Rate Of Metallothionein Synthesis Research Articles

Regulation of metallothionein in teleosts: Induction of MTmRNA and protein by cadmium in hepatic and extrahepatic tissues of a marine flatfish, the turbot ( Scophthalmus maximus)

Synthesis of the heavy metal-binding protein metallothionein (MT) was determined in the major organs of accumulation of Cd (liver, kidney and gills) of a marine flatfish, the turbot, after intraperitoneal (i.p.) administration of varying doses of Cd. Synthesis of MTmRNA and MT were linearly related to dose only at low Cd dosages (up to ca. 100 μgCd/kg). Induction of MTmRNA was rapid, peaking 1–2 days after Cd administration in gills and kidneys, at 4 days in liver. In all three tissues, at low doses of Cd, MTmRNA levels declined with an apparent half life of 5–7 days and for a given dose of Cd, similar MTmRNA concentrations were attained. Induction of MT levels temporally followed that of MTmRNA. Steady state levels of MT were attained more quickly at a low dose of Cd. At acute Cd doses of >200 μgCd/kg, MT gene transcription and protein translation appeared to be progressively reduced, inferring that the rate of MT synthesis was limiting due to cytotoxicity of the high acute Cd dosage. In contrast to MTmRNA levels which were induced to similar levels in the three tissues, MT levels decreased in the order liver > kidneys > gills implying differences in translational processes.

Zinc-induced metallothionein synthesis by Caco-2 cells

Caco-2 cells possess many morphological and biochemical characteristics of intestinal absorptive cells, including the ability to transport zinc. In the present study, metallothionein (MT) synthesis in response to increased levels of zinc was examined. Increased incorporation of [35S]cysteine into MTs was observed when excess ZnCl2 was added to the medium. The rate of MT synthesis was found to be concentration dependent. Also, induction of MT synthesis was greater early in the culture, before the cells were fully differentiated. Incubation of the monolayers with 65Zn and 200 microM zinc revealed that approximately 50% of the zinc incorporated into the cells was associated with MTs. The remainder was associated with large proteins as well as amino acids and small peptides. Actinomycin D and cycloheximide both inhibited the induction of MT synthesis, suggesting that the newly synthesized MTs are a result of expression of MT genes. Hence, Caco-2 cells, a model of intestinal absorptive cells, may be used to examine the role of MTs in zinc absorption.

Roles of synthesis and degradation in the regulation of metallothionein accretion in a chicken macrophage-cell line

Metallothionein (MT) is a metal-binding protein rapidly accreted in many tissues in response to trace elements or hormones. To gain an understanding of the regulation of MT accretion, rates of MT synthesis and degradation were determined by using a decay-kinetics technique. A chicken macrophage-cell line (HD11) that rapidly accretes incremental amounts of MT when stimulated with increasing concentrations of Zn2+ or Cd2+ was studied. The maximum rate of MT accretion occurred at 50 microM-Zn2+ or 20 microM-Cd2+. The absolute rate of MT accretion was less in macrophages incubated with 25 microM- as compared with 50 microM-Zn2+, owing to decreased and increased rates of MT synthesis and degradation respectively. The absolute rate of MT accretion was less in macrophages incubated with 10 microM- as compared with 20 microM-Cd2+, owing to a decreased rate of MT synthesis with no change in degradation. Compared with macrophages continually incubated with 50 microM-Zn2+, removal of Zn2+ from medium previously containing 50 microM-Zn2+ decreased the absolute rate of MT accretion, owing to decreased and increased rates of MT synthesis and degradation respectively. Removal of Cd2+ from medium previously containing 20 microM-Cd2+ also decreased the absolute rate of MT accretion in macrophages. Unlike Zn2+ removal, the decrease in MT accretion was due to a decreased rate of MT synthesis with no change in degradation. When macrophages incubated with 50 microM-Zn2+ were subsequently incubated with 20 microM-Cd2+, rates of MT synthesis and accretion were decreased as compared with cells continually incubated with 50 microM-Zn2+ or 20 microM-Cd2+. When macrophages incubated with 20 microM-Cd2+ were subsequently incubated with 50 microM-Zn2+, rates of MT synthesis and accretion were increased as compared with cells continually incubated with 50 microM-Zn2+ or 20 microM-Cd2+. Switching the metal in the incubation medium did not influence the rate of MT degradation. Our results indicate that the rate of MT accretion is determined by variations in the rates of MT synthesis and degradation, depending upon the inducing metal and the concentration of the metal.

