Comparisons of copper deficiency states in the murine mutants blotchy and brindled. Changes in copper-dependent enzyme activity in 13-day-old mice.

The activity of two copper-dependent enzymes, cytochrome c oxidase and copper, zinc-superoxide dismutase, was determined in six tissues of age-matched (13-day-old) copper-deficient mutant and normal mice. In the two mutants 'brindled' and 'blotchy', brain, heart and skeletal muscle had significant enzyme deficiencies. Cytochrome c oxidase was more severely affected than was superoxide dismutase. In these three tissues the degree of deficiency could be correlated with decreased copper concentration; however, enzyme activity was normal in liver, kidney and lung, despite abnormal copper concentrations in these tissues. In nutritionally copper-deficient mice, all six tissues showed decreased enzyme activity, which was most marked in brain, heart and skeletal muscle, the tissues which showed enzyme deficiencies in the mutants. Analysis in vitro of cytochrome c oxidase (temperature coefficient = 2) at a single temperature was found to underestimate the deficiency of this enzyme in hypothermic copper-deficient animals. Cytochrome c oxidase deficiency may therefore be sufficiently severe in vivo to account for the clinical manifestations of copper deficiency. An injection of copper (50 micrograms of Cu+) at 7 days increased cytochrome c oxidase activity by 13 days in all deficient tissues of brindled mice, and in brain and heart from blotchy mice. However, skeletal-muscle cytochrome c oxidase in blotchy mutants did not respond to copper injection. Cytochrome c oxidase activity increased to normal in all tissues of nutritionally copper-deficient mice after copper injection, except in the liver. Hepatic enzyme activity remained severely deficient despite a liver copper concentration three times that found in copper-replete controls. Superoxide dismutase activity did not increase with treatment in either mutant, but its activity was higher than control levels in nutritionally deficient mice after injection. This difference is probably due to sequestration of copper in mutant tissue such as kidney, but a defect in the copper transport pathway to superoxide dismutase cannot be excluded.     

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.     

Copper metabolism in mottled mouse (Mus musculus) mutants. Studies of blotchy (Moblo) mice and a comparison with brindled (Mobr) mice.

1. Copper concentrations were low in many organs of Moblo/Y mice, but very high in the gut. Absorption of 64Cu was seen to be very low when related to the absorption of cyano[57Co]cobalamin. The results in Moblo/+ mice were intermediate. 2. Copper therapy temporarily ameliorated many effects of the mutation in Moblo/Y mice, but did not improve the rate of weight gain as has been achieved previously in Mobr/Y mice. Lower capacity for a 'depot dose' effect at the site of injection may explain the difference. 3. The distribution of 64Cu after administration into the bloodstream of Moblo/Y mice altered from an initially normal state to one that resembled the abnormal distribution of pre-existing copper by 48 h. This indicated that the later mechanisms of copper distribution were at fault. Moblo/+ mice were equally affected. 4. The alteration of copper homoeostasis in blotchy mice was similar to that observed in brindled mice previously and in the present studies, although generally less severe. This is consistent with allelism of the two mutations.     

Copper metabolism in mottled mouse mutants: distribution of 64Cu in brindled (Mobr) mice.

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.     

Copper deficient rats and mice both develop anemia but only rats have lower plasma and brain iron levels

Iron homeostasis depends on adequate dietary copper but the mechanisms are unknown. Mice (Mus musculus) and rat (Rattus norvegicus) offspring were compared to determine the effect of dietary copper deficiency (Cu-) on iron status of plasma, liver, brain and intestine. Holtzman rat and Hsd:ICR (CD-1) outbred albino mouse dams were fed a Cu- diet and drank deionized water or Cu supplemented water. Offspring were sampled at time points between postnatal ages 13 and 32. Cu- rat and mouse pups were both anemic, but only rat pups had lower plasma and brain iron levels. Plasma iron was lower throughout the suckling period in Cu- rats but not Cu- mice. Cu- mice derived from dams restricted of Cu only during lactation were also severely anemic without hypoferremia. Intestinal metal analysis confirmed that Cu- pups had major reductions in intestinal concentration of Cu, increased Fe, and normal Zn. However, whole mouse (less the intestine) analysis demonstrated normal content of Fe indicating that the limitation in iron transport by intestinal hephaestin had no consequence to total iron reserves of the mouse. Further research will be needed to determine the reason Cu- mice were anemic since the "ferroxidase" hypothesis does not explain this phenotype.     

