Effect and possible role of Zn treatment in LEC rats,an animal model of Wilson's disease

The effect of oral zinc (Zn) treatment was studied in the liver, kidneys and intestine of Long-Evans Cinnamon (LEC) rats in relation to metals interaction and concentration of metallothionein (MT) and glutathione (GSH). We also investigated the change in the activity of antioxidant enzymes and determined the biochemical profile in the blood and metal levels in urine. We showed that the Zn-treated group had higher levels of MT in the hepatic and intestinal cells compared to both untreated and basal groups. Tissue Zn concentrations were significantly higher in the Zn-treated group compared to those untreated and basal, whereas Cu and Fe concentrations decreased. The antioxidant enzyme activities in the Zn-treated group did not change significantly with respect to those in the basal group, except for hepatic glutathione peroxidase activity. Moreover, the biochemical data in the blood of Zn-treated group clearly ascertain no liver damage. These observations suggest an important role for Zn in relation not only to its ability to compete with other metals at the level of absorption in the gastrointestinal tract producing a decrease in the hepatic and renal Cu and Fe deposits, but also to MT induction as free radical scavenger.     

Effect and possible role of Zn treatment in LEC rats,an animal model of Wilson's disease

The effect of oral zinc (Zn) treatment was studied in the liver, kidneys and intestine of Long–Evans Cinnamon (LEC) rats in relation to metals interaction and concentration of metallothionein (MT) and glutathione (GSH). We also investigated the change in the activity of antioxidant enzymes and determined the biochemical profile in the blood and metal levels in urine. We showed that the Zn-treated group had higher levels of MT in the hepatic and intestinal cells compared to both untreated and basal groups. Tissue Zn concentrations were significantly higher in the Zn-treated group compared to those untreated and basal, whereas Cu and Fe concentrations decreased. The antioxidant enzyme activities in the Zn-treated group did not change significantly with respect to those in the basal group, except for hepatic glutathione peroxidase activity. Moreover, the biochemical data in the blood of Zn-treated group clearly ascertain no liver damage. These observations suggest an important role for Zn in relation not only to its ability to compete with other metals at the level of absorption in the gastrointestinal tract producing a decrease in the hepatic and renal Cu and Fe deposits, but also to MT induction as free radical scavenger.     

Changes in plasma zinc,copper, iron,and hepatic metallothionein in adjuvant-induced arthritis treated with cyclosporin

The early changes in hepatic metallothionein (MT) and plasma zinc (Zn), copper (Cu), and iron (Fe) were investigated during the induction of adjuvant (AJ) arthritis in rats in conjunction with cyclosporin (CSA) treatment. Plasma Zn decreased after AJ injection (60% of control values at 8 h), and this was associated with a 4.5-fold increase in hepatic MT at 8 h. Plasma Zn was lowest at 16 h (40% of control), whereas hepatic MT concentrations increased to a maximum of 20-fold at 16 h. Changes in plasma Fe paralleled those of Zn, whereas plasma Cu levels were increased. Plasma metal and hepatic MT concentrations returned toward normal from d 1–7. At d 14, when marked paw swelling was apparent, hepatic MT and plasma Cu were again increased and plasma Zn decreased. Administration of CsA decreased MT induction in rats injected with AJ and also caused a marked recovery in plasma Zn and Fe levels. These changes were small but significant even in the early stages (up to 24 h) after AJ injection and were followed by a sustained improvement in all parameters, corresponding to the nonappearance of clinical arthropathy in CsA-treated rats. TNF-α and IL-6 production by peritoneal macrophages isolated from AJ-injected rats was significantly decreased by CsA treatment at d 7 and 14. The inhibition of hepatic MT induction during acute and chronic inflammation by cyclosporin emphasizes the role of the immune system in altered metal homeostasis in inflammation.     

Interactions between Zn and Cu in LEC rats, an animal model of Wilson's disease

The effect of oral Zn treatment was studied in the liver and kidneys of 26 male Long-Evans Cinnamon (LEC) rats (mutant animals, 5 weeks old) in relation to both the interaction between Zn and Cu and the localisation and concentration of metallothionein (MT). Rats receiving 80 mg zinc acetate daily by gavage and control rats receiving no treatment were killed after 1 or 2 weeks. By immunohistochemical and analytical chemical techniques we revealed that treated rats had higher levels of MT in the hepatic and renal cells compared to untreated ones. Tissue Zn concentrations were significantly higher in treated rats compared to untreated whereas Cu concentrations decreased in the liver and kidneys as indicated by analytical chemical analyses. MT levels also decreased with treatment period. A histochemical procedure, obtained using autofluorescence of Cu-metallothioneins, confirms these findings: after 2 weeks, the signal decreased in both the liver and kidney sections. This gives a greater understanding of the mechanism of Cu metabolism in the two tissues considered. These results suggest that Zn acts both to compete for absorption on the luminal side of the intestinal epithelium and to induce the synthesis of MT.     

