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

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

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

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

Cytosolic copper-binding proteins in rat and mouse hepatocytes incubated continuously with Cu(II).

The proteins that bind copper when it first enters cells are likely to play roles in its intracellular distribution and utilization. When hepatocytes were incubated with 64Cu(II), the time-dependence of the subcellular distribution of 64Cu was consistent with one or more cytosolic proteins distributing copper to the mitochondrial and nuclear fractions. Cytosolic copper was reproducibly distributed among four protein fractions from Sephadex G-150 columns at the earliest time (1 min) and at the lowest concentration used [2 microM-64Cu(II)] with both rat and mouse hepatocytes. Copper binding to proteins in these functions was sensitive to copper metabolic status. Hepatocytes from nutritionally copper-deficient rats or neonatal (9-30 days old) developing rats showed an inverse correlation between copper binding to metallothionein and copper binding to proteins in fraction I (approximately 88 kDa apparent) and fraction II (approximately 38 kDa apparent). The distribution of cytosolic 64Cu from the brindled-mouse model of Menkes disease indicated decreased binding by a protein in fraction I. Brindled-mouse hepatocytes also contain decreased levels of a approximately 55 kDa protein or subunit, which most likely represents a liver-specific secondary response to the primary defect. The results are consistent with one or more copper-binding proteins in fractions I and II having significant functions in intracellular copper metabolism.     

Influence of zinc on copper binding in tissue proteins of steers

Two experiments were conducted with steers fed diets containing 270 ppm copper either with or without 2050 ppm zinc. Liver biopsies were taken from steers biweekly for 10 wk for analysis. The steers were then killed; tissues were removed, homogenized, and centrifuged, and the pellets were extracted with mercaptoethanol (BME), and selected cytosols and extracts were subjected to gel filtration (Sephadex G-75). Copper and zinc were determined on the BME extracts, pellets after extraction, cytosols, and gel-filtration fractions. Copper accumulated at about the same rate in BME extract and in the extracted pellet, with the smallest amount in the cytosol. In contrast, over 70% of the zinc was present in the hepatic cytosols. Gel filtration of BME extracts revealed the greatest amount of copper in a low-molwt (MW) peak in addition to three minor peaks of copper. Within the hepatic cytosols, the greatest amount of copper accumulated in proteins of MW>75,000, the next greatest amount in 30,000-MW proteins, and the least amount with metallothionein (MT) of steers fed the diet with only copper added. In contrast, the greatest amount of copper was present with MT in hepatic cytosols of the steer fed a diet that included copper plus zinc. Hence the zinc status of steers influences the deposition of copper in the cytosolic proteins (as demonstrated by liver, kidney, and pancreas), but not in the intracellular fractions.     

Correction of a mouse model of Menkes disease by the human Menkes gene

The brindled mouse is an accurate model of the fatal human X-linked copper deficiency disorder, Menkes disease. Males carrying the mutant allele of the Menkes gene orthologue Atp7a die in the second week of life. To determine whether the genetic defect in the brindled mice could be corrected by expression of the human Menkes gene, male transgenic mice expressing ATP7A from the chicken beta-actin composite promoter (CAG) were mated with female carriers of the brindled mutation (Atp7a(Mo-br)). Mutant males carrying the transgene survived and were fertile but the copper defect was not completely corrected. Unexpectedly males corrected with one transgenic line (T25#5) were mottled and resembled carrier females, this effect appeared to be caused by mosaic expression of the transgene. In contrast, males corrected with another line (T22#2) had agouti coats. Copper concentrations in tissues of the rescued mutants also resembled those of the heterozygous females, with high levels in kidney (84.6+/-4.9 microg/g in corrected males vs. 137.0+/-44.3 microg/g in heterozygotes) and small intestine (15.6+/-2.5 microg/g in corrected males vs. 15.7+/-2.8 microg/g in heterozygotes). The results show that the Menkes defect in mice is corrected by the human Menkes gene and that adequate correction is obtained even when the transgene expression does not match that of the endogenous gene.     

