Article

Trace Metal Metabolism in Cultured Skin Fibroblasts of the Mottled Mouse: Response to Metallothionein Inducers

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Abstract

Menkes' kinky hair syndrome is a lethal X-linked disorder marked by tissue-specific increases in copper content. An animal model of kinky hair syndrome is provided by mice mutant at the X-linked mottled locus. The basic defect is unknown. In order to discriminate among potential etiologies, we asked whether the expression of the mottled mutation causes abnormalities in the metabolism of trace metals other than copper in hemizygous mottled (blotchy) cultured skin fibroblasts, and whether we can differentiate mutant and normal cells according to their response to metal inducers of metallothionein. Blotchy fibroblasts accumulated up to 12 times more 64Cu than control (littermate) cells, over time and over a range of 64Cu concentrations. A saturable high affinity component to 64Cu accumulation over a fixed time interval was revealed in these studies. While 64Cu uptake kinetics were indistinguishable in mutant and control cells, the patterns of 64Cu exit differed. In both cell types, the rate of release of a rapidly exchangeable fraction of newly acquired 64Cu was similar. However, in mutant cells, a larger fraction of recently accumulated 64Cu is retained. In contrast to the results for 64Cu, accumulation and exit of 65Zn and 109Cd were not distinguishable in mutants and controls. With exposure to either a strong (cadmium) or weaker (zinc) inducer of metallothionein, 64Cu accumulation was increased in normal cells, while there was no change from the already elevated level of 64Cu accumulation in blotchy cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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Chapter
Copper is a trace element of essential importance to humans. Our need for copper relates to its role as a cofactor for a number of enzymes whose activities are responsible for critical cellular processes. Numerous conditions can influence copper homeostasis and serum copper levels, including several genetic diseases described in this chapter. Spawned by identification of the genes responsible for Menkes disease and Wilson disease, the past 3 decades have witnessed a remarkable growth in our knowledge and understanding of eukaryotic copper metabolism. Appreciation of the basic pathways that guide cellular copper uptake, transport, and export has reached a reasonable level; however, considerably less is known about the precise mechanisms that underlie the neurological consequences of disturbed copper homeostasis and the ideal remedies. Mutations in the copper-transporting ATPase, ATP7A, yield three distinct X-linked syndromes—Menkes disease, occipital horn syndrome (OHS), and isolated distal motor neuropathy, whereas defects in ATP7B cause autosomal recessive Wilson disease. Advances in genomics have facilitated identification of previously unappreciated disorders of copper transport, as well as consideration of newborn screening using molecular approaches. Similar advances in viral gene therapy augur well for new treatment paradigms. An array of useful animal models provides opportunities for further exploration of human copper metabolism and evaluation of potential disease remedies for inherited copper transport disorders, including gene therapy.
Article
Copper is a trace element of essential importance to humans. Our need for copper relates to its role as a cofactor for a number of enzymes whose activities are responsible for critical cellular processes. Numerous conditions can influence copper homeostasis and serum copper levels, including several genetic diseases described in this chapter. Spawned by identification of the genes responsible for Menkes disease and Wilson disease, the past two decades have witnessed a remarkable growth in our knowledge and understanding of eukaryotic copper metabolism. Appreciation of the basic pathways that guide cellular copper uptake, transport, and export has reached a reasonable level; however, considerably less is known about the precise mechanisms that underlie the neurological consequences of disturbed copper homeostasis, and the ideal remedies. Mutations in the copper-transporting ATPase, ATP7A, yield three distinct X-linked syndromes—Menkes disease, occipital horn syndrome, and isolated distal motor neuropathy—whereas defects in ATP7B cause autosomal recessive Wilson disease. Numerous examples of infantile copper toxicosis have been reported globally. Environmental exposures to copper often contribute to this syndrome, for which the genetic bases remain obscure. An array of useful animal models provides opportunities for further exploration of human copper metabolism and evaluation of potential disease remedies for inherited copper transport disorders, including gene therapy.
Chapter
Heavy metals are both ubiquitous and long-lived in the environment (reviewed by GOyer 1991) and have enormously varied effects on cells. They are an absolute requirement for the function of both prokaryotic and eukaryotic cells (17 out of the 30 elements essential for life are metals; COtton and WIlkinson 1980), but are toxic to cells and organs through different pathways and to different degrees (reviewed in several chapters of this volume). Some metals have no known function in cells, but have toxic effects:cadmium and arsenic are examples. Cells have developed mechanisms to keep toxic metal species away from critical targets, and some of those mechanisms will be covered in this review. Others are essential for normal cellular function, but are toxic under certain circumstances and at particular concentrations:metals that fall into this category include copper (an essential cofactor for many oxidative enzymes, including catalase, peroxidase, cytochrome oxides, and others — but also a dangerous cellular toxin; HOrn 1984), cobalt (an essential cofactor for vitamin B12), manganese (a cofactor in many enzymatic reactions involving phosphorylation, cholesterol, and fatty acid synthesis), iron (required for haemoglobin), selenium (essential for glutathione peroxidase; HOGBERG and ALexander 1986), and molybdenum (an essential cofactor for xanthine oxidase and aldehyde oxidase, and required in plants for fixing atmospheric nitrogen by bacteria).
Article
Background: Direct association of trace and macro-elements in relation to human disease has been observed in many research studies. In many cases, an alteration in the metabolism of these minerals has been demonstrated. Methods: In this investigation, the hair levels of the trace elements zinc (Zn), copper (Cu), chromium (Cr), iron (Fe), nickel (Ni) and the toxic elements lead (Pb) and cadmium (Cd) were determined in 110 subjects (57 skin- diseased subjects and 53 controls). Samples were analyzed using atomic absorption spectrophotometric methods. Results: Analysis of hair samples revealed significantly lower levels of zinc, iron and copper in skin-diseased patients compared with normal controls, and significantly higher levels of chromium and nickel and of toxic elements, lead and cadmium. The same pattern was observed in males and females and in both age groups studied (6-15 and 16-30 years). Conclusions: These data can guide clinicians and other professionals investigating deficiencies in essential trace metals and excessive levels of toxic metals in biological samples.