Enhanced lipid peroxidation is not necessary for induction of metallothionein-I by oxidative stress

A study has been made of factors which may influence the induction of metallothionein-I (MT-I) synthesis by the superoxide radical generating agent, paraquat (PQ). Hepatic concentrations of zinc (Zn) and MT-I increased in rats injected with PQ (40 mg/kg, s.c.) or fasting, but were greater in the former. Renal concentration of MT-I increased in fasted rats but not in PQ-treated rats. The data suggest that the increase in MT-I concentrations in PQ-treated rats is not caused by reduction in food intake. Administration of PQ increased hepatic concentrations of Zn, MT-I and thiobarbituric acid-reactive substances (TBA-RS), indicating the occurrence of lipid peroxidation. Treatment of rats with vitamin E (400 mg/kg, s.c.) on 4 successive days before injection of PQ prevented only the enhancement of lipid peroxidation. The data indicate that the induction of MT synthesis by PQ is not correlated with enhancement of lipid peroxidation. Similar results were obtained in the liver of rats subjected to the radical-generating conditions, such as fasting and exposure to carbon tetrachloride. Free radicals may induce MT synthesis by direct or indirect mechanisms.     

Impaired hepatic regeneration in metallothionein-I/II knockout mice after partial hepatectomy

Although the translocation of metallothionein (MT) from cytoplasm to nucleus has been demonstrated in liver during times of high requirement for zinc (fetal development and the neonatal period), the role of MT in cellular growth is not well understood. In this study, a potential role of MT in liver regeneration was investigated in wild type (WT) and MT-I and MT-II gene knockout (MT-null) mice after 35% partial hepatectomy (PH) or sham laparotomy. Hepatic MT levels and proliferation index were measured at 0, 5, 15, 24, 36, 48, and 60 hrs after PH and 48 hrs after sham laparotomy (control). MT levels were increased in WT mice (peak at 24 hrs after PH) and declined to normal levels by 60 hrs after PH. Immunohistochemical staining for MT in WT mice indicated the presence of MT in both nucleus and cytoplasm of hepatocytes at 24 hrs after PH, whereas MT was present mainly in the cytoplasm at 36-60 hrs after PH and 48 hrs after sham laparotomy. Hepatic proliferation index in both WT and MT-null mice, as determined by argyrophilic nucleolar organizing region staining and proliferating cell nuclear antigen immunohistochemical staining, reached a peak at 48 hrs and declined by 60 hrs after PH. Cell proliferation was significantly less in MT-null mice as compared to WT mice during liver regeneration after PH. These results suggest that MT may play a positive role in hepatic regeneration after PH.     

Interaction of metallothionein and carbon tetrachloride on the protective effect of zinc on hepatotoxicity

To study the influence of hepatic metallothionein (MT) on the hepatotoxic response to carbon tetrachloride (CCl4), adult male rats were pretreated with a 10 mg X kg-1 dose of zinc (Zn) 24 h prior to CCl4 (i.p., l mL X kg-1) treatment. Zn pretreatment increased the hepatic MT concentrations markedly and reduced the magnitudes of the CCl4-induced reduction of cytochrome P450 concentration as well as elevation of serum alanine aminotransferase and aspartate aminotransferase activities when determined at 4 or 24 h following CCl4 treatment. Treatment of Zn-exposed animals with CCl4 also resulted in significant reduction of the concentrations of hepatic MT (as determined by the cadmium-saturation method) as well as cytosolic Zn. Sephadex G-75 chromatographic study of hepatic cytosols showed that MT-bound Zn was selectively depleted by CCl4 exposure. Moreover, it was demonstrated that CCl4, after metabolic activation, reduced the cadmium binding capacity of Zn-induced hepatic MT in vitro. To examine the possible protective effect of Zn independent of induction of MT synthesis, CCl4 was administered 2 h following Zn pretreatment and the hepatotoxic response was examined 4 h later. This study revealed limited protection by Zn prior to the induction of MT synthesis. These data further support a role of MT in the modulation of CCl4 hepatotoxicity.     

