Liver Metallothionein mRNA Research Articles

Gestational Cd Exposure in the CD-1 Mouse Sex-Specifically Disrupts Essential Metal Ion Homeostasis.

In CD-1 mice, gestational-only exposure to cadmium (Cd) causes female-specific hepatic insulin resistance, metabolic disruption, and obesity. To evaluate whether sex differences in uptake and changes in essential metal concentrations contribute to metabolic outcomes, placental and liver Cd and essential metal concentrations were quantified in male and female offspring perinatally exposed to 500 ppb CdCl2. Exposure resulted in increased maternal liver Cd+2 concentrations (364 µg/kg) similar to concentrations found in non-occupationally exposed human liver. At gestational day (GD) 18, placental Cd and manganese concentrations were significantly increased in exposed males and females, and zinc was significantly decreased in females. Placental efficiency was significantly decreased in GD18-exposed males. Increases in hepatic Cd concentrations and a transient prenatal increase in zinc were observed in exposed female liver. Fetal and adult liver iron concentrations were decreased in both sexes, and decreases in hepatic zinc, iron, and manganese were observed in exposed females. Analysis of GD18 placental and liver metallothionein mRNA expression revealed significant Cd-induced upregulation of placental metallothionein in both sexes, and a significant decrease in fetal hepatic metallothionein in exposed females. In placenta, expression of metal ion transporters responsible for metal ion uptake was increased in exposed females. In liver of exposed adult female offspring, expression of the divalent cation importer (Slc39a14/Zip14) decreased, whereas expression of the primary exporter (Slc30a10/ZnT10) increased. These findings demonstrate that Cd can preferentially cross the female placenta, accumulate in the liver, and cause lifelong dysregulation of metal ion concentrations associated with metabolic disruption.

Induction of autophagy by zinc during acute ethanol intoxication in mice

Recently, autophagy has emerged as an important player in the defense against ethanol toxicity. The potential role of zinc in the modulation of autophagy during ethanol stress in humans and animal models has not been examined previously. In this study, the effect of zinc deficiency, adequacy and excess on hepatic autophagic flux, liver injury, lipids accumulation and metallothionein expression was examined in C57BL/6 mice acutely exposed to ethanol. Mice were fed diets containing three different levels of zinc: low, adequate and excess for 30 days. After this, mice were given ethanol or water at a total accumulative dosage of 4.5‐g/kg body weight. Mice were sacrificed at 24h after the first gavage. Hepatic autophagic flux was estimated by measuring LC3II/LC3I ratio and Beclin1, p62/ SQSTM1 and Lamp2 levels. The dietary treatments did not affect food intake and body weight. Low zinc intake caused inhibition of autophagic flux in mice exposed or not to ethanol. In addition, zinc deficient mice exhibited increased liver injury and accumulation of lipids in liver. Conversely, high zinc intake was associated with increased autophagic flux and decreased accumulation of lipids in liver in mice exposed to ethanol. Lastly, ethanol consumption increased the expression of metallothionein mRNA in liver. The results of this study suggest that adequate zinc intake is critical to maintain autophagy. The protective effects of zinc against ethanol toxicity could be mediated by autophagy.Supported by NIH 1R03AA022451‐01

Influence of dietary methionine level on the liver metallothionein mRNA level in rats.

The effects of some methyl-containing compounds added to a choline-deficient diet on the metallothionein mRNA level in the rat liver were studied. The addition of choline or carnitine to the choline-deficient diet did not induce a gain in body weight, while the addition of either betaine or methionine to the choline-deficient diet, or of methionine to the choline-deficient diet with choline significantly increased the body weight. The metallothionein mRNA level in the liver of rats fed on the choline-deficient diet was similar to that of rats fed on the choline-deficient diet with choline, betaine or carnitine. However, the addition of methionine to the choline-deficient diet with or without choline caused a marked suppression in the metallothionein mRNA level in the liver. It is thus surmised that the metallothionein mRNA level in the liver might be regulated by the dietary content of methionine.