Biochemical Study on the Critical Period for Treatment of the Mottled Brindled Mouse

Hemizygous mottled brindled mice (Mobr/y mice) were treated by subcutaneous injection of copper and were decapitated on postnatal day 14. Cytochrome c oxidase (COX) activity of the brain mitochondria in the mice given 10 micrograms of copper/g on day 4 or 7 showed significant increases compared with that of untreated Mobr/y animals, and these mice had no neurological symptoms. Mice given 10 micrograms of copper/g on day 12 showed neither increases in COX activity nor clinical improvement. The brain levels of copper, noradrenaline, and dopamine in the mice treated on day 12 were the same as those in animals treated on day 4 or 7. The in vitro activities of dopamine-beta-hydroxylase of the brain were also the same among the treated mice, irrespective of the date of treatment. The results indicate that delays in copper treatment produce irreversible changes in COX activity of the brain and lead to clinical unresponsiveness to treatment.     

Rodent brain and heart catecholamine levels are altered by different models of copper deficiency

Limiting dopamine beta-monooxygenase results in lower norepinephrine (NE) and higher dopamine (DA) concentrations in copper-deficient Cu- tissues compared to copper-adequate Cu+ tissues. Mice and rat offspring were compared to determine the effect of differences in dietary copper Cu deficiency started during gestation or lactation on catecholamine, NE and DA, content in brain and heart. Holtzman rat and Hsd:ICR (CD-1) outbred albino mouse dams were fed a Cu- diet and drank deionized water or Cu supplemented water. Offspring were sampled at time points between postnatal ages 12 and 27. For both rat and mouse Cu- tissue, NE and DA changes were greater at later ages. Though Cu restriction began earlier in rats than mice in the gestational model, brain NE reduction was more severe in Cu- mice than Cu- rats. Cardiac NE reduction was similar in Cu- rodents in the gestation models. In the lactation model, mouse catecholamines were altered more than rat catecholamines. Furthermore, following lactational Cu deficiency Cu- mice were anemic and exhibited cardiac hypertrophy, Cu- rats displayed neither phenotype. Within a species, changes were more severe and proportional to the length of Cu deprivation. Lactational Cu deficiency in mice had greater consequences than in rats.     

Effects of copper deficiency on mouse yolk sac vasculature and expression of angiogenic mediators

BACKGROUND: Cu deficiency results in embryonic defects and yolk sac (YS) vasculature abnormalities. In diverse model systems, Cu treatment modulates angiogenesis, perhaps by influencing the activity of angiogenic mediators such as vascular endothelial growth factor (VEGF). Conversely, Cu chelators can suppress angiogenesis. METHODS: Gestation day (GD) 8.5 embryos from mice fed Cu-adequate (Cu+) or Cu-deficient (Cu-) diets were cultured in Cu+ or Cu- medium for 48 hr. Growth and development were evaluated, and YS vessel diameters were measured. Using RT-PCR and immunohistochemistry, the mRNA and protein expressions of VEGF, Flt-1, Flk-1, Angiopoietin-1 (Ang-1), and Tie-2 were analyzed. RESULTS: Cu+/Cu+ embryos developed normally, whereas Cu-/Cu- embryos showed a high incidence of developmental anomalies. Cu-/Cu- YS had a high proportion of vessels that were large in diameter compared to the Cu+/Cu+ YS. The mRNA expression of angiogenic mediators in Cu-/Cu- YS was similar to that in Cu+/Cu+ YS. The protein expression of VEGF in the Cu-/Cu- YS without any vessel defects, and Tie-2 in the Cu-/Cu- YS with both vessel defects and blood islands was significantly lower than that in the Cu+/Cu+ YS. The protein expression of Flt-1, Flk-1 and Ang-1 was similar among groups regardless of the presence, or type, of vessel defects. CONCLUSIONS: Results from the current study support the concept that Cu is required for the normal development of YS vasculature. Our data suggest that the impaired vascularization of Cu-deficient YS cannot be explained fully by the altered protein expression of the angiogenic growth factors reported here.     

