Mineral metabolism in the developing turkey embryo--II. The role of the yolk sac.

1. Turkey embryos were incubated in ovo or in long-term shell-less culture (ex ovo) for 14, 18, 22 or 26 days, at which time the concentrations of zinc, copper, iron, manganese and calcium in yolk and yolk sac membrane were determined. 2. Yolk manganese and calcium concentrations increased during incubation in ovo while the concentrations of zinc, copper and iron declined. The concentrations of zinc, copper and iron in yolk from ex ovo embryos did not decline. Yolk calcium concentration increased during incubation ex ovo, although to a much lesser extent than that observed in ovo. 3. The concentration of zinc, copper and iron declined in yolk sac tissue during incubation in ovo whereas no decline was observed for yolk sac tissue from ex ovo embryos. Yolk sac calcium and manganese concentrations increased during incubation in ovo and ex ovo, although the increase in calcium concentration for ex ovo yolk sac was much smaller than that observed in ovo. 4. A peak corresponding to metallothionein (MT) which bound both zinc and copper was isolated from yolk sac cytosol on day 14 of incubation in ovo using gel-permeation column chromatography. 5. Further fractionation of the MT peak by anion exchange chromatography revealed three metal-binding peaks designated MT-1, MT-2a and MT-2b. The majority of the zinc was bound to MT-2a and MT-2b whereas most of the copper was bound to a single peak (MT-2b). 6. The concentrations of zinc and copper in yolk sac cytosol reached a maximum on day 14 of incubation in ovo and declined through to day 28 (hatching).(ABSTRACT TRUNCATED AT 250 WORDS)     

The movement of zinc and copper from the fertilized egg into metallothionein-like proteins in developing chick hepatic tissue

The movement of copper and zinc from the fertilized egg into the developing embryo and the subsequent association of the two metals with metallothionein-like proteins (MT) of chick liver was investigated. Hepatic levels of MT in 1-d-old chicks, estimated from specifically bound^115mCd, were increased eightfold over endogenous levels of these copper- and zinc-binding proteins in adult liver. Copper was assimilated from the oocyte into the embryo more rapidly than zinc in the early stages of embryogenesis. Between 15 and 16 d of development, during the time that (MT) was detected, there was a significant increase in copper concentration, whereas hepatic zinc concentration remained relatively constant.     

Metabolism of copper and zinc in the liver and bone of the perinatal guinea-pig

Hepatic copper concentration in the guinea-pig increased markedly during the second-half of gestation, attaining a maximum shortly after birth; thereafter, concentration declined rapidly during the neonatal period. Changes in perinatal hepatic copper concentrations paralleled the binding of copper to a cytosolic metallothionein-like component, and the loss of hepatic copper in the neonates coincided with increases in serum copper concentrations. Zinc concentrations of the perinatal liver were low and showed no dramatic developmental changes. The humerus showed striking increases in zinc concentration with gestational age, attaining peak concentration before term and a marked depletion of tissue zinc during the neonatal period.     

Zinc, copper, and iron metabolism during porcine fetal development

Zinc, copper, and iron levels in maternal and fetal pig tissues and fluids were measured starting on d 30 of gestation and continuing to term (d 114) at 10-d intervals. Fetal hematocrit increased from a low of 19% on d 30 to 32% by d 50, after which it remained above 30% to term. Amniotic fluid zinc, copper, and iron all reached maximal levels by d 60 of gestation. Maternal serum zinc levels fluctuated little during gestation, but fetal serum zinc concentration was significantly elevated above maternal levels during the second trimester. Fetal serum copper levels were significantly lower than maternal values throughout gestation and this was also the case for ceruloplasmin oxidase activity. Maternal serum iron reached its lowest level by d 80 of gestation when rate of transfer of iron to the developing fetuses was high. Fetal serum iron declined throughout gestation, reaching its lowest level on d 100. In general, fetal liver concentrations of zinc, copper, and iron were higher than the corresponding maternal values throughout gestation. Distinct increases were noted for fetal hepatic zinc and copper concentrations during the second trimester of pregnancy and these were accompanied by increases in cytosolic and metallothionein-bound zinc and copper levels. Maternal hepatic iron declined during the second trimester, reaching its lowest point on d 80, indicative of the shunting of maternal iron reserves to fetal tissues. Fetal kidney metal levels did not demonstrate any distinctive developmental patterns with respect to zinc, copper, or iron concentrations, but a general accumulation of each metal was observed as gestation progressed. The results of this study highlight some of the distinct changes occurring in the metabolism of zinc, copper, and iron in both maternal and fetal tissues and fluids during gestation in the pig. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other suitable products.     