Metallothionein metabolism in the liver and kidney of the streptozotocin-diabetic rat

1. 1. Elevated levels of metallothionein (MT)-I and -II were identified in the liver and kidney of insulin-deficient diabetic rats. 2. 2. The relative rate of MT synthesis and the turnover of cytoplasmic MT were both accelerated in the liver of diabetic rats. 3. 3. The rate of synthesis of MT, but not its cytoplasmic turnover, was increased in diabetic kidney. 4. 4. Maximal relative rates of MT synthesis in liver and kidney were first observed at 4 and 10 days, respectively, after inducing the diabetic condition. 5. 5. The altered metabolism of hepatic MT in diabetic rats was attributed primarily to disturbances in endocrine status, while the altered metabolism of renal MT was largely due to accumulation of excessive dietary copper in the kidney.

Metallothionein gene regulation in the preimplantation rabbit blastocyst.

Expression of metallothionein (MT) genes in the preimplantation rabbit blastocyst was analysed by determination of the levels of MT mRNA and relative rates of MT synthesis. MT was found to be constitutively expressed at low levels in the blastocyst. Exposure of the day-6 blastocyst to zinc ions in vitro rapidly increased the level of MT gene expression in a dose-dependent manner, with a ten-fold induction in the relative rate of synthesis at 400 microM-Zn2+. Ion-exchange chromatography of pulse-labelled blastocyst protein showed that the relative rates of synthesis of both MT-I and MT-II were markedly increased following zinc treatment, with MT-I being the predominant isometallothionein. Zinc induction of MT synthesis in the blastocyst was also detected on day 4 of gestation just after the morula-to-blastocyst transition. In contrast to the zinc effects on MT, in vitro exposure to 10 microM-Cd2+ resulted in a large induction of MT mRNA but only a modest increase in the relative rate of MT synthesis. Cadmium was found to be toxic to the day-6 blastocyst, and 10 microM-Cd2+ induced an acute stress response as indicated by a dramatic induction of heat-shock protein (HSP-70) gene expression.

Induction of metallothionein synthesis in Menkes' and normal lymphoblastoid cells is controlled by the level of intracellular copper.

A study was carried out on the uptake of copper, zinc, or cadmium ions and their induction of metallothionein synthesis in Menkes' and normal lymphoblastoid cells. The main difference between Menkes' and normal cells in the uptake of these metal ions was an increased uptake of copper ions in Menkes' cells at a low concentration of CuCl2 (2.1 microM). The CuCl2 concentration necessary to induce metallothionein synthesis in Menkes' cells was 50 microM, whereas that in normal cells was about 200 microM. The levels of zinc or cadmium ions needed to induce metallothionein in Menkes' cells were similar to those in normal cells. At least four isomers of metallothionein were induced by copper, zinc, and cadmium ions in both types of cells. Metallothionein synthesis in Menkes' and normal cells was induced when the amounts of intracellular copper reached a threshold level of approximately 0.2 nmol/10(6) cells, and the rate of metallothionein synthesis in these cells was increased as a function of the amounts of intracellular copper (0.2-1.7 nmol/10(6) cells). These results indicate that the induction of metallothionein synthesis in lymphoblastoid cells is controlled by the level of intracellular copper, suggesting that the major defect in Menkes' cells is not due to the abnormal regulation of metallothionein synthesis but to an alteration of the copper metabolism in cells by which the levels of intracellular copper become larger than those in normal cells and just lower than the threshold level for induction of metallothionein synthesis.

Induction and Accumulation of Metallothionein in Liver and Pancreas of Chicks Given Oral Zinc: A Tissue Comparison

The present study was undertaken to compare apparent zinc metabolism in two organs, the liver and pancreas. Four-week-old chicks received an oral dose of saline or an equal volume of zinc solution containing 4, 8 or 16 mg zinc. The accumulation of zinc metallothionein (MT) was determined 24 hours postgavage in hepatic and pancreatic cytosol. The rate of MT synthesis was determined in both tissues by a pulse labeling technique. In response to orally administered zinc, plasma zinc increased sharply (by 3 hours) and in proportion to the amount of zinc given. At 12 hours there was little difference in plasma zinc. The concentration of zinc MT in pancreatic cytosol relative to liver was nearly fivefold higher in control chicks and increased at approximately twice the rate when chicks were given zinc. Despite this marked difference in both the rate and extent of zinc MT accumulation there was little evidence of a difference in the rate of MT synthesis. Quantitation of MT by anion-exchange chromatography demonstrated an apparent maximum in the rate of MT synthesis at 6 hours, which was apparently the same in pancreas and liver. Our data suggest that when compared to liver, the pancreas possesses a markedly higher concentration of MT-bound zinc and a greater propensity to accumulate zinc MT when zinc status is acutely elevated. This tissue difference appeared not be be due to a greater capacity to synthesize MT.