Ascorbic acid synthesis and concentrations in organs of copper-deficient and brindled mice

Copper deficiency was studied in mice to investigate an interaction between copper and ascorbic acid. Twelve-day-old mutant brindled mice that exhibited signs of copper deficiency were compared to their normal brothers as well as to age-matched suckling mice that were copper deficient (-Cu) because their dams were consuming a copper-deficient diet throughout gestation and lactation, and a fourth group of copper-supplemented ( + Cu) suckling mice that served as dietary controls. Dietary copper deficiency was also produced in older mice by beginning the treatment at birth and continuing for 7 wk. Organ ascorbate levels were determined by high performance liquid chromatography with electrochemical detection. Differences caused by diet and genetics were evident but age-dependent. Compared to controls, liver and kidney ascorbate levels did not change remarkably in young or old copper-deficient mice. Cardiac ascorbate levels were higher in 7-wk-old - Cu mice and lower in 12-d-old - Cu mice, despite hypertrophy in both cases. Spleen ascorbate levels were lower in older -Cu mice and higher in 12-d-old mice, but total spleen ascorbate reflected the hypertrophic and atrophic size in the older and younger -Cu mice, respectively. Brindled mutants had an extremely low level of ascorbate in spleen. Plasma ascorbate was lower in 7-wk-old - Cu mice. Reasons for the alterations in ascorbate levels are not known. Synthesis in liver from D-glucuronate was not altered by dietary copper deficiency in 7-wk-old mice. Synthesis was lower in livers from 12-d-old - Cu and brindled mice compared to control values. However, the difference correlated better with body weight of the mice rather than with degree of copper deficiency. Consequences of the altered organ levels of ascorbate in copper-deficient mice are not completely known.     

A comparison of phenotype and copper distribution in blotchy and brindled mutant mice and in nutritionally copper deficient controls

The murine mottled mutants brindled, Mo br, and blotchy, Mo blo, are valuable animal models for the study of mammalian copper metabolism. In this paper, we present data showing that a nutritionally copper deficient suckling mouse, Cu-, with strong phenotypic similarities to the brindled mutant can be produced by feeding genetically normal dams a copper deficient diet (0.1-0.4 ppm Cu2+) from the day of mating. Comparisons of copper distribution between the Cu- mice and brindled mutants indicate that when a small dose of copper (0.5-0.9 micrograms Cu2+) was administered by intracardiac injection, the copper was abnormally distributed, and that the pattern of tissue distribution was very similar in Cu- mice and brindled mutants 24 h after injection. When, however, a treatment dose (50 micrograms Cu2+) was injected subcutaneously, and tissues assayed 3 d after injection, copper distribution in Cu- mice and brindled mutants was clearly different. Copper deficiency in Cu- suckling mice is entirely derived from maternal effects. Evidence that maternal effects may also influence the survival and phenotype of the brindled and blotchy mutants was obtained by comparing the viability of mutants born to dams carrying mottled mutations on one or both X chromosomes.     

Equivalent affinity of aldosterone and corticosterone for type I receptors in kidney and hippocampus: direct binding studies

In studies from several laboratories evidence has been adduced that renal Type I (mineralocorticoid) receptors and hippocampal "corticosterone-preferring" high affinity glucocorticoid receptors have similar high affinity for both aldosterone and corticosterone. In all these studies the evidence for renal mineralocorticoid receptors is indirect, inasmuch as the high concentrations of transcortin (CBG) in renal cytosol make studies with [3H]corticosterone as a probe difficult to interpret, given its high affinity for CBG. We here report direct binding studies, with [3H]aldosterone and [3H]corticosterone as probes, on hippocampal and renal cytosols from adrenalectomized rats, in which tracer was excluded from Type II dexamethasone binding glucocorticoid receptors with excess RU26988, and from CBG by excess cortisol 17 beta acid. In addition, we have compared the binding of [3H]aldosterone and [3H]corticosterone in renal cytosols from 10-day old rats, in which CBG levels in plasma and kidney are extremely low. Under conditions where neither tracer binds to type II sites or CBG, they label an equal number of sites (kidney 30-50 fmol/mg protein, hippocampus approximately 200 fmol/mg protein) with equal, high affinity (Kd 4 degrees C 0.3-0.5 nM). Thus direct tracer binding studies support the identity of renal Type I mineralocorticoid receptors and hippocampal Type I (high affinity, corticosterone preferring) glucocorticoid receptors.     