Influence of egg production on zinc, copper and iron metabolism in the turkey hen (Meleagris gallopavo)

1. Serum concentrations of zinc, copper, iron, calcium and protein increased significantly with the onset of egg production in turkey hens. 2. Hepatic concentrations of zinc, copper and iron declined significantly with the onset of egg production, whereas the total hepatic content of zinc and copper increased significantly. 3. When plasma from laying and nonlaying hens was subjected to gel-permeation column chromatography, a high molecular weight protein fraction was detected in laying but not in nonlaying plasma which bound significant amounts of zinc, copper and iron and which coeluted with purified vitellogenin. 4. Vitellogenin purified from the plasma of laying hens is a metalloprotein which upon analysis was found to contain 0.54, 0.09 and 0.72 micrograms of zinc, copper and iron, respectively, per mg of protein.     

Developmental changes in hepatic metallothionein, zinc, and copper levels in genetically altered mice.

Using mice that either overexpress metallothionein 1 (MT-1*) or do not express metallothionein 1 and 2 (MT-null) and a control strain (C57BL/6), the essential metal storage function of hepatic metallothionein and its subcellular localization were investigated during development. Hepatic metallothionein, zinc, and copper levels were measured in all groups from gestational day 20 to 60 days of age. Hepatic metallothionein levels were maximal during the perinatal period in both MT-1* and C57BL/6 mice with levels approximately three times higher in MT-1* mice. MT-null mice had no detectable hepatic metallothionein throughout development. Hepatic zinc levels were highest in the neonatal period of MT-1* and C57BL/6 mice and declined to adult levels by 30 days of age, while hepatic zinc levels in MT-null mice did not vary markedly throughout development. Hepatic copper profiles were very similar in MT-1* and MT-null mice as compared with the C57BL/6 mice. Correlation analysis showed a strong positive correlation between hepatic metallothionein and zinc levels in MT-1* mice, moderate correlation between hepatic metallothionein and metals in C57BL/6 mice, but only a very weak correlation between hepatic metallothionein and copper levels in MT-1* mice. Immunohistochemical localization showed specific nuclear staining in both MT-1* and C57BL/6 mice during the neonatal period with a gradual shift to the cytoplasm. The results show that hepatic metallothionein is a major determinant of zinc but not copper levels during murine development. Additionally, hepatic metallothionein levels and localization are regulated in a similar manner in MT-1* and C57BL/6 mice. The MT-null mice maintain a basel level of zinc sufficient for development, which was found to be 15.9 micrograms/g. This value was similar to the levels of hepatic zinc that was not bound to metallothionein in MT-1* and C57BL/6 mice during development.     

Ontogenically regulated expression of metallothionein and its messenger RNA in chick liver

By electrophoretic and immunological assay the concentration of hepatic metallothionein in new born chick liver was found to be ontogenically modulated, reaching a peak accumulation per gram liver in fourth day of hatching and declining below the detection limit after second week postnatal. The protein was undetectable upto second week of incubation in egg-embryonic stage. The concentration of metallothionein mRNA shows drastic change during first few days after hatching. The greatest accumulation of metallothionein mRNA was detected in the one day new born chicks, which declined rapidly there after, and reduced to a barely detectable level. Metallothionein was also detected in the in vitro translated product of one day neonatal chick hepatic poly(A+) RNA by S-cysteine labelling and immunoprecipitation. The naturally occurring new-born chick liver metallothionein was found to be a zinc-metallothionein and the concentration of hepatic zinc in new-born chick was found to undergo drastic modulation during development, unlike some other chick tissues. Endogenous zinc ion mobilization can thus play a significant role in the developmental regulation of chick metallothionein expression.     