Expression of metallothionein in the liver and kidney of rats is influenced by excess dietary histidine

It is well known that excess dietary histidine induces the metabolic changes in copper and zinc. Therefore, this study was carried out to clarify whether excess dietary histidine alters the gene expressions of metallothionein-1 and metallothionein-2 in the liver and kidney. Male rats were fed the control (ad libitum and pair-fed) or histidine-excess (50 g of l-histidine per kg of diet) diet for 0, 1 and 3 days. The levels of liver metallothionein-1 and metallothionein-2 mRNA were markedly lower in the rats fed the histidine-excess diet as compared to those of the control (ad libitum and pair-fed) diet, when fed for 1 or 3 days. The levels of renal metallothionein-1 and metallothionein-2 mRNA in the rats fed the histidine-excess diet were higher or tended to be higher as compared with the rats fed the control (ad libitum and pair-fed) diet when fed for 1 or 3 days, respectively. At the same time, hepatic copper content was decreased and renal zinc content was increased by dietary histidine. It thus appears, that such a response on the level of liver metallothionein mRNA might be related to the contents of liver copper, but of kidney metallothionein mRNA might be due to the content of zinc.

Arctic char (Salvelinus alpinus) metallothionein: cDNA sequence, expression, and tissue‐specific inhibition of cadmium‐mediated metallothionein induction by 17β‐estradiol, 4‐OH‐PCB 30, and PCB 104

AbstractIn this study, we haveexamined the basallevel expression and tissue‐specificexpressionpatternsof metallothionein (MT) in Arctic char following metal and E2 (17β‐estradiol) treatment. To study the gene regulation in Arctic char, the two MT isoforms were isolated from a lambda‐ZAP hepatic cDNA library and characterized. Determination of basal MT mRNA and MT expression for 10 different tissues revealed a lack of correlation between MT mRNA and MT levels. The inducibility of MT mRNA and the correlation to resulting MT levels were then determined for liver and kidney. We found a more rapid and stronger induction of MT mRNA in liver than in kidney at day 1 and 3 postinjection, whereas the MT protein quantification showed higher MT levels in kidney than in liver at days 3 and 7 postinjection. These discrepancies indicate that differences in metal handling or posttran‐scriptional regulation of MT exists between tissues. Whereas metals induce MT synthesis, E2 inhibit the hepatic MT expression. To examine the tissue specificity of this inhibition, we determined the effect of 17pj‐estradiol (E2) and two estrogenic PCBs (4′‐OH‐PCB 30 and PCB 104) on Cd‐mediated MT induction in liver and kidney. Although E2 and the estrogenic PCBs inhibited cadmium‐mediated hepatic MT induction, these compounds did not interfere with renal MT induction.

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.

Subcutaneous NaAs 3+ exposure increases metallothionein mRNA and protein expression in juvenile winter flounder

We investigated the effect of NaAs 3+ exposure on liver metallothionein (MT) mRNA and protein expression in juvenile winter flounder ( Pleuronectes americanus). Sexually immature flounder (age 18 months) were subcutaneously implanted with silastic tubing containing 0 (vector control), 1.5, 3.0, 6.0 and 16.0 μmol g −1 fish wt. NaAs 3+. Liver MT mRNA and protein were significantly elevated within 24 h of exposure at the 1.5 μmol g −1 dose before there was significant liver As accumulation or mortality. All four NaAs 3+ treatments caused a significant rise in MT mRNA and protein and the mRNA demonstrated the clearest dose response and greatest fold change (19 fold at 16.0 μmol g −1 dose). MT protein reached its maximum (1.8 times the control) at the 1.5 μmol g −1 dose. This maximum was maintained across all four treatments and decreased slightly at the 16.0 μmol g −1 dose where there was 40% mortality. The level of liver As content among fish with maximal MT expression was similar to that of fish which recently succumbed to NaAs 3+ toxicity. This links saturation of the MT response with mortality and provides a framework for assessing the significance of MT measurements in feral flounder. The experiment was repeated (minus the 16.0 μmol g −1 dose) in a smaller (and younger) group of fish (12 months old) which expressed a two-fold lower liver Zn and MT protein content. This relatively low level of pre-existing MT theoretically widened the MT response window such that the MT maximum was not attained until the 6.0 μmol g −1 dose. These fish also did not show a significant rise in either MT mRNA or protein at the sublethal dose (1.5 μmol g -1). This lack of change appeared to reflect the higher level of baseline liver As which also did not increase at the 1.5 μmol g −1 dose. Reevaluation of the earlier experiment revealed a 1.5 fold increase in liver As content at the 1.5 μmol g −1 dose that was not significant due to high variance. This suggests that MT is a better biomarker of NaAs 3+ exposure because there is less variance associated with either MT mRNA or protein measurements. These results lend merit to MTOs usefulness as a biomarker of metal exposure and provides the foundation for linking NaAs 3+ exposure with MT expression and possibly metal-induced carcinogenesis in fish.