Effect of zinc supplementation on metallothionein,copper, and zinc concentration in various tissues of copper-loaded rats

The present study was designed to investigate the effects of Zn administration on metallothionein concentrations in the liver, kidney, and intestine of copper-loaded rats. Male CD rats were fed a diet containing 12 mg Cu and 67 mg Zn/kg body wt. They were divided into either acute or chronic experimental protocols. Rats undergoing acute experiments received daily ip injections of either Cu (3 mg/kg body wt) or Zn (10 mg/kg body wt) for 3 d. Chronic experiments were carried out on rats receiving Cu ip injections on d 1, 2, 3, 10, 17, and 24, Cu injections plus a Zn-supplemented diet containing 5 g Zn/kg solid diet, or a Zn-supplemented diet alone. Rats injected Zn or Cu had increased MT concentrations in liver and kidney. Zn produced the most important effects and the liver was the most responsive organ. Rats fed a Zn-supplemented diet had significantly higher MT concentrations in liver and intestine with respect to controls. Increased MT synthesis in the liver may contribute to copper detoxification; the hypothesis of copper entrapment in enterocytes cannot be confirmed.     

In Vitro Radiation Induced Alterations in Heavy Metals and Metallothionein content in Plantago ovata Forsk

Proton Induced X-ray emission (PIXE) and fluorescence-activated cell sorting (FACS) have been used to study the effects of gamma irradiation on heavy metal accumulation in callus tissue of Plantago ovata-an important cash crop of India. PIXE analysis revealed radiation-induced alteration in trace element profile during developmental stages of the callus of P. ovata. Subsequent experiments showed antagonism between Fe and Cu and also Cu and Zn and synergistic effect between Fe and Zn. FACS analysis showed significant induction of the metallothionein (MT) protein following gamma-irradiation, and maximum induction was noted at the 50-Gy absorbed dose. This indicated a progressive increment of MTs as a measure for protection against gamma-rays, to combat alteration in the homeostasis of heavy metals like Fe, Cu, Zn, and Mn.     

Copper-metallothionein induction in the liver of LEC rats.

Recently, copper (Cu) was found to be unusually accumulated, suggesting the induction of metallothionein (MT) in the liver of LEC rats (Long-Evans rats with a cinnamon-like coat color), which develop spontaneous jaundice with hereditary hepatitis. Thus, the direct relationship between the unusual Cu accumulation and the induction of Cu-MT was investigated by giving LEC rats Cu-overloaded or Cu-deficient diets. Results based on the determinations of Cu and MT levels in several organs, as well as the gel-filtration profiles of the cytosols of liver homogenates, showed that dietary Cu induced Cu-MT and development of hepatic injury associated with jaundice.     

Zinc supplementation decreases hepatic copper accumulation in LEC rat

The effect of dietary zinc (Zn) supplementation on copper (Cu)-induced liver damage was investigated in Long-Evans Cinnamon rats (LEC), a model for Wilson's disease (WD). Four-week-old LEC (N=64) and control Long-Evans (LE) (N=32) female rats were divided into two groups; one group was fed with a Zn-supplemented diet (group I) and the other was given a normal rodent diet (group II). LEC rats were killed at 6, 8, 10, 12, 18, and 20 wk of age; the LE control rats were killed at 6, 12, 18, and 20 wk of age. Cu concentration in the liver was reduced in LEC rats fed the Zn-supplemented diet compared with LEC rats on the normal diet between 6 and 18 wk of age. Metallothionein (MT) concentration in the livers of LEC rats in group I increased between 12 and 20 wk of age, whereas hepatic MT concentration in LEC rats from group II decreased after 12 wk. Hepatocyte apoptosis, as determined by TUNEL, was reduced in Zn-supplemented LEC rats at all ages. Cholangiocellular carcinoma was observed only in LEC rats in group II at wk 20. These results suggest that Zn supplementation can reduce hepatic Cu concentration and delay the onset of clinical and pathological changes of Cu toxicity in LEC rats. Although the actual mechanism of protection is unknown, it could be explained by sequestration of dietary Cu by intestinal MT, induced by high dietary Zn content.     

The survival of mice and the metallothionein content of their livers and kidneys as criteria for assessing exposure to pulsed neutron radiation

The effect of pulsed neutron radiation was studied in comparison with continuous neutron radiation and continuous gamma-radiation. Animal survival and induction of metallothionein (MT) synthesis in liver and kidney of mice exposed to equivalent doses were chosen as criteria for evaluation of radiation effects. It was found that the level of MT in liver and kidney of mice exposed to neutron radiation decreased 24 hours after irradiation and then continued decreasing in kidney for 48 hours after irradiation. This is evidence of more intensive free-radical processes initiated by pulsed neutron radiation. At the same time, RBE values of pulsed neutrons did not differ significantly from that of continuous neutron radiation.     