Article
We present64Cu uptake studies in cultured muscle cells from a one-year-old patient with Menkes' disease. The cultured muscle cells from the patient showed a five-fold higher64Cu uptake than control muscle cells. Copper uptake in muscle cells was of the same magnitude as that found in fibroblasts from the patient and also from other Menkes' patients. The copper content of a muscle biopsy from the patient was twice that of a control biopsy. The enhanced uptake is probably copper specific, since zinc uptake was unaltered in both muscle cells and fibroblasts from the patient. Cytochrome c oxidase in the muscle of the patient was reduced to one-third of the value for controls, which is in agreement with the hypothesis that in Menkes' disease copper accumulates in a biologically non-active form. However, in cultured muscle cells and fibroblasts from the patient the cytochrome c oxidase activity was in the normal range, probably because of the relatively large amount of copper already available in the culture medium.
Article
Menkes' kinky hair syndrome (KHS) is a lethal x-linked neurodegenerative disorder of copper metabolism, with low serum copper concentrations, tissue-specific copper sequestration, and decreased activities of cuproenzymes in a number of cell types. Although liver copper accumulation is abnormal in KHS, the actual defect in hepatic copper metabolism has not been elucidated. Our studies of liver copper metabolism were conducted in the mottled (blotchy) mouse, an animal model of KHS. After implantation of central venous and biliary catheters in both blotchy and control mice, we measured biliary copper excretion, hepatic copper uptake, and tissue copper contents over an 8-h period after i.v. bolus administration of radioactive 64Cu. Under the experimental conditions used, bile flow and biliary bile acid excretion were held constant, and control and blotchy hepatic 64Cu concentrations were similar in the face of the expected differential in control and mutant kidney 64Cu contents. Biliary excretion of radiocopper was 24.7 +/- 1.5% of injected 64Cu over 8 h in control animals, whereas heterozygotes excreted 6.5 +/- 1.3% and a single hemizygote excreted less than 2%. The pattern of biliary copper excretion was different, with sharp increase and steady decline in control biliary 64Cu excretion but consistently low excretion in mutant mice. No differences were observed in control or mutant hepatic uptake of 64Cu. These data show a reduced biliary excretion of copper in the blotchy mouse, in the absence of a defect in hepatic copper uptake. We suggest that defective copper transport from hepatocyte to bile represents the hepatic expression of the mottled mutation and speculate that a similar defect occurs in human KHS.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
Both deficiency and excess of copper induce toxic effects on mammalian cell systems in vivo and in vitro. The effects can be related to the affinities of Cu(II) ions for specific cell components. The nucleus is a potential site for temporary Cu storage while primary targets for free Cu(II) ions are the thiol groups which reduce the ions to Cu(I). Cu(II) ions show a high affinity for nucleic acids, binding with DNA both at intrastrand and interstrand levels, possibly through intercalation between GC pairs. The ability to chelate Cu(II) ions is seen to be of the order: purine greater than purine ribonucleotides greater than purine ribonucleoside greater than pyrimidine ribonucleotides. Copper is an integral part of enzyme activation and enters into the molecular structure of several proteins, like ceruloplasmin. Cu(II) ion is a potential mutagenic agent as seen by its property of inducing infidelity in DNA synthesis in vitro. Teratogenic activities of copper have been reported but carcinogenicity is not yet confirmed. Copper is an essential component of chromatin and is known to accumulate preferentially in the heterochromatic regions. External application of higher doses, however, induces both clastogenic effects and spindle disturbances. In certain forms, inorganic copper enhances the clastogenic activity of other agents. The most widely studied human genetic maladies linked with copper metabolism are Menkes' and Wilson's diseases. Several mutations are known which influence Cu homeostasis in mammals. Such mutations in mice have been used extensively for biochemical studies.
Article
Chorionic villus samples with copper contents of 1·91, 4·2, 5·6, and 6·3 ng/mg were observed in four cases with male karyotypes. These values were outside the range for unaffected males (0·30–0·85 ng/mg), and three of them were outside the control range (0·20–2·39 ng/mg). But these three values were below the values previously observed for affected Menkes fetuses (12·0–24·8 ng/mg). Follow-up by 64Cu uptake studies on the amniotic fluid cells was performed in three of these cases. A combination of 64Cu uptake and chase experiments on the amniotic fluid cells showed more convincingly than 64Cu uptake per se the direct copper values of 4·2 and 5·6 ng/mg to correspond to affected fetuses. Amniotic fluid cells from the male fetus with the CV copper value of 1·9 ng/mg showed normal results. The CV copper value of 6·3 ng/mg was considered pathognomonic for Menkes disease. The pregnancy was terminated, and the diagnosis was confirmed on fetal fibroblasts. Maternal deciduum prepared from the placentae showed in one of the cases with an affected fetus copper values ranging from 1·5 to 5·7 ng/mg. In six additional diagnostic cases, the copper content was determined in both CV samples and maternal deciduum. In three of these cases with normal CV sample copper, maternal decidua values of 4·85–7·8 ng/mg copper were observed. These results show that maternal deciduum contamination of a CV sample could cause a false-positive diagnosis.
Article
Menkes kinky hair syndrome is an X-linked neurodegenerative disorder, causing tissue-specific increases in copper and metallothionein content. A mouse model is provided by hemizygotes for mutant alleles at the X-linked mottled locus. Herein we test the possibility that the primary defect in both species is in metallothionein gene regulation. We show that metallothionein-I messenger RNA (mRNA) (mouse) and metallothionein-II mRNA (human) are elevated in mutant fibroblasts. However, comparable dose-response curves in mutant and control cells are generated when mouse metallothionein-I mRNA concentrations are measured in cells exposed to varying concentrations of cadmium or copper (metallothionein inducers). Furthermore, when mutant and control cells are grown to achieve overlapping intracellular copper concentrations in the two cell types, metallothionein-I (mouse) and metallothionein-II (human) mRNA levels are proportional to the intracellular copper concentrations. Finally, in paired determinations in blotchy hemizygote and littermate kidneys containing comparable copper levels, metallothionein-I mRNA contents are very similar. The observations suggest that elevated intracellular copper in these mutants induces metallothionein synthesis by normal regulatory mechanisms.
