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.

Obesity and hyperleptinemia in metallothionein (-I and -II) null mice.

Metallothionein (MT) has several putative roles in metal detoxification, in Zn and Cu homeostasis, in scavenging free radicals, and in the acute phase response. Mice of mixed 129/Ola and C57BL/6J background with targeted disruption of MT-I and MT-II genes are more sensitive to toxic metals and oxidative stress. We noted that these animals were larger than most strains of mice, and we systematically studied aspects of their physiology and biochemistry relating to energy metabolism. During the first 2 weeks after weaning, the growth rates of MT-null and C57BL/6J mice were similar, but the transgenic mice became significantly heavier at age 5-6 weeks. At age 14 weeks, the body weight and food intake of MT-null mice was 16 and 30% higher, respectively, compared with C57BL/6J mice. Most 22- to 39-week-old male MT-null mice were obese, as shown by increased fat accretion, elevated obese (ob) gene expression, and high plasma leptin levels, similar to those recorded in Zucker fatty (fa/fa) rats. Seven-week-old MT-null mice also had significantly higher levels of plasma leptin and elevated expression of ob, lipoprotein lipase, and CCAAT enhancer binding protein alpha genes as compared with age-matched C57BL/6J mice. These observations indicate that abnormal accretion of body fat and adipocyte maturation is initiated at 5-7 weeks of age, possibly coincident with sexual maturation. Targeted disruption of MT-I and MT-II genes seems to induce moderate obesity, providing a new obese animal model. A link between MT and the regulation of energy balance is implied.

Research communication copper-1,10-phenanthroline induces internucleosomal DNA fragmentation in HepG2 cells, resulting from direct oxidation by the hydroxyl radical.

In view of the current speculation regarding the possible role of reactive oxygen species (ROS) in apoptosis, both under physiological conditions and in response to chemicals that promote their intracellular formation, the present investigation was undertaken to examine whether DNA fragmentation during oxidative stress results from endonuclease activity (apoptosis) or from direct attack by ROS. We report that the incubation of HepG2 cells (a human-derived hepatoma cell line) with the copper(II) complex of 1,10-phenanthroline, CuII(OP)2, results in internucleosomal DNA fragmentation, which is widely recognized as being a hallmark of apoptosis. DNA fragmentation did not occur at low temperature, but activity was restored by the addition of ascorbic acid. It is proposed that DNA fragmentation results from the direct attack of hydroxyl radicals upon DNA. Hydroxyl radicals are produced from oxygen by the redox-cycling of CuII(OP)2, which is supported by metabolic processes at normal temperature. At low temperature ascorbic acid provides an artificial cellular reducing environment, thereby restoring hydroxyl radical formation. These findings were confirmed by the detection of internucleosomal DNA fragmentation following the exposure of isolated chromatin to a biomimetic CuII(OP)2 redox-cycling system. We conclude that DNA laddering, the widely employed hallmark of apoptosis, is not unique to endonuclease activity and may also result from direct attack upon DNA by the hydroxyl radical.

Generation of 5,5-dimethyl-1-pyrroline N-oxide hydroxyl and scavenger radical adducts from copper/H2O2 mixtures: effects of metal ion chelation and the search for high-valent metal-oxygen intermediates.

A metal-catalyzed nucleophilic addition mechanism for the formation of radical adducts of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has been described recently (K. Makino, T. Hagiwara, A. Hagi, M. Nishi, and A. Murakami, 1990, Biochem. Biophys. Res. Commun. 172, 1073-1080; P. M. Hanna, W. Chamulitrat, and R. P. Mason, 1992, Arch. Biochem. Biophys. 296, 640-644). In the present investigation, we have demonstrated that the recently reported inhibition of copper-dependent hydroxyl radical formation by the complexing agent 1,10-phenanthroline (OP), which appears to contradict the well-known chemical nuclease properties of CuI(OP)2, is an artifact resulting from an inhibition of the nucleophilic addition of water to DMPO by OP (A. C. Mello-Filho and R. Meneghini, 1991, Mutat. Res. 251, 109-113). Copper bound to OP was found to be a good catalyst of hydroxyl radical formation: the CuII(OP)2 complex can be reduced by H2O2 and the CuI(OP)2 generated reacts with the peroxide to form .OH. In contrast, no evidence could be obtained for oxidant formation from the CuII(aq)/H2O2 reaction system, despite the detection of a prominent signal from the DMPO hydroxyl radical adduct (DMPO/.OH) (the formation of which was due solely to the nucleophilic addition of water to DMPO). The failure to generate an oxidant in this reaction mixture was attributed to the failure of hydrogen peroxide to reduce CuII(aq), as hydroxyl radical formation did occur when CuI(aq) was added directly to H2O2. However, in order to account for the high concentration of alpha-hydroxyethanol radicals detected when ethanol was included in the CuI(aq)/H2O2 reaction, the possibility that an oxidant in addition to .OH (e.g., CuO+) is generated is discussed.

