Ian Bremner

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.

Nutritional and Physiological Significance of Metallothionein

The biological function of metallothionein (MT) is still a matter of considerable conjecture. Convincing evidence has accrued that the protein can play an important role in the detoxification of certain heavy metals but a more fundamental role may be concerned with the metabolism of the essential elements zinc and copper. In addition the effects of physical and inflammatory stress and of infection on MT production imply an involvement in defence mechanisms against these conditions. MT may also act as a general detoxifying agent against certain xenobiotics.

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.

The influence of dietary iron and molybdenum on copper metabolism in calves.

1. Twenty heifer calves were allocated to four groups and maintained for 32 weeks on a diet based mainly on barley and straw and containing 4 mg copper/kg. The diet was supplemented with 0 or 800 mg iron/kg and 0 or 5 mg molybdenum/kg. 2. Liver and plasma Cu concentrations, erythrocyte superoxide dismutase (EC 1.15.1.1) and plasma caeruloplasmin (EC 1.16.3.1) activities decreased greatly and rapidly in all calves given the Fe or Mo supplements or both. Levels indicative of severe Cu deficiency were attained within 16 weeks. There were no significant differences in values in animals given Fe, Mo or Fe plus Mo. 3. Clinical signs of Cu deficiency developed after 20 weeks in the calves given the Mo supplement. Growth rates were reduced, skeletal lesions developed and hair texture and colour were affected. No such effects were observed in the calves given only the Fe supplement. 4. Plasma and liver Fe concentrations increased in calves given the Fe supplement but were not greatly affected by Mo, even when the calves were severely Cu-deficient. 5. The significance of the effects of Fe and Mo on Cu metabolism are discussed with special regard to the influence of soil ingestion on Cu availability and to the frequent lack of correlation between the Cu status of animals and their clinical condition.

Copper and zinc metabolism in health and disease: speciation and interactions

It has long been evident that the metabolism of trace elements cannot be considered in isolation. A wide range of nutritional and physiological factors can influence their uptake, transport and storage, with subsequent enhancement of susceptibility to deficiency or toxicity states. Interactions occur with other trace elements and these can be classified as competitive or non-competitive, direct or indirect. However, their physiological or toxicological significance is sometimes debatable, partly because they were demonstrated under extreme experimental conditions where massive doses of the antagonistic metal were administered, often by parenteral routes and to animals whose trace element status was already severely compromised. Our understanding of the mechanisms of the interactions is frequently limited and indeed in only a few cases has it been possible to describe these on a molecular basis. It is almost traditional to cite the early work of Hill & Matrone (1970) as the first serious attempt to describe trace element interactions on a rational basis. They postulated that elements with similar physical or chemical properties will act antagonistically to each other biologically. The implication was that such metals could compete for binding sites on transport proteins or on enzymes requiring metals as co-factors. Over subsequent years, much evidence was produced in support of this view but rarely was the specific site of interaction identified, other than at tissue level. In part this reflected somewhat embarrassing limitations in our knowledge of the proteins involved in the intracellular and extracellular transport and storage of trace metals.

Protective effect of zinc supplementation against copper toxicosis in sheep

1. A study has been made of the effects of dietary zinc supplementation on the development of copper toxicosis in three groups each of eight 12-week-old lambs. 2. None of the lambs receiving 420 mg Zn/kg diet developed Cu toxicosis in the 24-week experimental period, compared with three in the control group receiving 43 mg Zn/kg and possibly one in the group receiving 220 mg Zn/kg. 3. Liver Cu concentrations were reduced by up to 40% in the Zn-supplemented animals, with concomitant reductions, especially in the early stages of the experiment, in the extent of liver damage, as assessed by measurement of plasma aspartate aminotransferase (EC 2.6.1.1) and arginase (EC 3.5.3.1) activities. 4. Plasma and liver Zn concentrations were increased only slightly in the lambs receiving the Zn-supplemented diets, and the only indication of possible toxic effects of the Zn supplements was the development of a slight anaemia in those animals receiving 420 mg Zn/kg diet. 5. The results suggest that the incidence of Cu toxicosis in sheep may be controlled by increasing their dietary Zn intake.

Distribution of copper and zinc in the liver of the developing sheep foetus.

1. A study has been made of the copper- and zinc-binding proteins in the livers from sheep foetuses of 80-142 d gestational age. 2. Metallothionein was found to constitute the major Zn-binding component in the cytosol at all times and to be identical to Zn-thionein from adult sheep liver. 3. Zn also occurred in two fractions, not normally found in sheep liver, with approximate molecular weights of 28000 and 47000. The relative proportions of these were age-dependent. 4. Between 15 and 35% of the hepatic Cu, corresponding to most of the Cu in the cytosol, also occurred in the metallothionein-containing fraction.

Effect of zinc, copper and glucocorticoids on metallothionein levels of cultured neurons and astrocytes from rat brain

The knowledge of brain metallothionein (MT) regulation and especially of MT presence in specific cell types is scarce. Therefore, the effect of several well-known MT inducers, measured by radioimmunoassays using antibodies that cross-react with MT-I and MT-II or specific for MT-I and which do not cross-react with human growth inhibitory factor (GIF or MT-III), has been studied in primary cultures of neurons or astrocytes obtained from rat cerebrum. MT-I levels in glial cells were about ten times higher than those in neuronal cells (538 +/- 194 vs. 49 +/- 16 pg MT-I/micrograms protein, mean +/- S.D. from three separate cell preparations). Increasing the concentration of Zn in the bovine serum albumin (BSA)-containing culture medium up to 50 microM significantly increased MT-I levels by up to 3.5-fold in neurons and 2.5-fold in astrocytes. In contrast, Cu up to 50 microM increased MT-I levels in a saturable manner in both neurons (up to 5-fold) and astrocytes (up to 1.5-fold), the maximum effect occurring at 5 microM Cu. In general, the combination of Zn and Cu further increased MT-I levels. The effect of the metals on MT-I appeared to reflect metal uptake, since MT-I induction was less marked when the BSA concentration in the medium was increased from 2 to 10 mg/ml. Dexamethasone increased MT-I levels in both neurons and astrocytes in vitro in a concentration-dependent manner. Endotoxin, IL-1 and IL-6 did not have a significant effect on glial MT levels at the concentrations studied. The administration of dexamethasone to rats increased MT-I levels in non-frontal cortex, cerebellum, pons+medulla, midbrain and hippocampus, but not in hypothalamus, frontal cortex and striatum. Endotoxin increased liver but not brain MT-I levels. Immunocytochemical studies in adult rat brain preparations with a polyclonal antibody that cross-reacts with MT-I and MT-II indicated that immunostaining was always nuclear in glial cells, whereas in neurons it was nuclear in the cerebral cortex, hippocampus and the granular layer of the cerebellum, and nuclear plus cytoplasmic in Purkinje cells in the cerebellum, hypothalamic nuclei and gigantocellular reticular nucleus in the brain stem. Meninges, choroidal plexus, ependymal and endothelial cells were also MT-immunoreactive.

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.