The effects of trace metal nutrition on the fetus and neonate have been described. Since very little research has been done with the human fetus and neonate, much of our knowledge must be extrapolated from animal studies. In addition, most of the work centres around the effects of copper and zinc nutrition. Nutritional requirements (when known) for both enteral and total parenteral feeding of certain trace metals, as well as the bioavailability, have been discussed. Finally, methods of assessment of trace metal nutritional status have been discussed. These include direct measurement of metal concentrations and determination of biochemical indicators, such as metal-dependent enzyme activities--both of which are static indicators of nutritional status, and functional assessment of nutritional status which is a dynamic measure of trace metal nutrition and includes tests measuring the effects of metal nutrition on the function of cells, tissues, organs, and the host in general.
Article
An animal model for Menkes' kinky hair syndrome is provided by mice mutant at the X-linked mottled locus. Two mechanisms have been invoked to explain disease manifestations in mottled and in kinky hair syndrome: relative tissue copper deficiencies and corresponding reductions in cuproenzyme activities; or defective intracellular copper utilization, with impaired intracellular translocation to cuproenzymes or to copper-dependent processes. We addressed the second possibility through measurements of soluble superoxide dismutase (SOD-1) in cytosol extracts of confluent mottled (blotchy) cultured skin fibroblasts. At comparable intracellular copper concentrations over a broad range, SOD-1 specific activities in the mutant cells were not distinguishable from those in controls, or, in some instances, were actually higher. These data suggest that the excess copper anomalously sequestered in a cell expressing the mutation remains available for binding to a cytosolic cuproenzyme. When taken together with data in other systems, the results are consistent with the thesis that the basic lesion in blotchy may primarily affect copper transport or delivery to specific copper transport systems.
Article
The precise nature of the variation in cellular copper load against medium copper concentration is defined using a comprehensive logarithmically incremented series of medium copper concentrations ranging from low levels (4.8 p.p.b.) through 'normal' to toxic levels (40 p.p.m.) in which fibroblasts were grown followed by determination of intracellular content. Menkes' fibroblasts showed an unexpected plateau region of stable intracellular copper content against a change in medium concentration of over 100-fold, albeit only when sufficient copper was present in the medium (0.08-8.0 p.p.m.). Thus, Menkes' cells are clearly capable of balancing uptake/efflux providing copper availability allows. Simultaneous analysis of cellular copper and zinc load at various medium copper concentrations shows an indistinguishable intracellular copper:zinc ratio between the two cell lines. The nature of non-labeled copper uptake by fibroblasts over a 40 min and 7 day period is reported. During the 40 min period copper uptake (20 p.p.m.) was essentially the same in both cell lines. However, copper absorbed was superimposed upon large pre-existing copper pools in the case of Menkes' cells only. Advantages of techniques determining non-labeled copper in copper uptake/efflux experiments are discussed in the light of these results. Fibroblast growth studies showed that, compared with normal cells, Menkes' cells are significantly (P < 0.01) more growth sensitive to extended exposure to low copper concentrations. Thus, Menkes' disease appears to be not only a result of copper maldistribution but also a direct result of an inability of Menkes' cells to function normally in low copper environments.
Article
Full-text available
Menkes syndrome in humans is an X-linked disorder characterized in part by abnormal copper transport, cellular copper sequestration, and defective crosslinking of collagen and elastin. A decrease in the functional activity of lysyl oxidase, a cuproenzyme, is thought in part to be responsible for the decreased crosslinking of collagen and elastin. It has also been suggested that low levels of lysyl oxidase activity may occur secondarily to disturbances in intracellular copper translocation and consequently impaired incorporation of copper into lysyl oxidase. Herein, we examine the expression and accumulation of selected extracellular matrix proteins in fibroblasts from a Menkes patient, as well as fibroblasts from the tortoiseshell (MoTo/y) mouse. The MoTo mutation is an allele of the mottled (Mo) locus, which is considered to be a murine analog of the human Menkes locus. In both Menkes and tortoiseshell fibroblasts, levels of lysyl oxidase mRNA transcripts were less than 15% of levels for corresponding controls. The level of elastin mRNA transcripts was also markedly lower in both cell lines in comparison to controls. In contrast, the levels of procollagen Type I mRNA were similar or enhanced in Menkes and MoTo/y fibroblasts compared to their respective controls. Consequently, we conclude that the connective tissue defects associated with Menkes syndrome and those occurring in mottled mouse mutants involve more than abnormal copper utilization in the formation of lysyl oxidase holoenzyme. Based on the present studies in cell culture, the production of essential enzymes and matrix proteins, such as lysyl oxidase and elastin, appear to be altered at the level of transcription or mRNA turnover.
Article
Menkes disease is an X-linked disorder of copper transport characterized by progressive neurological degeneration and death in early childhood. We have isolated a candidate gene (Mc1) for Menkes disease and find qualitative or quantitative abnormalities in the mRNA in sixteen of twenty-one Menkes patients. Four patients lacking Mc1RNA showed rearrangements of the Menkes gene. The gene codes for a 1,500 amino acid protein, predicted to be a P-type cation-transporting ATPase. The gene product is most similar to a bacterial copper-transporting ATPase and additionally contains six putative metal-binding motifs at the N-terminus. The gene is transcribed in all cell types tested except liver, consistent with the expression of the Menkes defect.
Article
Cellular copper transport processes are required by all organisms for correct utilization in cell biochemical processes and avoidance of the toxicity of copper excess. Copper import into bacterial, yeast, and mammalian cells requires the coordinate function of proteins with both metal-binding and catalytic domains in mediated transport steps. Following entry, detoxification mechanisms found across species include the binding of copper to specific proteins (e.g. metallothioneins) and the transfer of copper into isolated cell compartments (e.g. periplasmic space, lysosome). Multiple proteins mediate intracellular transfers in bacteria, and glutathione may play a major role in cytosolic copper delivery to cuproenzymes in mammalian cells. Study of two human disorders of copper transport, Menkes disease and Wilson disease, led to the identification of an important category of proteins mediating cell copper export. The Menkes and Wilson disease gene products are copper-transporting ATPases of the P type, with ATPase domains and N-terminal metal-binding amino acid motifs that are evolutionarily conserved in unicellular and mammalian organisms. These observations suggest that yeast and bacterial copper transport proteins, or individual domains of these proteins, may generally have homologues in mammalian systems.