Biliary copper excretion in the neonatal rat: role of glutathione and metallothionein.

Metallothionein (MT) and glutathione (GSH) have been implicated as two major copper-binding agents involved in the hepatobiliary copper metabolism in the adult rat. This study was designed to explore their potential role in facilitating copper export from the copper-laden hepatocyte of the newborn rat. Biliary and hepatic copper, GSH, and immunoreactive MT-I concentrations were measured in Sprague-Dawley rats at 1, 2, 3, 4, and 8 weeks of age. Bile was collected by duct cannulation for 90 minutes before the rats were killed. Liver was removed, weighed, and freeze-dried. The bile flow rate (BFR) doubled and the liver weight increased 14-fold during maturation. Hepatic and biliary copper and MT-I concentrations were significantly higher in the suckling than in the weanling. The total biliary output of copper per 24 hours was low at 1 week and increased significantly by 8 weeks of age. MT-I-bound copper represented a maximum of only 3.4% of biliary copper at 1 week and 0.5% at 8 weeks. GSH was not detected in bile until 2 weeks and then increased 15-fold by 8 weeks, while hepatic GSH levels remained unchanged. Therefore, GSH levels did not correlate with the high biliary copper concentration at week 1, although there was a close correlation between the total daily biliary excretion of copper and GSH at 2 weeks and beyond. We conclude that the net biliary copper excretion per day is relatively low in the first week of life and is independent of MT and GSH secretion. It increases significantly once GSH is available in bile.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The intestinal true absorption of 65Zn in rats is adversely affected by diets containing a faba bean (Vicia faba L.) nonstarch polysaccharide fraction.

The effects on the absorption of 65Zn by two varieties of raw faba bean (Vicia faba L., minor) or seed components that may interfere with mineral metabolism in the gut, have been studied in growing rats. In bean diets all protein was supplied by the meals, and the fractions were tested by incorporating them in control diet at the same levels as they occur in the seeds. Absorption of 65Zn was also measured in rats fed dephytinized bean meal produced by including phytase in the diet. Rats were pair-fed diets supplemented with amino acids and minerals to target requirements and containing 40 mg Zn/kg diet. True absorption of Zn was 50-70% lower in rats fed diets containing both cultivars of faba bean meals than in those fed the control diet. Although soluble nonstarch polysaccharides caused a significant reduction in the absorption of Zn, this effect disappeared after the removal of phytate by demineralization. In contrast, despite its negligible content of phytate, the insoluble nonstarch polysaccharides in the cell wall fraction of the cotyledon accounted for most of the reduction in Zn absorption in rats fed the faba bean diets. Addition of phytic acid to the control diet significantly reduced the absorption of 65Zn but only from 44 to 36%. Moreover, the increase in the absorption of Zn was similarly small, from 21% to 29%, with the addition of phytase to the faba bean diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of metallothionein concentrations in rat brain: effect of glucocorticoids, zinc, copper, and endotoxin.

The effects of known inducers of liver metallothionein (MT) synthesis on MT concentrations in the rat brain have been determined using antibodies that are specific for MT I and II and do not cross-react with MT III. There were substantial differences in the MT concentrations in different areas of the brain. Dexamethasone increased MT levels after 24 h in the frontal cortex, cortex, medulla oblongata plus pons, midbrain, striatum, hippocampus, and cerebellum but not in the hypothalamus. Corticosterone produced similar results except in the hippocampus. Long-lasting adrenocorticotropic hormone increased MT concentrations after 12 h in midbrain and striatum but not in the liver. Adrenalectomy decreased MT concentrations after 6 days in the medulla oblongata plus pons, striatum, hippocampus, and hypothalamus but increased concentrations in the liver and kidneys; these effects were reversed by corticosterone. The role of glucocorticoids in the regulation of MT levels therefore differs between tissues and within specific areas of the brain. Injection of zinc or copper intracerebroventricularly and the use of a zinc-deficient diet increased and decreased MT levels, respectively, in some but not all brain areas. Endotoxin increased liver MT but not brain MT I levels after 8 h.