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.     

Metabolic consequences of the cytochrome c oxidase deficiency in brain of copper-deficient Mo(vbr) mice

Biochemical micromethods were used for the investigation of changes in mitochondrial oxidative phosphorylation associated with cytochrome c oxidase deficiency in brain cortex from Mo(vbr) (mottled viable brindled) mice, an animal model of Menkes' copper deficiency syndrome. Enzymatic analysis of cortex homogenates from Mo(vbr) mice showed an approximately twofold decrease in cytochrome c oxidase and a 1.4-fold decrease in NADH:cytochrome c reductase activities as compared with controls. Assessment of mitochondrial respiratory function was performed using digitonin-treated homogenates of the cortex, which exhibited the main characteristics of isolated brain mitochondria. Despite the substantial changes in respiratory chain enzyme activities, no significant differences were found in maximal pyruvate or succinate oxidation rates of brain cortex homogenates from Mo(vbr) and control mice. Inhibitor titrations were used to determine flux control coefficients of NADH:CoQ oxidoreductase and cytochrome c oxidase on the rate of mitochondrial respiration. Application of amobarbital to titrate the activity of NADH:CoQ oxidoreductase showed very similar flux control coefficients for control and mutant animals. Alternately, titration of respiration with azide revealed for Mo(vbr) mice significantly sharper inhibition curves than for controls, indicating a more than twofold elevated flux control coefficient of cytochrome c oxidase. Owing to the reserve capacity of respiratory chain enzymes, the reported changes in activities do not seem to affect whole-brain high-energy phosphates, as observed in a previous study using 31P NMR.     

Plasma peptidylglycine alpha-amidating monooxygenase (PAM) and ceruloplasmin are affected by age and copper status in rats and mice

In an attempt to identify a sensitive and improved marker of mammalian copper status during neonatal development experiments compared two plasma cuproenzymes, peptidylglycine alpha-amidating monooxygenase (PAM ), an enzyme involved in peptide posttranslational activation, to ceruloplasmin (Cp), a ferroxidase involved in iron mobilization. Dietary Cu deficiency (Cu-) was studied in dams and offspring at postnatal age 3 (P3), P12, and P28. Rodent Cp activity rose during lactation whereas PAM activity fell. Reduction in Cp activity was more severe than reduction in PAM activity in Cu- offspring and dams. Cp activity was greater in rats than mice whereas PAM activity was similar in adults but greater in mouse than rat pups. Both cuproenzymes changed during neonatal development and when dietary copper was limiting. With proper controls, each enzyme can be used to assess copper status.     

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.     

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)     

Intracellular distribution of hepatic copper in macular mutant mice

To evaluate the role of lysosomes in copper-mediated hepatocellular injury, copper was administered, sc, to both normal and macular mutant mice at doses of 4.5, 9.0, and 18 mg Cu/kg, and the subcellular distribution of copper has been investigated in the liver of normal and mutant mice 24h after injection. The amount of copper in all fractions of copper-treated mutant mice was markedly lower than those in copper-treated normal mice, with the exception of microsomal fraction. However, there were no distinct differences in the proportion of copper in subcellular fractions between normal and mutant mice.     

生物耐铜的分子机理及铜污染环境的生物联合修复

铜是动植物和人类必需的微量元素,缺乏或过多都将产生不良影响。随着社会经济的发展,人类活动对环境的干扰日益加剧,工业和农业生产活动常可导致土壤铜污染,铜已成为土壤重金属污染的主要元素之一。总结了铜在植物体内的自发内稳态调节机制,在细菌和真菌体内的吸收、分布、解毒和调节因子,同时以蚯蚓为例简要阐述了土壤动物对铜的解毒机理;从分子生物学角度对重金属铜在生物体内的代谢机理及生物对环境中过量铜的联合修复研究进展进行了综述,以期为铜污染环境的植物、微生物和动物联合修复的分子机理研究提供借鉴。     