Article
To define the function of the Cu-transporting ATPase in Menkes disease, Menkes and normal fibroblasts were incubated with 67Cu before and after brief exposure to -SH reagents, p-chloromercuribenzoate (PCMB) and dithiothreitol (DTT). Accumulation and retention were compared among these cells, BeWo cells, and rat C6 glioma cells similarly treated. The Michaelis constant for influx of 67Cu into normal and Menkes fibroblasts was practically the same (0.21 +/- 0.07 vs. 0.24 +/- 0.06 microM). The PCMB treatment stimulated 67Cu accumulation in C6 cells, inhibited accumulation in normal and Menkes fibroblasts, and did not affect BeWo cells. DTT stimulated 67Cu uptake in all cells but BeWo cells. DTT treatment after PCMB further enhanced 67Cu accumulation in normal fibroblasts and C6 cells but had no enhancing effect on Menkes fibroblasts or BeWo cells. Menkes fibroblasts and BeWo cells released 67Cu at rates considerably slower than normal fibroblasts (0.06 and 0.09 vs. 0.22%/min, respectively). The PCMB blocked 67Cu release from normal fibroblasts but did not affect Menkes fibroblasts or BeWo cells. Reverse transcription-polymerase chain reaction analysis of total RNA from BeWo cells failed to show a predicted 943-base pair fragment representing a partial transcript of the Menkes factor. The fragment was present in extracts from normal fibroblasts. We conclude that the mechanism underlying Cu homeostasis varies among different cell types. As exemplified by BeWo and Menkes cells, failure to efflux Cu ions may be linked with the failure to express a functional Cu-transporting ATPase, namely, the Menkes protein.
Article
Full-text available
Cultured skin fibroblasts from patients with Menkes disease, an X-linked disorder involving a defect in copper metabolism, were analyzed for copper concentration by means of atomic absorption spectrophotometry. These cultures consistently exhibited elevated copper concentrations (mean = 335.5 ng of copper per mg of protein) when compared to control fibroblast cultures (mean = 59.2 ng of copper per mg of protein). External factors that could influence the copper content of cultures were found not to affect the differences in copper concentration between control and Menkes cells. Furthermore, Menkes cells could be differentiated from cultured fibroblasts of controls, of presumed heterozygotes, and of Wilson's disease patients by copper concentration. These observations led to the conclusion that the increased copper content of cultured Menkes cells was characteristic of Menkes disease, resulting from the expression of the genetic abnormality. This provides a genetic marker, a defect in metal metabolism demonstrated in human fibroblasts, that should prove valuable in both the diagnosis of Menkes disease and in the study of the fundamental defect of this genetic disorder.
Article
Full-text available
Cultured lymphoblasts derived from infants with Menkes' disease exhibit the same increased avidity for copper as do fibroblasts and most extrahepatic tissues from these patients. The Menkes' cells preferentially take up not only copper but also, on exposure to elevated metal concentrations, the other metallothionein-binding metals, zinc and cadmium. Menkes' lymphoblasts contain larger amounts of metallothionein than normal cells following exposure to each of these metals; the amount bound to this protein quantitatively accounted for the total cellular increment in metal in Menkes' cells. Induction of metallothionein synthesis caused both normal and Menkes' cells to subsequently take up increased amounts of 67Cu. These observations suggest that an enhanced capacity of Menkes' cells to accumulate metallothionein may be responsible for their increased uptake and retention of copper.
Article
Natural selection more often favors status quo rather than adaptive changes. Reflecting this extreme conservatism, the X linkage group appears to have been conserved in toto throughout mammalian evolution. It is proposed that the particular form of dosage compensation mechanism which relies on always keeping the X chromosome in functionally haploid state developed because the ancestral X chromosome of mammals happened to carry a large number of regulatory gene loci disproportionate to its size (5% of the genome). The functionally haploid state appears advantageous to regulatory gene loci. Cattanach's 'controlling element' situated in the middle of the mouse X linkage map appears to represent the operator site of the X involved in the X activation inactivation process. When a mutated form (O(hv)) is coupled with the wild type (O+) in the female, the preferential activation of the X carrying O(hv) is observed. O(hv) in the hemizygous state (i.e., XY) appears to repress the activity of nearby genes, including the Tfm regulatory locus. The wild type O+ might also exercise this type of cis effect although to a lesser extent. Such repressive cis effect upon X linked regulatory gene loci might also be of advantage since regulatory gene products should be made in exceedingly small amounts; i.e. several thousand molecules per cell.
Article
Defective copper metabolism was demonstrated in male mice bearing the blotchy (Moblo/y) allele at the mottled locus on the X-chromosome. Copper absorption from the gut was only 64% of that found in normal mice and hepatic copper levels were only 56% of the controls. Ceruloplasmin and heart cytochrome c oxidase activities were normal, yet lysyl oxidase activity from cultured fibroblasts was only 45% of control levels. Copper accumulated in fibroblasts cultured from these mutants to values that were five times normal. The accumulation of copper in the fibroblasts was associated with a protein of approximately 12,000 molecular weight.
Article
The incorporation of 64Cu into cultured fibroblasts from 7 boys with Menkes' steely hair disease and from 9 controls was examined. The median 20 h incorporation was 74-4 ng 64Cu/mg protein for the patients and 26-1 ng 64Cu/mg protein for the controls (P less than 0.01). Thus, the defect in copper metabolism in the disease was expressed in cultured cells. The method was applied to amniotic fluid cell-cultures of 2 pregnancies at risk and to amniotic fluid cell-cultures with a male karyotype of 8 normal pregnancies. The median 20h 64Cu incorporation into the normal cell-cultures was 19.2 ng 64Cu/mg protein, into the cell-culture from one pregnancy at risk was 48-6 ng 64Cu/mg protein, and from the other, 12-5 ng 64Cu/mg protein. The first case was aborted and the copper distribution in the fetus was found to be abnormal--consistent with Menkes' disease. The second case at risk was born in May, 1975, and no signs of Menkes' disease have developed. These cases demonstrate that prenatal diagnosis of Menkes' steely hair disease might be feasible.