Variation in the amounts of hepatic copper, zinc and metallothionein mRNA during development in the rat.

Amounts of hepatic metallothionein mRNA were assessed in RNA from foetal and neonatal rat livers by using dot-blot hybridization. Metallothionein mRNA began to increase about day 15 of gestation and reached a foetal maximum of 5-fold higher than adult values between 18 and 21 days of gestation. The amounts fell significantly for the first 3 days after parturition, and rose again to 6-fold above adult values 6 days after birth. By 15 days after birth the metallothionein mRNA had declined to adult amounts. In comparison, amounts of ornithine transcarbamoylase mRNA did not vary greatly during development. Hepatic zinc concentrations increased from day 14 of gestation to a maximum just before birth, and remained above adult values until 30 days after birth. From 14 days of gestation to 8 days after birth, hepatic copper concentrations were about 4-fold higher than in the adult, but a substantial increase (to about 9-fold higher than in the adult) occurs between 10 and 15 days after birth. CdCl2 administered to pregnant rats on day 18 of gestation was shown to block placental transfer of zinc, and we found decreased foetal hepatic zinc concentration after the CdCl2 treatment, but this failed to cause a significant decrease in metallothionein mRNA, suggesting that zinc may not be the primary inducer of hepatic metallothionein mRNA during foetal life.     

Cadmium and lead toxicity effects on zinc, copper, nickel and iron distribution in the developing chick embryo

The teratologic effects on chicken embryos induced by the load of 100 micrograms of cadmium or lead nitrate salts injected into the yolk on day 5 of incubation as well as their effects upon zinc, copper, nickel and iron distribution were studied. Despite a significant teratologic influence of lead, cadmium administration did not induce significant effects. A week after injections, embryo lead content, but not cadmium concentration, increased; this differential action suggested an active uptake of lead by the embryo, not observed for cadmium. Both cadmium and lead affected significantly the iron, copper and zinc homeostasis in the egg. Cadmium induced these changes probably through alterations in the metallic ion transport processes towards the embryo.     

Post-hatching parental care behaviour and hormonal status in a precocial bird

In birds, the link between parental care behaviour and prolactin release during incubation persists after hatching in altricial birds, but has never been precisely studied during the whole rearing period in precocial species, such as ducks. The present study aims to understand how changes in parental care after hatching are related to circulating prolactin levels in mallard hens rearing ducklings. Blood was sampled in hens over at least 13 post-hatching weeks and the behaviour of the hens and the ducklings was recorded daily until fledging. Contacts between hens and the ducklings, leadership of the ducklings and gathering of them steadily decreased over post-hatching time. Conversely, resting, preening and agonistic behaviour of hens towards ducklings increased. Plasma prolactin concentrations remained at high levels after hatching and then fell after week 6 when body mass and structural size of the young were close to those of the hen. Parental care behaviour declined linearly with brood age, showed a disruption of the hen-brood bond at week 6 post-hatching and was related to prolactin concentration according to a sigmoid function. Our results suggest that a definite threshold in circulating prolactin is necessary to promote and/or to maintain post-hatching parental care in ducks.     