Differential effect of adrenalectomy on rat liver metallothionein mRNA levels in basal and stress conditions.

Liver metallothionein (MT) mRNA and serum MT levels of adrenalectomized (ADX) and sham-ADX rats in basal and stress (1, 3 or 6 h of restraint) conditions have been measured. Serum MT levels were overall lower in ADX than in sham-ADX rats. Basal liver MT mRNA levels were increased in ADX rats, suggesting that glucocorticoids have an inhibitory role on the regulation of liver MT synthesis. In contrast, liver MT mRNA levels were increased by stress in sham-ADX but not in ADX rats, suggesting a stimulatory role for glucocorticoids. These results suggest that glucocorticoids have a different role in liver MT regulation depending on the physiological situation.

Exposure to different forms of cadmium in mice: Differences in metallothionein and alphafetoprotein mRNA induction in liver and kidney

Cadmium (Cd) exposure in mice induces transcription of metallothionein (MT) mRNA and protein accumulation in both liver and kidney. Resistance to hepatotoxicity through chronic exposure to heavy metals is the result of this induction. However, the same chronic exposure results in damage to kidney. We report here that acute exposure of mice to Cd as cadmium sulfate (CdSO4), which resulted in preferential accumulation of metal in liver, or Cd-metallothionein (CdMT), which resulted in preferential metal accumulation in kidney, induced MT mRNA accumulation in both liver and kidney. However, MT mRNA accumulated to a level twofold higher in liver than in kidney in response to CdSO4. Equivalent doses of CdMT induced MT mRNA accumulation to an equal degree in kidney and liver. While MT mRNA accumulation in kidney was directly proportional to the amount of cadmium in the organ, this was not the case in liver. There, liver MT mRNA was elevated in the absence of elevated tissue cadmium levels. Interestingly, CdMT induced alphafetal protein (AFP) mRNA accumulation in kidney, but not liver. It appears that (a) maximal MT mRNA accumulation in kidney is less than in liver, and (b) liver is capable of accumulating MT mRNA in response to even very low cadmium exposure that may not result in elevated tissue cadmium.

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.

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.

Levels of Metallothionein Messenger RNA in Foetal, Neonatal and Maternal Rat Liver

We have used translation in vitro of hepatic polyadenylated RNA to characterize the levels of metallothionein mRNA in foetal, neonatal, pregnant and nulliparous rats. The translation products of foetal hepatic metallothionein mRNA increased relative to other mRNA translation products from day 18 of gestation to birth and attained a maximum, maintained throughout suckling, which is tenfold above 17-day foetal hepatic levels and fourfold above adult levels. Maternal liver metallothionein mRNA decreased fivefold between 17 days and 20 days of gestation, rose sharply immediately before birth, and was low throughout lactation.

Induction of rat liver metallothionein mRNA and its distribution between free and membrane-bound polyribosomes.

Total, membrane-bound and free polyribosomes were purified from livers of Zn2+-treated and control rats. Polyadenylated RNA was separated from the polyribosomal RNA extracts by oligo(dT)--cellulose chromatography and translated in a wheat-germ cell-free translation system. Newly synthesized 35S-labelled metallothionein was isolated from the other [35S]methionine-labelled translation products by activated-thiol--Sepharose 4B chromatography. The purity of the 35S-labelled metallothionein product was substantiated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Zinc administration resulted in an elevation of metallothionein mRNA activity to 11% of the total polyribosomal mRNA activity. The vast majority of biologically active metallothionein mRNA was localized in the free polyribosomal pool, at least 94% and 97% in control and zinc-treated rats respectively. The increase in the percentage of polyribosomal mRNA coding for metallothionein after zinc administration was 3-fold, whether measured directly in total polyribosomal mRNA or as a combination derived from membrane-bound and free polyribosomal mRNA. These data indicate that the induction of metallothionein mRNA by zinc involves only free polyribosomes and suggest that the function of metallothionein is limited to intracellular processes.