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.     

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.     

Regulation of liver metallothionein and plasma zinc by the glucocorticoid dexamethasone.

Administration of the glucocorticoid dexamethasone to adrenalectomized rats significantly decreased the serum zinc concentration within 14 hr. Dexamethasone did not detectably alter the liver zinc content, but markedly increased the proportion of zinc associated with liver metallothionein. The rate of incorporation of ³⁵S-cystine into this protein was stimulated to a maximal extent 7 hr after administration of the glucocorticoid. Poly(A)⁺ mRNA from liver polysomes was isolated and translated in a cell-free protein synthesizing system. Nearly twice as much polysomal metallothionein mRNA was found 7 hr following treatment with dexamethasone. These results suggest that glucocorticoids can regulate the plasma zinc concentration by a process that is related to the biosynthesis of the hepatic zinc-binding protein, metallothionein.     

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.     

A comparison of cadmium-induced metallothionein gene expression and Me2+ distribution in the tissues of cadmiumsensitive (rainbow trout; Salmo gairdneri) and tolerant (stone loach; Noemacheilus barbatulus) species of freshwater fish

1. The accumulation of cadmium in the liver, kidney and gills of rainbow trout and stone loach was measured during exposure of the fish to the metal at 3 smg/l in their aquarium water. The pattern of accumulation of the toxic metal in the individual organs was different between the two species.2. The tissue concentrations of metallothionein-specific mRNA and metallothionein protein were also determined in these organs from the same fish. In rainbow trout, the induction of metallothionein gene expression resulted in a gradual increase in metallothionein concentration in gill over the course of the experiment whereas increases in metallothionein in the liver and kidney were detected only at the later time points of analysis (beyond 19 weeks). By contrast, in the same tissues from stone loach, relatively minor changes were quantified in specific mRNA and metallothionein concentrations.3. Throughout the experimental period, tissue concentrations of zinc and copper were determined in the liver, kidney and gills of the rainbow trout and stone loach. Subtle decreases were observed in the zinc concentration of gills in rainbow trout and substantial increases were observed in the hepatic copper concentrations in both species at the later time points of analysis.4. The ability of cadmium to induce metallothionein gene expression and its subsequent ability to compete for the sequestration sites on the newly-synthesized protein is discussed with regard to the relative levels of cadmium, zinc and copper in the organs studied and differing regimes of cadmium administration.     

The biosynthesis of metallothionein in rat liver and kidney after administration of cadmium

The biosynthesis of the cadmium-binding protein, metallothionein, was studied in rat liver and kidney after injection of cadmium chloride. A simplified procedure for the isolation of metallothionein from liver and kidney tissues was devised. It was found that the concentration of a subcutaneously injected dose of 30 μmoles of ¹⁰⁹CdCl₂/kg in the liver reached the maximum within 36 h. Thereafter, a slow decrease in the concentration of the isotope was noted during the 3 week period. In the kidney, the isotope was taken up in two phases. During the first phase the uptake was faster and lasted for about 4 days. The second phase of ¹⁰⁹Cd accumulation showed a slower increase in the concentration of the isotope. In both liver and kidney tissues 75–80% of the ¹⁰⁹Cd was associated with metallothionein. Amino acid incorporation studies revealed that active biosynthesis of metallothionein took place in the kidney as well as in the liver of cadmium-exposed rats. The turnover of ³⁵S-labeled metallothionein was also investigated and the half-lives of the hepatic and the renal metallothionein were found to be 2.8 and 5 days, respectively.     

Plasma lipoproteins, tissue cholesterol overload, and skeletal muscle apolipoprotein A-I synthesis in the developing chick