Liberation and analysis of protein-bound arsenicals

Protein-bound arsenicals were liberated from binding sites on liver cytosolic proteins by exposure to 0.1M CuCl at pH 1. This method released greater than 90% of the arsenicals associated with biological matrices. Ultrafiltrates of CuCl-treated cytosols were subjected to thin-layer chromatography to speciate and quantify inorganic and methylated arsenicals. For rat liver cytosol in an in vitro methylation assay and for liver and kidney cytosols from arsenite-treated mice, most inorganic arsenic was protein bound. Appreciable fractions of the organoarsenical metabolites present in these cytosols were also protein bound. Therefore, CuCl treatment of cytosols releases protein-bound arsenicals, permitting more accurate estimates of the pattern and extent of arsenic methylation in vitro and in vivo.     

Reduction of copper and metallothionein in toxic milk mice by tetrathiomolybdate, but not deferiprone.

Copper is both essential for life and toxic. Aberrant regulation of copper at the level of intracellular transport has been associated with inherited diseases, including Wilson's disease (WND) in humans. WND results in accumulation of copper and the copper and zinc-binding protein metallothionein (MT) in liver and other tissues, liver degeneration, and neurological dysfunction. The toxic milk (TX) mutation in mice results in a phenotype that mimics human WND, and TX has been proposed to be a model of the disease. We characterized TX mice as a model of altered metal ion and MT levels during development, and after treatment with the metal ion chelators tetrathiomolybdate (TTM) and deferiprone (L1). We report that hepatic, renal and brain copper and MT are elevated in TX mice at 3 and 12 months of age. Zinc was significantly higher in TX mouse liver, but not brain and kidney, at both time points. Nodules appeared spontaneously in TX mouse livers at 8-12 months that maintained high copper levels, but with more normal morphology and decreased MT levels. Treatment of TX mice with TTM significantly reduced elevated hepatic copper and MT. Transient increases in blood and kidney copper accompanied TTM treatment and indicated that renal excretion was a significant route of removal. Treatment with L1, on the other hand, had no effect on liver or kidney copper and MT, but resulted in increased brain copper and MT levels. These data indicate that TTM, but not L1, may be useful in treating diseases of copper overload including WND.     

Influence of maternal mineral deficiency on the hepatic metallothionein and zinc in newborn rats

The effects of maternal Zn, Cu, or Fe deficiencies during late gestation on hepatic levels of metals and metallothionein (MT) and the binding of Zn and Cu to protein fractions were investigated in newborn rats. Timed pregnant rats were fed one of the following diets: Zn deficient (Zn-D), Cu-D, Fe-D, or control from day 12 of gestation until birth. The specific nutritional deficiency status of the dams was confirmed by low plasma levels of the deficient metal. Livers from pups were analyzed for MT, metal content, and metal-protein binding. Maternal Zn-D resulted in a greater than 50% reduction of hepatic MT levels in pups, whereas Cu-D and Fe-D had no significant effects. Pups in each deficient group showed a significant decrease in the hepatic levels of the respective metals. Fractionation of hepatic cytosols from the pups by Sephadex G-75 gel filtration showed that in both Fe-D and Cu-D pups the respective metals were depleted from the high molecular weight protein fraction, whereas in Zn-D pups the Zn was depleted mainly from the MT fraction (Ve/V0 approximately 2). Incorporation of [35S] cysteine into MT fractions was significantly lower in Zn-D pups as compared with control pups. These results indicate that there is a specific effect of the maternal Zn-D on the hepatic storage of Zn as MT in newborn rats.     

Primary biochemical defect in copper metabolism in mice with a recessive X-linked mutation analogous to Menkes' disease in man

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.     

Degradation of 35S-labeled metallothionein in the liver and the kidney of Brindled mice: Model for Menkes' disease

An amino acid analysis of the renal copper-binding protein of heterozygous Brindled mice indicated that the protein labeled with L-[³⁵S]cystine was metallothionein.The metabolism of ³⁵S-labeled hepatic and renal metallothionein of adult normal (Mo^+/+) and heterozygous (Mo^br/+) Brindled mice was investigated without prior induction with metals. After incorporation of L-[³⁵S] cysteine into hepatic and renal metallothionein, ³⁵S-labeled metallothionein is normally degraded with two half-lives (liver: 11.6 ± 1.3 hours and 3.1 ± 0.3 days; kidney: 8.22 ± 0.08 hours and 3.5 ± 1.2 days). However, ³⁵S-labeled renal metallothionein of the heterozygous Brindled mice is exclusively degraded with a half-life of 3.1 ± 0.2 days.The results imply that the mutation in Brindled mice causes an impaired renal reabsorption of copper (transport of copper from the tubular cells into the blood circulation).     

