently positive Cu balances. There may be subtle effects of a low dietary intake of Zn on Cu metabolism which were not revealed by the methods used in this study.

Copper thionein in the kidneys of copper-poisoned sheep.

A study has been made of the distribution of copper in the kidneys of copper-poisoned sheep, containing up to 240 microgram copper/g fresh cortex. About 64% of the copper in the cortex was present in the cytosol and 75% of this occurred in a form with molecular weight of approx. 12 000. This was partially purified by gel filtration on Sephadex G-75 and Bio-Gel P.10 and ion exchange chromatography on DEAE Sephadex A-25 to give three sub-fractions, which also contained zinc. The amino acid composition, copper content and chromatographic behaviour of these proteins indicated that they were copper-thioneins. No significant amounts of the proteins were detected in the plasma or erythrocytes of the copper-poisoned sheep when they were undergoing the haemolytic crisis typical of this syndrome. It is concluded that metallothionein constitutes the major copper-binding protein in the kidneys of copper-poisoned sheep. However the rapid accumulation of the protein in the kidney, and the development of kidney damage, are unlikely to have arisen from the release of the intact copper-protein from the liver and its transport via the blood to the kidneys.

The protective role of metallothionein in copper overload: I. Differential distribution of immunoreactive metallothionein in copper-loaded rat liver and kidney.

The cytotoxicity of copper is probably determined by its molecular association and subcellular localisation rather than its concentration within tissues. Metallothionein (MT) is a copper binding protein distributed between the particulate and soluble cellular components. The role of MT in conferring protection to the copper-loaded rat has been investigated by comparing the distribution of the immunoreactive protein between the soluble and particulate fractions of liver and kidney during the development of copper tolerance. Young male Wistar rats were fed a high copper (1 g/kg) diet for 16 weeks and killed sequentially during this period; liver and kidneys were retained. Pellet and supernatant preparations from homogenised, pooled samples of liver and kidney were subjected to chromatographic separation. Copper and zinc were analysed in whole tissue, homogenates and eluant fractions and MT identified likewise using an enzyme-linked immunoassay. Copper accumulated for 5 weeks in the liver falling subsequently accompanied by similar changes in MT content. Kidney copper and MT rose to maximum concentrations at 8 weeks and were maintained thereafter. Substantial differences were apparent in the relative distribution of MT between the two organs. MT was the major, predominantly cytosolic, copper-binding protein in the kidney but in the liver immunoreactive MT was pelleted and present in lower concentration than the high molecular weight cuproproteins. It was concluded that whilst MT plays a role in the detoxification and adaptation of rats to copper-loading the regulatory functions of liver and kidney may differ significantly in this respect.