In the present study we investigated the changes of plasma lipids, lipoproteins, and tissue lipids that occur during the late embryonic life (5 days before hatching) and the postnatal period (0, 2, 7, 14, and 30 days after hatching) of the chick. The chick emerges from the egg with extreme hypercholesterolemia associated with a high level of cholesterol-rich VLDL + IDL. The density gradient profile of plasma lipoproteins showed that the concentrations of VLDL + IDL and LDL decreased during the first week of postnatal life, whereas HDL concentration increased sharply around hatching and remained stable afterwards. All plasma lipoprotein classes of the newborn chick (2 days from hatching) were enriched in cholesterol and cholesteryl esters; 2 weeks after hatching, the relative amount of cholesterol and cholesteryl esters decreased. In the newborn chick, plasma VLDL + IDL consisted of two populations of cholesteryl ester-rich lipoproteins: the main one (designated apoB-VLDL) contained apoB and no apoA-I; the other (designated apoA-I-VLDL) contained predominantly apoA-I. In the newborn chick there was an accumulation of free and esterified cholesterol in the liver and, to a lesser extent, in the skeletal muscle. These cholesterol deposits were depleted 2 to 7 days after hatching. The depletion in skeletal muscle was preceded by and associated with a striking increase in the synthesis of apoA-I in this tissue, as demonstrated by immunological methods and apoA-I mRNA measurements. In addition, apoA-I-containing HDL were secreted in vitro by explants of skeletal muscle of the newborn chick. The synthesis of apoA-I in the skeletal muscle decreased to the level found in the adult animal 1 week after hatching. It is likely that the rise of HDL and apoA-I in plasma observed 1-2 days after hatching reflects the production of apoA-I-containing HDL by skeletal muscle. We suggest that the cholesterol overload in skeletal muscle might stimulate the production of apoA-I which, in turn, would promote the removal of cholesterol from this tissue. The hypothesis that metabolic stimuli play a role in inducing apoA-I synthesis in skeletal muscle is supported by the observation that feeding the newborn chick a diet rich in proteins and lipids and free of carbohydrates delays the fall of apoA-I mRNA which normally occurs 1 week after hatching.     

Regulation of the ontogeny of rat liver metallothionein mRNA by zinc

To investigate the role of metals in the regulation of the ontogenic expression of rat liver metallothionein (MT) mRNA, the concentrations of zinc, MT and MT mRNA were determined in livers of fetal and newborn rats from dams which were fed with a control or zinc-deficient or copper-deficient or iron-deficient diet from day 12 of gestation. The liver samples were analyzed for MT-mRNA levels using a mouse MT-I cRNA probe. Although the newborn hepatic levels of each metal (zinc or copper or iron) was specifically reduced corresponding to the respective mineral deficiencies, the hepatic concentrations of total MT and MT-I mRNA were significantly decreased only in pups born from zinc-deficient dams. Injection of the zinc-deficient newborn pups with 20 mg Zn as ZnSO4/kg restored with MT-I mRNA levels to slightly above control values within 5 h of injection. The hepatic zinc, MT and MT-I mRNA levels were observed to increase significantly in control fetal rat liver on days 17-21 of gestation but there were little changes in either zinc or MT in fetal livers from zinc-deficient dams during the late gestational period. The MT-I mRNA level also did not show an increase on days 18 and 20 of gestation in zinc-deficient fetal liver as compared to controls. These results demonstrate a direct role of zinc in hepatic MT gene expression in rat liver during late gestation. Immunohistochemical localization of MT using a specific antibody to rat liver MT showed that the staining for MT in zinc-deficient pup liver was mainly in the cytosol in contrast to the significant nuclear MT staining observed in control newborn rat liver. The results suggest that maternal zinc deficiency has a marked effect not only in decreasing the levels of hepatic MT and MT-I mRNA but also in the localization of MT in newborn rat liver.     

Iodothyronine 5''-deiodinase activity as an early event of prenatal brown-fat differentiation in bovine development.

The regulation of metallothionein (MT) biosynthesis in rainbow-trout liver was studied after a single intraperitoneal injection of oestradiol-17 beta. Sampling was performed after 2, 7, 14, 21, 28 and 35 days. Following induction of vitellogenin synthesis in the liver, liver somatic index (LSI) rose from 1.25 to 2.00 in 14 days. Associated with the increase in LSI was an elevation of hepatic vitellogenin mRNA and zinc concentrations. The vitellogenin mRNA concentrations peaked at 7 days after treatment. The zinc concentrations increased to a peak at day 14. MT was analysed by using differential pulse polarography and a rainbow-trout MT RNA probe. The MT mRNA concentrations rose after 14 days and remained elevated at 21 and 28 days. The MT concentrations increased after 14 days and remained elevated throughout the experimental period. The concentrations of MT-bound zinc increased in association with the elevation in MT concentrations in the oestradiol-treated rainbow trout. These findings indicate that MT is involved in the regulation of zinc during the period of vitellogenin induction and that MT may function by maintaining the pool of available zinc at an appropriate concentration.     