Article
Primary cultures of rat liver parenchymal cells maintained as a monolayer in serum-free culture medium were used to investigate the characteristics of zinc accumulation in vitro. Liver parenchymal cells accumulated zinc by a temperature-dependent, saturable process that was inhibited by cyanide, azide, oligomycin, N-ethylmaleimide and iodoacetamide. Cadmium reversibly inhibited zinc accumulation in both serum-free and serum-containing media. Gel filtration chromatographic studies showed that recently accumulated intracellular zinc was present as a low molecular weight complex smaller than metallothionein, the zinc storage protein, but larger than individual amino acids. The quantity of zinc accumulated was affected by preincubation of the cells with various hor?ONES. Dexamethasone, prednisone and prednisolone each increased zinc uptake by 40--50% when either insulin or glucagon was also present. Hydrocortisone, cortisone and sex steroids did not influence zinc accumulation. Removal of the polypeptide hormones from the medium abolished the stimulatory effect of the synthetic glucocorticoid steroid hormones on zinc accumulation.
Article
In contrast to the situation for copper, the levels of cadmium, nickel and zinc in various tissues from mice hemizygous for the neonatallethal mottled allele are entirely normal. A developmental study of copper levels in the semi-viable has revealed that the accumulation of copper in the kidney and the deficiency in the brain are maintained at about the same level throughout the first 4 weeks of life. In contrast, the accumulation in the gut mucosa disappears in older animals, and the deficiency in the liver is much reduced by 21–30 days. A reduced gut accumulation and liver deficiency is also seen in adult /+ heterozygotes. The deficiency in the liver and the accumulation in the kidney of male mice is associated with a protein fraction of about 14000 daltons. This fraction isolated from liver tissue has been further divided by ion-exchange chromatography into 4 sub-fractions, with each sub-fraction from mutant tissue showing an approximately proportionate reduction in copper content.
Article
Metallothionein biosynthesis is not induced by extracellular copper in Menkes Kinky hair disease (MKHD) or in normal cultured fibroblasts under the conditions of these experiments. In the presence of copper, MKHD fibroblasts also incorporated less cysteine than did normal fibroblasts. Extracellular cadmium greatly enhanced the uptake of cysteine in both normal and MKHD cultures. By the technique of polyacrylamide gel electrophoresis, it was demonstrated that metallothionein is induced by cadmium in normal and MKHD-cultured fibroblasts.
Article
Copper metabolism was analyzed in Brindled (Mobr) male mice and in heterozygous Brindled (Mobr) adult female mice. The intestines of Mobr mice suckling dams injected with ⁶⁴Cu contained 72% of the radioactivity absorbed from the mother's milk and the liver contained <3% of the label obtained via the milk. The intestines from normal pups suckling labeled dams never contained more than 25% of the label taken up from the milk and approximately 50% of the absorbed ⁶⁴Cu was found in the liver of these pups. The copper concentration in the kidneys from Mobr males was significantly greater than the concentration of copper in the kidneys from normal pups. When Mobr male pups and normal pups were given two injections of 10 µg Cu, approximately 2.6% of the injected copper was recovered in the liver of the Mobr pups at autopsy but approximately 38% of the copper injected into the normal pups was present in the liver at autopsy. The copper injections produced a 3.5-fold increase in the kidney copper concentration of Mobr mice but there was no significant increase in the copper content of the kidneys from normal mice. These results demonstrate that both intestinal copper absorption and hepatic copper uptake are impaired in Mobr male mice. When either 1.0 µg or 10 µg of ⁶⁴Cu was administered orally to adult female mice, the Mobr females absorbed approximately 30% less copper than the normal females. The rate of copper excretion was decreased in adult Mobr females and the kidney copper concentration of the Mobr was significantly greater than that of normal females. These results demonstrate that copper homeostasis is altered in both adult Mobr female mice and their neonatal Mobr male offspring.
Article
The defect in Menkes' disease in man is identical to that in Brindled mice. The defect manifests itself in a accumulation of copper in some tissues, such as renal, intestinal (mucosa and muscle), pancreatic, osseous, muscular, and dermal. Hence a fatal copper deficiency results in other tissues (e.g., hepatic). The copper transport through the intestine is impaired and copper, which circumvents the block in the copper resorption, is irreversibly trapped in the above-mentioned, copper accumulating tissues where it is bound to a cytoplasmatic protein with molecular weight 10,000 daltons, probably the primary cytoplasmatic copper transporting protein. This protein shows a Cu-S absorption band at 250 nm, and the copper:protein ratio is increased. Such copper rich protein was found neither in the kidneys of the unaffected mice nor in the liver of the mice that do have the defect. Three models of the primary defect in Menkes disease are proposed.
Article
1. Duodenal injection of 64Cu in treated adult mutant mice (Mobr/y) revealed severe malabsorption of copper. In suckling mutants, malabsorption was less severe, owing to delayed absorption between 2 and 5 h after injection. Pinocytosis at the distal small intestine seems the likely explanation for this difference, and this is supported by results of ileal injection of radioisotope in the suckling mice. 2. The distribution of 64 Cu in various organs was measured in suckling normal, mutant and heterozygote mice and in adult normal and mutant mice during 48 h after intracardiac injection. Excessive accumulation of radioisotope was observed in most extrahepatic organs of mutant and heterozygote mice and was most pronounced in kidney. This could not be explained by initial copper deficiency. The livers of suckling mutant and heterozygote mice lost radioisotope rapidly after normal initial uptake. This pattern was not seen in adult mutants.
Article
Copper therapy was applied to brindled mouse mutants, which suffer from lethal hypocupraemia, by using cuprous and cupric solutions. The method of treatment was a single subcutaneous injection of 50 microgram of copper at 7 days of age. Early effects of the dose were: prevention of the tremors and spasms seen in untreated mutants, raising to normal and near-normal of caeruloplasmin oxidase and lysyl oxidase activities and pigmentation of skin and fur. Growth of mutants was retarded up to 23 days of age, but thereafter they rapidly gained weight to be nearly normal by 60 days of age. At 3 days after injection, copper concentrations in previously deficient mutant organs apart from liver were at least as much as those of treated normals, which had remained unchanged. Copper in mutant livers had increased only slightly in comparison with the normal control. A state of copper deficiency recurred in mutant tissues by 25 days after injection. A solution of Cu+, retained as such by an alkyl polyether, and sebacic acid resulted in greater growth rates after 23 days than did three other copper treatments. Cu+ may have resulted in an improved growth response owing to it being more readily metabolized than C12+. Delayed release of copper from the site of injection may have played an important role.