Analysis of renal and hippocampal type I and type II receptors by fast protein liquid chromatography

Type I and Type II adrenal steroid receptors from rat renal and hippocampal cytosols were studied by the technique of Fast Protein Liquid Chromatography. Type I receptors were labelled with [3H]aldosterone plus excess RU26988, and Type II receptors with [3H]dexamethasone. On a Mono Q anion exchange column the molybdate-stabilized renal and hippocampal Type I receptors both eluted as single symmetrical peaks at 0.27 M NaCl, with a recovery of approximately 90% and 60-fold purification (renal) and 10-15-fold (hippocampal). Molybdate-stabilized Type II binding sites from both hippocampal and renal cytosols co-eluted with the Type I sites. On Superose gel filtration renal Type I receptor-steroid complexes consistently eluted two fractions later than hippocampal Type I complexes, suggesting that the renal complexes are smaller; Type II receptor-steroid complexes from both cytosols co-eluted, consistently one fraction behind hippocampal Type I sites. Sequential gel filtration and anion exchange chromatography achieved a 1000-fold purification of renal Type I binding sites, with an overall recovery of 10%.     

The distribution of copper in neonatal mottled mutant mice after exposure to copper and penicillamine

Tissue copper levels of brindled ($\text{Mo}$^br) mice and normal litter-mates after single and repeated dosing with CuCl₂ and/or D-penicillamine are examined, together with a study of the cytosol distribution of copper after CuCl₂ treatment. The results confirm that the mutant mouse kidney is capable of extensive copper accumulation in association with the low MW copper-binding protein. Deficient tissues such as brain, heart and spleen are able to sequester sufficient of the exogenous copper to raise their levels to the normal control level, whereas mutant liver levels, even after copper treatment, remain below normal, indicating that the $\text{Mo}$ gene affects the ability of the liver to retain copper.     

Genetic diseases of copper metabolism

There are several known examples of mutations which influence copper homeostasis in humans and animals. Pleiotropic effects are observed when the mutant gene disturbs copper flux. In some cases, the mutation alters the level of a specific copper ligand (enzyme) and the clinical consequences are unique. The two most widely studied genetic maladies in humans are Menkes' and Wilson's diseases. Menkes' disease is an X-linked fatal disorder in which copper accumulates in some organs (intestine and kidney) and is low in others (liver and brain). Wilson's disease is an autosomal recessive disorder in which copper accumulates, if untreated, in liver and subsequently in brain and kidney. Pathophysiological consequences of copper deficiency and toxicity characterize these two disorders. Specific mutations of human cuproenzymes include overproduction of copper-zinc superoxide dismutase in Down's syndrome, absence of tyrosinase in albinism, hereditary mitochondrial myopathy due to reduction in cytochrome c oxidase, and altered lysyl oxidase in X-linked forms of cutis laxa and Ehlers-Danlos syndrome. Mutations altering copper metabolism are also known in animals. Several murine mutants have been studied. The most extensively investigated mutants are the mottled mice, in particular brindled mice, which have a mutation analogous to that of Menkes' disease. Another recently described murine mutation is toxic milk (tx) an autosomal recessive disorder that is characterized by copper accumulation in liver. Two other mutants, crinkled and quaking, were once thought to exhibit abnormal copper metabolism. Recent data has not confirmed this. A mutation in Bedlington terriers has been described which is very similar to Wilson's disease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deficiencies in sex-regulated expression and levels of two hepatic sterol carrier proteins in a murine model of Niemann-Pick type C disease.

Hepatic sterol carrier protein-2 (SCP2) and sterol carrier protein-X (SCPx) levels in normal and in mutant Niemann-Pick Type C mice were determined by immunoblotting with antiserum against rat SCP2. A 14-kDa protein (SCP2) was detected in the cytosol fraction and a 58-kDa protein (SCPx) was found in both cytosolic and organellar fractions. Expression of hepatic SCPx protein was developmentally regulated in a sex-specific pattern. The amounts of organelle-associated SCPx increased 4-fold during sexual development of normal males but decreased dramatically during development of normal females. Levels of hepatic SCP2 increased much less dramatically during sexual maturation of normal males and females. Adult Niemann-Pick Type C mice were deficient in both hepatic SCPx and SCP2. The deficit in SCPx in affected males reflected a failure to increase hepatic SCPx levels during sexual maturation. In affected males SCPx remained at levels found in immature mice. Affected male and female mice were also unable to maintain levels of hepatic SCP2. The level of SCP2 was near normal in affected immature males and subnormal in affected immature females. During sexual maturation hepatic SCP2 declined in affected animals.     