Posthatching evolution of in vivo mevalonate metabolism in chick liver

The in vivo mevalonate incorporation into total nonsaponifiable lipids by chick liver was minimal after hatching and drastically increased between 1-5 days. The hepatic synthesis of different cholesterol precursors emerged sequentially after hatching. Between 1-5 days increased strongly the conversion of mevalonate into squalene and also the formation of oxygenated lanosterol derivatives from squalene. The conversion of squalene became completely active at day 8. Cholesterol formation from lanosterol derivatives was completely activated between 8-11 days. Results in this paper demonstrate for the first time the accumulation of a fraction of nonsaponifiable lipids identified as lanosterol derivatives and cholesterol precursors formed from [5-14C]mevalonate in experiments carried out in vivo. Postnatal evolution of these oxysterols may explain the great increase of 3-hydroxy-3-methylglutaryl-CoA reductase activity found in chick liver between 5-11 days, simultaneous or posterior to the diminution of the oxygenated cholesterol precursors.     

Changes in chick liver and brain mevalonate kinase, mevalonate-5-phosphate kinase and mevalonate-5-pyrophosphate decarboxylase during development

Phosphorylation and decarboxylation of mevalonate in chick liver and brain was investigated during early post hatching stages of development. In chick liver, both mevalonate kinase and mevalonate-5-phosphate kinase increased their activity from day 5 of age while pyrophosphate decarboxylase activity remained low during the first days after hatching, increased sharply up to day 9 of age, and remained practically unchanged thereafter. The developmental pattern obtained in brain shows a slight decrease in the phosphorylation and decarboxylation of mevalonate after the first week of postnatal development. Further studies were performed using the specific substrate of mevalonate-5-pyrophosphate decarboxylase, corroborating the results obtained using mevalonate as substrate. Changes in hepatic decarboxylase were more pronounced than those observed in mevalonate-phosphorylating enzymes, thus suggesting an important role for decarboxylase in the control of cholesterogenesis during postnatal development.     

Age-dependent variation for inducibility of metallothionein genes in mouse liver by cadmium

Cadmium is a toxic metal that induces the expression of metallothionein genes in many tissues and that binds avidly to metallothionein, a soluble transition metal binding protein. The present study examined the temporal pattern and magnitude of accumulation of metallothionein mRNA in liver of C57BL/6J mice of various ages treated with cadmium. In adult female mice, accumulation was dependent on the dosage level of cadmium and related to the concentration of this metal in liver. The accumulation of metallothionein mRNA in liver depended on age at exposure to cadmium. Intraperitoneal administration of 2 mg of cadmium per kg provoked small increases (two- to threefold) in levels of metallothionein mRNA in livers of 7- and 14-day-old mice. In contrast, cadmium treatment of 28- and 56-day-old mice resulted in 12- to 19-fold increases in levels of metallothionein mRNA in liver with maximum increases occurring 3 to 4 hr after treatment. Because similar patterns for the accumulation of cadmium of liver were found in 7-, 28-, and 56-day-old mice, observed age-dependent differences in induction of metallothionein mRNA in liver were probably not due to differences in the accumulation of cadmium in this organ. Taken together, these data suggest that tissue-specific factors controlling the expression of metallothionein genes may account for developmental variation in the inducibility of these genes by cadmium. Ontogenic variation in accumulation of metallothionein mRNA after cadmium treatment may be a factor in developmental variation in the acute lethality of cadmium in C57BL/6J mice.     

The levels of liver metallothionein and zinc in plaice, Pleuronectes platessa L., during the breeding season, and the effect of oestradiol injection

Wild plaice from two locations were examined for liver metallothionein and liver and serum zinc content, before, during and after the breeding season. During the early stages a number of females had very high liver metallothionein and zinc levels. Egg formation and ripening were accompanied by a reduction in serum zinc. In males, the metallothionein levels did not reach such high values and were not correlated with gonad development.
There was a correlation between the zinc concentration of the liver and the metallothionein concentration of the liver. Above the threshold value for metallothionein formation, approximately half the additional zinc was found in the metallothionein and half in non-metallothionein pool(s).
Intramuscular injection of oestradiol 3-benzoate into immature females caused a significant increase in liver weight but a depression in metallothionein concentration relative to the controls.     