Suboptimal levels of dietary copper vary immunoresponsiveness in rats

The effects of severe, moderate, and mild copper deficiencies on cellular and humoral immunity were studied. Fifty male Sprague-Dawley rats, 5 wk of age, were fed diets containing 0.5, 2.0, 3.5, or 5.0 micrograms Cu/g for either 4 or 8 wk. Ten of the rats were fed the control diet, but were pair-fed with the 0.5-micrograms/g treatment group. All rats were immunized once with sheep red blood cells. Mean plasma-copper concentration reflected the dietary levels of copper, and ceruloplasmin activity correlated highly to plasma copper. Rats consuming suboptimal levels of copper responded differently to the deficiencies, so copper status varied among those animals. After 8 wk, cell proliferation, when stimulated by phytohemagglutinin, was dependent on the copper status of the animal. Severely deficient rats had consistently lower lymphocyte stimulation indexes for phytohemagglutinin and concanavalin A, but specific antibody response was not reduced. Immunoglobulin G (IgG) concentrations were variable for all rats, and immunoglobulin M (IgM) concentrations were lower for the severely deficient rats. Suboptimal dietary copper may alter immune function in rats, depending on the ensuing effect on copper status.     

Effect of copper on antioxidant ability and nutrient metabolism in broiler chickens stimulated by lipopolysaccharides

The aim of the experiment was to investigate the interaction between Cu intake, inflammatory challenge and oxidative stress in broiler chickens. Furthermore, it was tested whether plasma ceruloplasmin (Cp) could be a sensible parameter for dietary Cu. One hundred forty-four day old chickens were raised on a basal control diet without added copper (Group Cu-6.5, basal Cu content 6.5 mg/kg diet) or a diet supplemented with Cu at 8 or 50 mg/kg (Groups Cu-14.5 and Cu-56.5, respectively) with four replicates of 12 animals for each treatment. Starting on day 21, chickens were injected intraperitoneally with lipopolysaccharides (LPS) once a day for 3 days. Before this challenge, Group Cu-14.5 had the lowest gain and the feed to gain ratio was the highest. After injection of LPS, however, chickens of Group Cu-14.5 had the best zoo-technical performance. For chickens of Group Cu-6.5, LPS injection resulted in elevated rectal temperature, and lower erythrocyte superoxide dismutase (CuZn-SOD) activity, compared with the other groups. LPS injection increased plasma uric acid in Group Cu-6.5 significantly, but was without influence in Group Cu-56.5. At all Cu-levels, LPS injection increased erythrocyte CuZn-SOD activity and decreased thiobarbituric acid reacting substances. No significant difference in plasma Cp was found in chickens fed different dietary Cu. LPS injection significantly increased plasma Cp in Group Cu-56.5. The results suggest that varying dietary Cu levels seem to modulate the parameters involved in immunological responses and oxidant stress and that plasma Cp is not a reliable parameter for dietary Cu.     

Biological activities of partially purified elastase produced by Flavobacterium meningosepticum

Injection of viable cells of Flavobacterium meningosepticum at doses of 10(6) colony-forming units/eye caused ophthalmia in rabbits' corneas. Elastase, free of protease activity, was partially purified from broth cultures of F. meningosepticum ATCC12535 and TMS516. The optimum pH of the elastase activity was about 8.0. The activity was suppressed by Ni, Zn, Co, EDTA, and o-phenanthroline, and accelerated by Cu and Fe. The minimum dose of the elastase causing ophthalmia in the eyes of rabbits was 5 micrograms/eye (0.065 elastase units). The minimum lethal dose of the elastase for suckling mice by intracerebral injection was 0.4 micrograms (0.0052 elastase units)/suckling mouse, and its minimum lethal dose for adult mice by intraperitoneal injection was 740 micrograms/mouse (9.52 elastase units).     

Altered copper metabolism in cultured cells from human Menkes' syndrome and mottled mouse mutants

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 hundredfold 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.This work was supported by grants from the Australian National Health and Medical Research Council, the McPherson/Shutt Trust, and the Apex Foundation.