Effects of copper aspirinate and aspirin on tissue copper,zinc, and iron concentrations following chronic oral treatment in the adjuvant arthritic rat

Concentrations of copper, zinc, and iron were analyzed and compared in a number of tissues of adjuvant arthritic rats following 22 d of chronic treatment (per os) with either vehicle, aspirin or copper aspirinate, at doses of 100 mg/kg, 200 mg/kg, or 400 mg/kg. Such chronic treatment resulted in a negative balance in copper, zinc, and iron in many tissues. Among the tissues examined, liver and kidney exhibited the greatest changes in metal concentrations; brain and skeletal muscle exhibited the least. Arthritis-induced changes in the concentrations of all three metals in the liver were reversed upon treatment with aspirin. Treatment with copper aspirinate, on the other hand, resulted in an extremely high accumulation of copper in the liver. Arthritis-induced changes in copper, zinc, and iron concentrations in the pancreas and copper concentration in the plasma were generally not reversed upon treatment with either aspirin or copper aspirinate. Among the three metals examined, the degree of change observed as a result of drug treatments was greatest for iron and least for zinc. Finally, it appeared that the effects of aspirin and copper aspirinate on tissue metal concentrations were independent of the antiarthritic effects of these compounds.     

Alaskan malamute chondrodysplasia--VIII. Incorporation of [14C]glucosamine and [3H]serine in hepatic metal-binding proteins of Canis familaris.

1. Hepatic proteins isolated from control kennel dogs bound small quantities of zinc and iron and the peptide fraction contained neither metal. 2. Zince loading of kennel dogs stimulated an hepatic uptake of five times more zinc and three times more iron than an equivalent copper load. The increase in metal concentration was noted in the 10,000 dalton protein. 3. Both the 12,000 and 10,000 dalton proteins isolated from kennel dogs contained more binding sites specific for zinc than for either copper or iron. All three proteins isolated from Alaskan Malamutes showed a smaller affinity for zinc than copper or iron. 4. Both copper and zinc loading stimulated an uptake of [14C]glucosamine and [3H]serine from the peptide fraction of control kennel dogs into the 10,000 dalton protein.     

Effect of chronic inflammation on copper and zinc metabolism

The effect of chronic inflammation on serum, blood and hepatic copper and zinc concentrations has been studied in arthritic female Sprague-Dawley rats, for a six week period. Metals were determined by atomic-absorption spectrophotometry and by inductively-coupled argon plasma spectrometry. Blood measurements evidenced hypercupremia and normal zinc levels. Serum and hepatic copper content were found to be increased. Serum zinc values were reduced whilst hepatic zinc concentration was higher. Serum alterations of both metals remained throughout the studied period. Good correlations were found between systemic alteration and changes in metal values.     

Laser ablation inductively coupled plasma mass spectrometry imaging of metals in experimental and clinical Wilson's disease

Wilson's disease is an autosomal recessive disorder in which the liver does not properly release copper into bile, resulting in prominent copper accumulation in various tissues. Affected patients suffer from hepatic disorders and severe neurological defects. Experimental studies in mutant mice in which the copper‐transporting ATPase gene (Atp7b) is disrupted revealed a drastic, time‐dependent accumulation of hepatic copper that is accompanied by formation of regenerative nodes resembling cirrhosis. Therefore, these mice represent an excellent exploratory model for Wilson's disease. However, the precise time course in hepatic copper accumulation and its impact on other trace metals within the liver is yet poorly understood. We have recently established novel laser ablation inductively coupled plasma mass spectrometry protocols allowing quantitative metal imaging in human and murine liver tissue with high sensitivity, spatial resolution, specificity and quantification ability. By use of these techniques, we here aimed to comparatively analyse hepatic metal content in wild‐type and Atp7b deficient mice during ageing. We demonstrate that the age‐dependent accumulation of hepatic copper is strictly associated with a simultaneous increase in iron and zinc, while the intrahepatic concentration and distribution of other metals or metalloids is not affected. The same findings were obtained in well‐defined human liver samples that were obtained from patients suffering from Wilson's disease. We conclude that in Wilson's disease the imbalances of hepatic copper during ageing are closely correlated with alterations in intrahepatic iron and zinc content.     