Synthesis and degradation of hepatic metallothionein in mice differing in susceptibility to cadmium mortality

We have confirmed the observations of Tsunoo et al. (Toxicol. Lett. 4:253, 1979) that (a) DBA/2 mice are resistant to cadmium mortality than C3H mice and (b) DBA/2 mice accumulate more 109Cd into hepatic metallothionein than do C3H mice in response to an injection of 30 mumol CdCl2/kg, a dose of CdCl2 which is lethal to C3H mice. We now report, using a nonlethal dose of 8 mumol CdCl2/kg, that the rates of both the synthesis and the degradation of cadmium-induced hepatic metallothionein are increased in C3H mice. The rate of metallothionein synthesis, measured 6 hr after cadmium administration and expressed as the percentage of injected [35S]cysteine incorporated into metallothionein/g liver, was 0.33 +/- 0.04% (SD) in C3H mice, compared to 0.19 +/- 0.06% in DBA/2 mice (significantly different rates by Students' t test. P less than 0.01). Also, at this dose, hepatic 35S-labeled metallothionein was degraded with a half-life of 22.5 +/- 0.7 hr in C3H mice, compared to 30.1 +/- 2.5 hr in DBA/2 mice (significantly different half-lives by F test, within 95% confidence limits). The increased accumulation of metallothionein in resistant DBA/2 mice compared to sensitive C3H mice after cadmium exposure appears to be due primarily to a difference in metallothionein degradation, rather than metallothionein synthesis.

Metallothionein synthesis in foetal, neonatal and maternal rat liver.

The synthesis of hepatic metallothionein relative to other cytosol proteins was measured by [35S]cysteine incorporation in foetal, neonatal and pregnant rats. The relative rate of hepatic metallothionein synthesis reached a maximum in foetal liver on days 18-21 of gestation. Metallothionein synthesis then declined until weaning, when adult levels were established. The rate of metallothionein synthesis was greater in pregnant rats at term than in nulliparous rats. To determine if circulating inducing agents could play a role in the regulation of metallothionein synthesis in foetal liver we treated pregnant rats with inducers at a time prior to the normal rise in foetal liver metallothionein synthesis. Injections of copper, cadmium or hydrocortisone to 17-day-pregnant dams failed to induce foetal metallothionein synthesis. In contrast, zinc injection to the dam was an effective inducer in the foetuses. Maternal laparotomy (performed to expose the foetus for direct injection of inducers) induced foetal metallothionein synthesis. Metallothionein synthesis in the livers of 17-day-gestation dams was induced by all metal injections and laparotomy but, surprisingly, not by hydrocortisone injection. Maternal adrenalectomy did not influence the subsequent normal elevation in foetal or maternal metallothionein synthesis. These results, in conjunction with previous reports, suggest that mobilization of zinc in serum during late gestation may regulate foetal and maternal changes in metallothionein synthesis.

736 EFFECT OF EXTRACELLULAR COPPER (Cu) ON METALLOTHIONEIN METABOLISM IN NORMAL AND MENKES FIBROBLASTS

Previously, we demonstrated that the abnormal accumulation of Cu in Menkes disease (MD) fibroblasts (Fb) is associated with an increased amount of metallothionein (MT) (2-3 times normal) (Pediat. Res., in press). To define the basic defect in MD, we investigated the effect of varying concentrations of extracellular Cu (exCu) on the regulation of CuMT metabolism informal (N) and MD Fb. Relative levels of MT were measured by 35S-cysteine incorporation into the 10,000 MW Cu-binding cytosolic protein. Total intracellular Cu was measured by atomic absorption spectrometry. After exposure of N and MD Fb to 2 ug/ml Cu (as CuCl2) maximal levels of MT synthesis were attained within 8 h and remained elevated for at least 56 h. Additional Cu (up to 20 ug/ml) did not increase the rate of MT synthesis above that observed with 2 ug/ml. Pulse-chase experiments demonstrated no difference in the half-life (T1/2=48-60 h) of MTs in N or MD Fb in the presence or absence of exCu. These results demonstrated that MT was an inducible protein in both N and MD Fb. Also, the rate of degradation of the MTs was the same in both the N and MD Fb. Although the mechanism of MT induction by exCu appears normal in MD Fb, the suppression of MT synthesis in the absence of excess Cu may be defective. This regulatory defect results in abnormally high constitutive levels of MT in MD Fb. Whether this defect in regulation of MT synthesis is transcriptional or translational is currently under investigation.