Metallothionein and the development of the mottled disorder in the mouse

Copper accumulates in kidney tissue of mottled (Mo) mice largely in association with a low MW cytosol protein, and the reduced copper levels in neonatal mutant liver are largely the result of a reduction in the amount of copper associated with this same protein. On the basis of ion-exchange chromatographic profile, heat stability, absence of a 280nm absorption peak, and the binding of Cd109 and Zn65 the protein mutants in the kidney is identified as metallothionein (MT). Amino acid analysis, however, failed to confirm this, and it is suggested that the high copper content of the mutant protein results in its oxidative degradation during purification, even when normal anaerobic precautions are taken. Estimates of thionein protein content of tissues from mutant and normal mice demonstrated that the levels are significantly elevated in both young and adult mutant kidney and depressed in young mutant liver, in parallel therefore with the changes in tissue copper levels. In adult mutant liver tissue, however, thionein levels are significantly raised, even though tissue copper content is normal. The synthesis and degradation of MT was examined in some detail. Incorporation of S35-cysteine in kidney MT was significantly raised in both young and adult mutant mice, while in adult tissue the rate of degradation of MT was significantly depressed. The elevated kidney MT levels arise therefore in young mutant mice from an increased rate of synthesis and in adult mice from the combined effects of increased synthesis and reduced degradation.     

Decreased hepatic triglyceride accumulation and altered fatty acid uptake in mice with deletion of the liver fatty acid-binding protein gene

Liver fatty acid-binding protein (L-Fabp) is an abundant cytosolic lipid-binding protein with broad substrate specificity, expressed in mammalian enterocytes and hepatocytes. We have generated mice with a targeted deletion of the endogenous L-Fabp gene and have characterized their response to alterations in hepatic fatty acid flux following prolonged fasting. Chow-fed L-Fabp-/- mice were indistinguishable from wild-type littermates with regard to growth, serum and tissue lipid profiles, and fatty acid distribution within hepatic complex lipid species. In response to 48-h fasting, however, wild-type mice demonstrated a approximately 10-fold increase in hepatic triglyceride content while L-Fabp-/- mice demonstrated only a 2-fold increase. Hepatic VLDL secretion was decreased in L-Fabp-/- mice suggesting that the decreased accumulation of hepatic triglyceride was not the result of increased secretion. Fatty acid oxidation, as inferred from serum beta-hydroxybutyrate levels, was increased in response to fasting, although the increase in L-Fabp-/- mice was significantly reduced in comparison to wild-type controls, despite comparable induction of PPAR alpha target genes. Studies in primary hepatocytes revealed indistinguishable initial rates of oleate uptake, but longer intervals revealed reduced rates of uptake in fasted L-Fabp-/- mice. Oleate incorporation into cellular triglyceride and diacylglycerol was reduced in L-Fabp-/- mice although incorporation into phospholipid and cholesterol ester was no different than wild-type controls. These data point to an inducible defect in fatty acid utilization in fasted L-Fabp-/- mice that involves targeting of substrate for use in triglyceride metabolism.     

Ah receptor in primate liver: binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin and carcinogenic aromatic hydrocarbons

Ah receptor in hepatic cytosols from adult cynomolgus monkeys (Macaca fasicularis) was identified and quantitated by its binding of the highly toxic chemical 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the carcinogens 3-methylcholanthrene, benzo[a]pyrene, and dibenz[a,h]anthracene. The concentration of Ah receptor in cynomolgus hepatic cytosols (approximately 10 fmol/mg cytosol protein) was about one-quarter of that typically detected in rodent hepatic cytosols. Receptor concentrations were equal in male and female cynomolgus. [3H]TCDD bound to cytosolic receptor with high affinity (Kd approximately 3 nM). In rodents, Ah receptor is known to play a central role in toxicity caused by halogenated aromatic compounds and in carcinogenesis caused by polycyclic aromatic hydrocarbons. Existence of Ah receptor in monkeys indicates that the receptor also may mediate such responses in primates.