Chronological changes in tissue copper, zinc and iron in the toxic milk mouse and effects of copper loading

The toxic milk (tx) mouse is a rodent model for Wilson disease, an inherited disorder of copper overload. Here we assessed the effect of copper accumulation in the tx mouse on zinc and iron metabolism. Copper, zinc and iron concentrations were determined in the liver, kidney, spleen and brain of control and copper-loaded animals by atomic absorption spectroscopy. Copper concentration increased dramatically in the liver, and was also significantly higher in the spleen, kidney and brain of control tx mice in the first few months of life compared with normal DL mice. Hepatic zinc was increased with age in the tx mouse, but zinc concentrations in the other organs were normal. Liver and kidney iron concentrations were significantly lower at birth in tx mice, but increased quickly to be comparable with control mice by 2 months of age. Iron concentration in the spleen was significantly higher in tx mice, but was lower in 5 day old tx pups. Copper-loading studies showed that normal DL mice ingesting 300 mg/l copper in their diet for 3 months maintained normal liver, kidney and brain copper, zinc and iron levels. Copper-loading of tx mice did not increase the already high liver copper concentrations, but spleen and brain copper concentrations were increased. Despite a significant elevation of copper in the brain of the copper-loaded tx mice no behavioural changes were observed. The livers of copper-loaded tx mice had a lower zinc concentration than control tx mice, whilst the kidney had double the concentration of iron suggesting that there was increased erythrocyte hemolysis in the copper-loaded mutants.     

Influence of dietary cadmium on the distribution of the essential metals copper,zinc and iron in tissues of the rat

The effect of long-term dietary cadmium treatment upon the distribution of the metals copper, iron and zinc has been compared in various organs of male and female rats. The renal accumulation of cadmium was similar in both sexes without a plateau being reached. In contrast, the hepatic accumulation of cadmium was higher in the female than in the male rat and a plateau was observed after 30–35 weeks of dietary cadmium treatment. Most of the cadmium which accumulated in these organs was recovered in the metallothionein fraction and the concentration of hepatic cadmiumthionein in the female rat was correspondingly higher than in the male rat. Accumulation of cadmium was associated with an increased zinc concentration in the liver and an increased copper concentration in the kidney; these increases were correlated with increases in liver and kidney metallothioneins induced by cadmium. Uptake of cadmium into organs other than liver and kidney occurred to a small extent but was not associated with changes in the concentration of copper and zinc. Cadmium also accumulated in the intestinal mucosa where it could be recovered in a fraction corresponding to metallothionein. A loss of iron from the liver and kidney was also observed following dietary cadmium treatment and involved mainly a loss of iron from ferritin.     

Presence of acute phase changes in zinc, iron, and copper metabolism in turkey embryos

Acute phase changes in trace mineral metabolism were examined in turkey embryos. An endotoxin injection resulted in increased concentrations of serum copper and liver zinc and decreased concentrations of serum zinc in embryos incubated either in ovo or ex ovo. Changes in zinc and copper metabolism occurred when endotoxin either was injected intramuscularly, into the amnionic fluid, or administered onto the chorioallantoic membrane. Unlike poults, embryos did not respond to an inflammatory challenge with decreased serum iron concentrations. Acute phase changes in embryo serum zinc and copper as well as liver zinc concentrations were similar to those in poults. Increased liver zinc concentrations were associated with increased zinc in metallothionein (MT). An injection of a crude interleukin 1 preparation into embryos resulted in similar increases in hepatic zinc and MT concentrations as an endotoxin injection, suggesting a role for this cytokine in mediating the acute phase changes in embryonic zinc metabolism.     