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.     

Induction of metallothionein mRNA in rat liver and kidney after copper chloride injection.

The kinetics of the increase of metallothionein mRNA in rat liver and kidney after CuCl2 injection was determined by cell-free translation and dot-blot hybridization of total RNA isolated at various times after the injection. Both assay procedures gave essentially the same result: a 16-fold increase in hepatic metallothionein mRNA was observed 7h after CuCl2 injection, with a decline to basal values by 15 h. The response in the kidney was less dramatic, with a 6-fold increase in metallothionein mRNA 5 h after injection, and basal values were attained by 12h. The rise in Cu2+ concentration in both organs was closely correlated with the increase in metallothionein mRNA; hepatic Cu2+ was increased 5.9-fold by 5h after injection and renal Cu2+ was increased 4.3-fold 5h after injection. The Zn2+ concentration in the liver had not risen significantly within 5h of Cu2+ injection. Renal Zn2+ concentrations did not alter appreciably in the Cu2+-treated animals. These results support the conclusion that Cu2+ is acting as a primary inducer of metallothionein mRNA in the rat.     

Dietary beta- and gamma-cyclodextrins stimulation of hepatic metallothionein gene expression in rats

This study investigated whether hepatic metallothionein gene expression is affected by dietary cyclodextrins. Young male Wistar rats were fed a basal diet or cyclodextrin-supplemented (50 g of cyclodextrin per kg diet) diets for 7 d. Copper content in the liver did not show any significant changes among rats fed the basal, beta- and gamma-cyclodextrin diets. There were no differences in liver or serum zinc among groups. Copper content in serum was markedly decreased in rats fed the gamma-cyclodextrin-supplemented diet. Liver metallothionein mRNA levels were significantly elevated in both beta- and gamma-cyclodextrins-fed rats, but not in alpha-cyclodextrin-fed rats. Thus, the increase in hepatic metallothionein mRNA levels might be due to this mechanism except for the contents of copper and zinc in the liver.     

Hepatic zinc,copper, and iron in the developing turkey embryo and newly hatched poult

The ontogeny of hepatic tissue growth and trace metal deposition was examined in the developing turkey embryo and newly hatched poult. Hepatic concentrations of zinc and iron in the embryo declined by about twofold between day 16 of incubation and hatching. Hepatic copper concentration increased approximately fourfold by day 23 of incubation and then declined rapidly through hatching. During the post-hatching period, hepatic zinc concentration increased twofold by day 10, whereas a small increase in hepatic iron concentration occurred just prior to hatching and continued through the third day post-hatching. A significant positive correlation existed between hepatic zinc and iron concentrations in the developing embryo. The concentrations of both these metals were inversely correlated with hepatic copper concentration during the same time. Total hepatic zinc and iron content increased throughout the entire time studied, whereas total copper content increased up to hatching and then declined during the first week post-hatching. The most rapid phase of hepatic metal accretion differed for each metal, with zinc being rapidly accumulated during the post-hatching period, copper during the last half of incubation and iron at about the time of hatching and the first few days post-hatching. Each of these metals demonstrated a specific relationship to hepatic tissue growth that changed between the embryonic and neonatal periods of development.     

Cadmium accumulation and metabolism by rat liver parenchymal cells in primary monolayer culture.

Primary cultures of adult rat liver parenchymal cells, isolated by the collagenase perfusion technique and maintained as a monolayer, were used to investigate the characteristics of hepatic cadmium accumulation and metabolism. Cadmium accumulation was found to be a temperature- and concentration-dependent process that required sulfhydryl groups and was significantly stimulated by the addition of dexamethasone to the medium. Once taken up, cadmium was less available for exit-exchange processes than its biologically required congener, zinc. Moreover, cadmium influx enhanced zinc efflux. While most of the intracellular cadmium was located in the cytosol, its distribution within this fraction was altered with time. Initially the metal was bound to both high molecular weight species (less than 50 000) and metallothionein. As the incubation period increased, the cytosol concentration of cadmium and the percentage of this metal associated with metallothionein was likewise increased. [3H]Amino acid incorporation studies indicated that the accumulation of cadmium resulted in de novo synthesis of the 1 and 2 forms of metallothionein.