Article
The amount of radioactivity accumulated in normal skin fibro-blasts cultured in the presence of 64Cu increased during the first few hours of incubation, and reached a plateau after 18 hr. Fibroblasts from patients with Menkes syndrome continued to accumulate 64Cu and after 48 hr, the mutant cells contained over 3 times more radioactivity than the normal cells. Normal skin fibroblasts grown in the presence of 64Cu for 24 hr and chased for 6 and 24 hr with 64Cu-free medium released 78% and 91% of the radioactivity, respectively, whereas Menkes fibroblasts grown under similar conditions released only 22% and 51%, respectively. The amount of radioactivity incorporated by fibroblasts increased with increasing concentrations of 64Cu in the culture medium, but the Menkes fibroblasts incorporated more 64Cu than normal fibroblasts. A slight reduction in the incorporation and efflux of 64Cu was seen with several metabolic inhibitors, but there was no difference between normal and mutant cells. Treatment of labeled cells with trypsin did not affect the amount of 64Cu picked up by the cells. Most of the radioactive copper incorporated by the cells was not precipitated by trichloroacetic acid or phosphotungstic acid, although the percentage of precipitated 64Cu was consistently higher in normal than in Menkes fibroblasts. Most of the 64Cu was bound to a molecule with a molecular weight of about 10,000, whereas a small fraction, proportionally higher in normal cells, was bound to a large molecular weight component(s). The amount of 64Cu bound to the small molecular weight species was significantly greater in Menkes fibroblasts than in normal cells. Menkes fibroblasts were more sensitive to high nonphysiologic levels of nonradioactive copper than were normal cells. These findings demonstrate pronounced metabolic differences between normal and Menkes fibroblasts and indicate the need for further studies before proper treatment of this disease can be instituted.
Article
The dose response as well as kinetics of uptake and retention of copper and cadmium of normal and Menkes kinky hair disease (MKHD) cultured fibroblasts are described. In basal culture medium, intracellular copper concentration in MKHD fibroblasts was approximately 3 times that of control cultures. The intracellular copper concentration of MKHD cells was significantly higher than that of normal fibroblasts at medium copper concentrations below 20 microgram/ml. Death of MKHD cells occurred at medium copper concentrations between 15 and 20 microgram/ml with an intracellular copper level 3 times that at basal medium. Normal cells died at medium copper concentration above 30 microgram/ml with an intracellular copper concentration 19 times that at basal medium. These observations suggested the existence of a regulatory mechanism for maintenance and control of intracellular copper in normal fibroblasts which is effective at medium copper concentrations below 30 microgram/ml. This system is defective in MKHD fibroblasts. In basal medium MKHD and normal fibroblasts had similar intracellular cadmium concentrations; however, at higher medium cadmium concentrations MKHD cells had increased intracellular cadmium levels. The uptake of both 64Cu and 109Cd was significantly higher in MKHD cells than in normal cells, indicating that the uptake of 64Cu and 109Cd is not impaired in MKHD cells. A higher retention of 64Cu was observed in MKHD cells at both 37 degrees C and 4 degrees C. No obvious trend, however, was observed in the difference of retention of 109Cd between MKHD and normal cells. An impairment of egress of copper in MKHD cells is implicated by these results.
Article
The kinky hair syndrome (KHS) is an X-linked defect of copper transport in man. An animal model is available in mutants at the X-linked mottled locus in mice. The defect does not involve the uptake of copper from the intestinal lumen but rather the transport of copper from intestinal cells. The reduced activity of several copper-dependent enzymes and the lower copper content of serum, liver, and probably brain account for the manifestations of the disorder which are evident at, or shortly after, birth. Intrauterine involvement is likely but prenatal diagnosis is not yet possible. Although the delivery of iron to the erythropoietic system, and its utilization, are impaired in nutritionally induced copper deficiency, as is neutrophil production, these processes appear normal in KHS. thus, adequate copper to carry them out is available in KHS. While there may be more than one transport system for copper (only one of which is affected in KHS) it is also possible that the hematopoietic tissue in KHS, like the intestinal cells, has abnormally high afficity for copper. The presence of multiple alleles at the KHS locus (and at other genetic loci) in man, which cause different degrees of reduction in copper transport, could account for variations in the susceptibility to copper deficiency observed in infant populations.
Article
This article has no abstract; the first 100 words appear below. Copper, an extremely important trace element in animal and human metabolism,¹²³ is necessary for the normal function of many enzymes. The effects of nutritional deficiency have been studied in sheep, rats, mice, pigs, chickens and turkeys and include changes in integument, bone, arteries, pigmentation and brain.¹²³ Copper deficiency in human infants has been studied extensively in Peru⁴ and has recently been reported in a few babies fed on synthetic diets,⁵,⁶ or nourished intravenously.⁷ The disturbance of nutrition has always been complex, and assignment of particular effects to copper deficiency has depended upon serial replacement of other nutrients — a rather . . . David M. Danks, M.D. Royal Children's Hospital Research Foundation Parkville, 3052, Victoria, Australia
Article
Copper deficiency has been demonstrated in seven babies with Menkes' kinky-hair syndrome. Gross changes in free sulphydryl groups in hair keratin, consistent with copper deficiency, probably explain the kinky hair. Copper deficiency also explains the important changes in the elastic fibres in the arterial walls, the scorbutic bone changes, and the hypothermia observed in these babies. Evidence for a defect in the intestinal absorption of copper is given. Treatment of Menkes' syndrome may become possible as a result of these findings.
Article
Duodenal mucosa obtained from two patients with Menkes' syndrome contained abnormally large amounts of copper. The defect in copper absorption in this disease must lie in the process of intracellular handling or of transport across the serosal cell membrane. Fibroblastic cells cultured from the skin of patients and of heterozygous females show intense metachromasia in primary culture which disappears in subculture. These cells may be useful for the study of copper transport in vitro and for the identification of heterozygotes in affected families.