Analysis of hepatic copper, zinc, metallothionein and metallothionein-Ia mRNA in developing sheep

The concentrations of zinc, copper, metallothionein and metallothionein-Ia mRNA in sheep livers during development was determined. It was found that early sheep foetuses (30-40 days gestation) had very high concentrations of hepatic zinc (2305 +/- 814 micrograms/g dry mass), and that these levels declined steadily to 644 +/- 304 micrograms/g near to term. The copper concentrations in the foetal livers were not higher than those in the adult. The concentrations of metallothionein and metallothionein-Ia mRNA were also very high in the foetal livers and declined steadily during gestation from 261 +/- 94 molecules/pg RNA to 71 +/- 18 molecules/pg near to term. Metallothionein-Ia mRNA concentrations were closely correlated with hepatic zinc concentrations but not with copper. Metallothionein concentrations also decreased during gestation: e.g. 3044 micrograms/g (wet mass) in one foetus on day 34 of gestation to 862 micrograms/g on day 125. After birth, however, the concentrations of metallothionein declined to less than 100 micrograms/g and this decline occurred despite the presence of significant quantities of mRNA. The ratio of metallothionein/metallothionein-Ia mRNA decreased from 1.3 to 3.2 x 10(5) molecules metallothionein/molecule of metallothionein-Ia mRNA during gestation to between 0.28-0.64 x 10(5) molecules/molecule in the postnatal animals. We conclude that the major function of metallothioneins in the foetal liver is protection of the liver against the potentially toxic accumulation of zinc. In the postnatal sheep there appears to be a decreased synthesis or increased degradation of metallothionein.     

Effect of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on the Hepatic Distribution of Iron,Copper, Zinc,and Magnesium in Rats

The distribution of iron, copper, zinc, and magnesium in hepatic subcellular fractions of male and female rats treated with 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) was determined. Animals received 40 μg TCDD per kilogram per day for three days by mouth (PO) or the vehicle and were killed seven or nine days posttreatment. Iron, copper, zinc, and magnesium were determined by atomic absorption spectroscopy. The iron content of liver from female animals was twofold higher than male animals. The administration of TCDD increased the iron content of mitochondria in female and male rats and decreased iron content of microsomes of both sexes. Significant increases occurred in the copper content of whole liver, mitochondria, and cytosol of male rats and in whole liver and cytosol of female rats. Decreases in the copper content of the microsomes of male rats were observed following TCDD treatment; however, TCDD produced no changes in the zinc content of hepatic subcellular fractions of either sex. The magnesium content of female TCDD-treated rats increased in whole liver, mitochondria, and cytosol, while the magnesium content of microsomes was not altered. With respect to the subcellular distribution of iron, copper, zinc, and magnesium, TCDD produces differential effects. The altered distribution of some cations may contribute to the broad range of effects of TCDD.     

Effect of transition metals on stress, lipid peroxidation and antioxidant enzyme activities in tobacco cell cultures

to-baccoBright Yellow 2 (BY-2) suspension culture to understand the mechanisms of metal resistance in plant cells.We have analysed superoxide dismutase, catalase, and ascorbate peroxidase enzyme activities and superoxidedismutase-isoforms by isoelectric focusing gels in tobacco cells grown at two different toxic concentrations ofeach of the transition metals: copper, iron, manganese and zinc. Exposure of tobacco cells to these metals causedchanges in total superoxide dismutase activity in a different manner, depending on the metal assayed: after cop-perand manganese treatments, total superoxide dismutase activity was enhanced, while it was reduced after ironand zinc exposure. Superoxide dismutase-isoforms were affected by the metal used, and a Fe-SOD band with thesame isoelectric point as a Cu, Zn-SOD from non-treated cells, was induced after iron and zinc treatments. Cu,Zn-SODs were present in all metal-treatments whereas Mn-SOD was not detected in any case. Concerning otherantioxidant enzymes tested, such as catalase and ascorbate peroxidase, the latter showed a remarkable increase inactivity in response to copper treatments and catalase activity was enhanced after iron and with the lowest man-ganeseconcentration. Lipid peroxidation was increased after each metal treatment, as an indication of the oxi-dativedamage caused by metal concentration assayed in tobacco cells. These results suggest that an activation ofsome antioxidant enzymes in response to oxidative stress induced by transition metals is not enough to confertolerance to metal accumulation.