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.

Quantification of metallothionein isoforms using an enzyme-linked immunosorbent assay (ELISA) with two specific antisera.

The specificity of three populations of antibodies to the two metallothionein (MT) isoforms has been characterized using a competitive enzyme-linked immunosorbent assay (ELISA). A polyclonal sheep anti-MT-1 antibody (PAb 1) showed higher affinity to MT-1 than to MT-2 with I50 (concentrations required to achieve 50% inhibition) being 18 and 480 ng for MT-1 and MT-2, respectively. Conversely, a polyclonal rabbit anti-MT-2 antibody (PAb 2) showed higher affinity to MT-2 than to MT-1 (I50 being 250 and 15 ng for MT-1 and MT-2, respectively). A third antibody (PAb 3), isolated by removal of anti-MT-1 antibodies in PAB 2 by immunoaffinity purification, showed no cross-reactivity with MT-1. This result suggests that there are two populations of antibodies which cross-react specifically with the MT isoforms. Rat tissue MT concentrations were quantified by ELISA using the three different antibodies and silver-saturation method. The sum of MT isoform concentrations (ELISA estimation using antibodies PAb 1 and PAb 3) were not significantly different (p < 0.05) from the total MT concentrations obtained by silver-saturation method. Therefore, the ELISA can be used to estimate the relative quantities of the two MT isoforms in rat tissues. The predominant isoform in rat tissues was MT-2 and the proportion of MT-1 increased with induced synthesis of MT by metal injection.

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.

Plasma 65Zn kinetics in the rat.

The amount of 65Zn in the plasma of rats after intravenous injection was found to decline following closely two-compartment kinetics over a period of 90 min. Comparative analysis of the amount of 65Zn present in the two kinetic pools at various time-intervals post-injection with the actual physiological location of the 65Zn, revealed that the initial pool (Qa) is primarily the blood plasma, while the second pool (Qb) is primarily within the liver. The plasma Zn concentration and Qa were both found to fall reproducibly during Zn depletion, whereas Qa and Qb increased following injection of Escherichia coli endotoxin in contrast to the decline in plasma Zn concentration. Further investigation of the nature of Qb indicates that it represents in part a metabolic pool within the liver which varies substantially in response to Zn status in a manner similar to metallothionein.

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.

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.

The protective role of metallothionein in copper-overload: II. Transport and excretion of immunoreactive MT-1 in blood, bile and urine of copper-loaded rats.

The regulation of copper homeostasis in copper overloaded animals occurs by excretion of excess of the metal in bile and urine, which may be facilitated by metallothionein (MT) a copper binding protein. The role of MT in the mobilisation and excretion of copper excess has been studied in copper-loaded rats during the development of tolerance. Young male Wistar rats were fed a high copper (1 g/kg) diet for 16 weeks during which period they were killed after prior collection of bile, blood and urine for analysis for copper and immunoreactive MT-1. In addition bile was separated chromatographically and the eluant fractions were assessed likewise for copper and MT-1. Biliary excretion of copper and MT-1 rose to a maximum after 6 weeks, falling subsequently as the rats became copper tolerant. Early increases in circulating copper and MT-1 occurred likewise but whereas MT-1 fell subsequently during the recovery period, serum copper remained elevated. By contrast, urinary copper and MT-1 maintained an increased output throughout. Chromatographic separation of bile revealed the presence of a range of immunoreactive MT-1 degradation products. It was concluded that the close correspondence between bile and serum MT reflected their hepatic derivation and implicated liver MT as an export protein in the early stages of copper overload. By contrast, urine MT, maintained independently of circulating MT levels, established the active secretory participation of the kidney in promoting the continued depletion of excess copper.

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.