Article
THE results presented here show that a primary defect in copper transport underlies the mottled syndrome in the mouse. The X-linked mottled mutants thus offer an excellent system for the study of mammalian copper metabolism. They also provide an animal model of the inherited human copper deficiency, Menkes kinky hair disease1,2, which is also X-linked.
Article
An animal model for Menkes' kinky hair syndrome is provided by mice mutant at the X-linked mottled locus. Two mechanisms have been invoked to explain disease manifestations in mottled and in kinky hair syndrome: relative tissue copper deficiencies and corresponding reductions in cuproenzyme activities; or defective intracellular copper utilization, with impaired intracellular translocation to cuproenzymes or to copper-dependent processes. We addressed the second possibility through measurements of soluble superoxide dismutase (SOD-1) in cytosol extracts of confluent mottled (blotchy) cultured skin fibroblasts. At comparable intracellular copper concentrations over a broad range, SOD-1 specific activities in the mutant cells were not distinguishable from those in controls, or, in some instances, were actually higher. These data suggest that the excess copper anomalously sequestered in a cell expressing the mutation remains available for binding to a cytosolic cuproenzyme. When taken together with data in other systems, the results are consistent with the thesis that the basic lesion in blotchy may primarily affect copper transport or delivery to specific copper transport systems.
Article
We have assigned the structural gene (Mt-1) coding for the murine metal-binding protein metallothionein I (MT-1) to mouse chromosome 8 by using a cloned DNA probe for mouse Mt-1 in combination with a panel of Chinese hamstermouse somatic cell hybrid clones segregating mouse chromosomes. Analysis of hybrid cell extracts for the presence of mouse Mt-1 or MT-1 mRNA revealed concordant segregation of Mt-1 with mouse glutathione reductase, an enzyme marker for mouse chromosome 8, but discordant segregation with enzyme markers for 14 other mouse chromosomes. Karyotype analyses of seven informative hybrid clones confirmed the assignment of mouse Mt-1 to chromosome 8. Menkes' disease in man and the mottled mutation (Mo) in the mouse, which provides an animal model of Menkes' disease, are both X-linked degenerative neurologic disorders involving abnormal copper metabolism and increased levels of intracellular metallothionein protein. Fibroblasts from Mo male mice have increased amounts of MT-1 mRNA, suggesting that both Mo and Menkes' disease may be due to a metallothionein gene mutation. However, our assignment of Mt-1 to mouse chromosome 8, rather than the X chromosome, demonstrates that a mutation in mouse Mt-1 or a closely linked regulatory gene is not the primary defect in Mo, and implies that a metallothionein gene mutation is not the genetic defect in human Menkes' disease.
Article
Studies of uptake and release of 64Cu, 109Cd, and 65Zn in suckling C57BL/6J male mice revealed kinetics and distributions that differed for each metal both within and among the organs analyzed, suggesting distinct, albeit overlapping, mechanisms for transport and binding of each metal. In mutants, there were tissue-specific increases in copper-binding capacity. In hemizygotes (Moblo/y) accumulation of 64Cu was increased in kidney, lung, and duodenum. In heterozygotes (Moblo/+), 64Cu content was increased in kidney, with a smaller increase in lung, and no change in duodenal Cu. Decreased 64Cu accumulation was seen in liver in both hemi- and heterozygotes. In contrast, 64Zn and 109Cd accumulation in organs of heterozygote mice was not significantly distinguishable from normal. In skin and connective tissues there is excessive accumulation of 64Cu in Moblo/+ and Moblo/y, no abnormality in heterozygote 65Zn accumulation, but a clear decrease in heterozygote 109Cd content. In both mutant kidney and liver, there was an aberrant subcellular distribution of 64Cu, with the major fraction of sequestered 64Cu in the cytosol. Our studies establish that in spite of the ubiquity of metallothioneins and the structural similarities of those that have been characterized, there is specificity and functional heterogeneity in metal binding among tissues. The aggregate data suggest that there are unique regulatory mechanisms for the metabolism of copper and zinc, while there exists, in part, an inverse relationship between the binding of copper and cadmium. Our data further suggest that the blotchy mutation involves a specific cytosolic copper storage or transport protein also capable of binding cadmium.
Article
Proteins of approximately 10,000 daltons (presumably metallothionein) and greater than 75,000 daltons bound 64Cu when this metal was added to fibroblast lysates. Treatment with either 2-mercaptoethanol or the disodium salt of ethylenediamine tetraacetic acid demonstrated that the high molecular weight copper-binding proteins in lysates prepared from both normal and Menkes fibroblasts exhibited a relatively low affinity for copper compared to the 10,000 dalton protein(s). No difference was detected in the affinity of the low molecular weight protein(s) of normal and Menkes fibroblast lysates for copper. The amount of 64Cu bound to the 10,000 dalton protein(s), however, was approximately two to three times greater in lysates prepared from Menkes fibroblasts than from normal fibroblasts. Mixing experiments indicated that the increased binding of 64Cu to the 10,000 dalton protein(s) in lysates of Menkes fibroblasts did not result from the deficiency of a factor that effects the cleavage of copper from this protein(s), from the presence of a soluble inhibitor, or from the lack of an activator. In addition, the use of lysates, rather than whole cells, demonstrated that the observed differences in copper binding between the normal and the Menkes fibroblasts were not caused by an abnormality in the membrane transport of copper in the mutant cells. Thus the findings suggest that the increased accumulation and the reduced efflux of copper previously observed in cultured Menkes fibroblasts result either from an increased amount of the 10,000 dalton copper-binding protein(s) or from an increased capacity of this molecule(s) for copper.
Article
The metabolism of copper by cultured fibroblasts from brindled male (Mobr/Y) and normal male (+/Y) mice was studied using radioactive copper (64Cu). Brindled fibroblasts accumulated more 64Cu than normal fibroblasts. The accumulation of 64Cu by both brindled and normal fibroblasts was enhanced if the cells were preincubated in a copper-supplemented medium or if fetal calf serum was omitted from the medium. Brindled fibroblasts also retained more 64Cu on continued incubation. The greater accumulation might, therefore, represent an effect of reduced copper efflux. The demonstrated expression of the inherited defect in copper metabolism of brindled mice in cultured fibroblasts emphasizes the value of this model for further study of copper metabolism and its genetic control.
Article
A metallothionein-like protein (MTP) is synthesized in normal diploid human skin fibroblasts cultured in Zn- or Cu-supplemented medium. Synthesis of MTP is not detected in cells cultured without metal supplementation of complete tissue-culture medium. Cultured fibroblasts from patients with Menkes' disease accumulate excess Cu which chromatographs both with high-molecular-weight protein(s) and with a Cu-MTP. Under normal culture conditions, the Menkes' MTP incorporates [35S]-cystine, but not appreciable amounts of 65Zn. However, Menkes fibroblasts retain the ability to incorporate 65Zn into MTP in response to Zn supplementation of the medium. The results do not support the idea that Menkes' disease results from a failure of Cu to bind to MTP, but rather that an elevated intracellular Cu concentration in Menkes' disease fibroblasts leads to association of excess Cu with high-molecular-weight protein, stimulating synthesis of a Cu-binding MTP.
Article
Menkes fibroblasts contain a significantly greater amount of cysteine-rich 10,000 dalton copper-binding protein(s) (metallotheionein) than normal cells. Mutant fibroblasts incorporated 30 to 40% more tritiated amino acids into 10,000 dalton protein(s) than normal cells. The protein(s) was deficient in aromatic amino acids The amount of 35S-cysteine incorporated by the same protein(s) in Menkes fibroblasts was twice that of normal fibroblasts. Comparison of the 35 S:3H isotopic ratios of chromatographic fractions of both normal and Menkes cell lysates showed that only the proteins eluted in the 10,000 dalton peak were enriched in 35S-cysteine, and this ratio was always greater than in Menkes than in normal cells. The 10,000 molecular weight 35S-cysteine- and 3H-amino acid-labeled peaks coincided with the 64Cu peak in both cell strains. The copper-labeled peak was always greater in Menkes than in normal cells. No difference in the 64Cu:35S isotopic ratio in the 10,000 dalton peak was observed between normal and Menkes fibroblast strains. This finding shows the direct relationship between the amount of cysteine-rich 10,000 dalton protein(s) and the amount of 64Cu bound by this protein(s) in both Menkes and normal fibroblasts. DEAE-cellulose ion-exchange chromatography resulted in a further two-fold enrichment of the 10,000 dalton, sulfur-rich proteins that were eluted from the Sephadex G-75 column. Most of the labeled proteins from both normal and Menkes fibroblasts were eluted from the ion-exchange column in a single peak at a chloride concentration of approximately 30 mM. Polyacrylamide disc gel electrophoresis of pooled fractions of the 10,000 dalton proteins eluted from the G-75 column and the DEAE-cellulose ion-exchange column showed no consistent differences in the staining pattern between normal and mutant fibroblast strains. When th acrylamide gels were sliced and subsequently counted for radioactive content, no band showed a further increase in the 35 S:3H isotopic ratio when compared to the electrophoresed samples that were eluted from the Sephadex G-75 or the ion-exchange columns. Also, no significant increase in the amount of radioactivity associated with a specific protein band could be demonstrated between the Menkes and the normal fibroblast strains.
Article
The characteristics of hepatic copper accumulation and metabolism were studied using primary monolayer cultures of adult rat liver parenchymal cells. Accumulation of copper from serum-free medium was temperature dependent and strongly inhibited by cyanide and N-ethylmaleimide. Addition of various concentrations of zinc to the medium did not alter copper accumulation by the cells. Furthermore, it was found that supplementation of the cell cultures with dexamethasone significantly stimulated zinc accumulation without affecting the accumulation of copper. Cycloheximide substantially stimulated accumulation of copper from the culture medium, whereas actinomycin D had no effect. Effux experiments showed that copper is rapidly sequestered by intracellular components and becomes unavailable for exchange soon after it is transported into the cells. Gel chromatography of liver cytosol demonstrated that most of the copper that is initially accumulated is bound to the low molecular weight cytoplasmic protein metallothionein.
Article
Cultured cells of a variety of different types from human Menkes' syndrome patients and brindled mouse mutants exhibit similarly altered responses to changes in extracellular copper concentration. This suggests that the mutations in the mouse and human are very similar and that mutant gene expression is occurring in many different tissues. Intracellular copper levels are markedly elevated in mutant cells in normal medium and in medium containing a hundred-fold higher copper. Some cell lines from heterozygotes possess elevated copper levels. Elevated extracellular copper and zinc are significantly more toxic to mutant cells. Mutant cells exhibit normal rates of uptake of copper-64 over a 10-min period but abnormally high accumulation over 24 hr and low rates of efflux. Menkes' fibroblasts become saturated with copper-64 at lower extracellular concentrations than for normal fibroblasts. These data support the idea of enhanced intracellular binding in mutant cells.
Article
Menkes fibroblast cultures were established and copper metabolism was investigated. Menkes fibroblasts contained four to six times higher concentrations of copper than control cells. In Menkes cells more than 90% of the intracellular copper was present in cytosol (105,000 x g supernatant fraction); in control fibroblasts the corresponding value was about 67%. During cultivation of fibroblasts in medium supplemented with 100 ng/ml of copper (270 ng CuCl2·2H2O /ml), the amount of copper increased continuously in Menkes cells, at least up to 4 days, while in control cells in reached a maximum after 24 h, followed by a gradual decrease. When the medium was replaced with one without copper chloride, copper concentrations in Menkes cells returned to the original level in three days, whereas those in control cells returned to the normal level in one day. Using Sephadex G-75 column chromatography of cytosol, two copper-containing peaks were observed (peaks 1 and 3, corresponding to the peaks from rat liver cytosol). Approximately 75% of the copper in the cytosol from Menkes cells was eluted in peak 3. The corresponding copper peak was very small in control cells. Copper peaks 1 and 3 from both cells increased after treating cells with copper chloride and the increase was inhibited by cycloheximide, an inhibitor of protein biosynthesis. Metallothionein purified from human kidneys was eluted as a single copper-protein from a Sephadex G-75 column in the same fractions as peak 3. SDS polyacrylamide-slab gel electrophoresis of the purified metallo-thionein and the material eluted in peak 3 from Menkes fibroblasts showed single peaks for copper at identical migration distances.
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