Molecular characterization of two metallothionein isoforms in avian species: evolutionary history, tissue distribution profile, and expression associated with metal accumulation

To characterize avian MTs, MT cDNAs were cloned from liver of cormorant (Phalacrocorax carbo) and mallard (Anas platyrhynchos). Expression profiles of MT isoforms and relationships between metal accumulation and MT mRNA expression in tissues were also investigated. We succeeded in cDNA cloning of MT1/2 from cormorant and MT1 in mallard. DNA sequence of chicken MT1 was obtained from chicken (Gallus gallus) genomic database. Considering previous reports on avian MTs, birds possess at least two distinct MT isoforms. Comparison of genomic synteny among vertebrates and phylogenetic analysis of MT amino acid sequences revealed that avian MT1/2 are evolutionarily close to mammalian MT3. Messenger RNAs of both MT isoforms were detected in all the tissues/organs in cormorant and mallard. Liver was the primary organ for cormorant MT1/2, and mallard MT2, whereas MT1 was dominant in mallard heart. Interspecies comparison of tissue distribution of MT mRNA expression between cormorant and mallard indicated that MT2 profile was similar, but MT1 was not. Significant positive correlations of mRNA expression levels between MT1 and MT2 were observed in the liver and kidney of cormorants, whereas no correlation was found in mallards. Expression levels of cormorant MT1/2 showed significant positive correlations with hepatic Cu and Zn concentrations, suggesting that both MT isoforms were induced by Cu and Zn in livers. Cormorant MT2 expression level exhibited a significant positive correlation with hepatic Ag, and a negative correlation with Rb, indicating that Ag and Rb concentrations depend on the expression of MT2 by Cu and Zn. In mallard, MT1 had no correlation with any metal concentration, and MT2 expression was positively correlated only with Cu, even though hepatic Cu and Zn concentrations in mallard were much higher than in cormorant. This may indicate that cormorant is a more susceptible species than mallard in terms of MT induction. These findings suggest tissue-, species-, and MT isoform-specific responses to metal stresses in these aquatic birds.     

Effects of Chronic Cadmium Poisoning on Zn, Cu, Fe, Ca, and Metallothionein in Liver and Kidney of Rats

An experiment was conducted to invest effects of chronic cadmium poisoning on Zn, Cu, Fe, Ca, and metallothionein gene expression and protein synthesis in liver and kidney in rats. Forty rats, 6?weeks old, were randomly allocated into two groups. A group was given CdCl(2) (1?mg/KgCd(2+)) by intraperitoneal injection once a day. The other group was treated with normal saline in the same way. Liver and kidney were collected for analysis at the end of the third week. Results showed that Cd exposure increased Cd (P?相似文献   

Involvement of Oxidative Stress in Paraquat-Induced Metallothionein Synthesis Under Glutathione Depletion

The inhibition of glutathione (GSH) synthesis by -buthionine-SR-sulfoximine (BSO) causes aggravation of hepatotoxicity of paraquat (PQ), an oxidative-stress inducing substance, in mice. On the other hand, synthesis of metallothionein (MT), a cysteine-rich protein having radical scavenging activity, is induced by PQ, and the induction by PQ is significantly enhanced by pretreatment of mice with BSO. The purpose of present study is to examine whether generation of reactive oxygens is involved in the induction of MT synthesis by PQ under inhibition of GSH synthesis. Administration of PQ to BSO-pretreated mice increased hepatic lipid peroxidation and frequency of DNA single strand breakage followed by manifestation of the liver injury and induction of MT synthesis. Both vitamin E and deferoxamine prevented MT induction as well as lipid peroxidation in the liver of mice caused by administration of BSO and PQ. In cultured colon 26 cells, both cytotoxicity and the increase in MT mRNA level caused by PQ were significantly enhanced by pretreatment with BSO. Facilitation of PQ-induced reactive oxygen generation was also observed by BSO treatment. These results suggest that reactive oxygens generated by PQ under inhibition of GSH synthesis may stimulate MT synthesis. GSH depletion markedly increased reactive oxygen generation induced by PQ, probably due to the reduced cellular capability to remove the radical species produced.     

Effect of surgically induced cholestasis on the levels of hepatic zinc and metallothionein in rat liver

Early effects of experimental cholestasis on the homeostasis of zinc (Zn) and metallothionein (MT) were studied in rats which had undergone bile duct ligation for 0, 3, 6, 9, 12, 16, 20, and 24 h. Transient increases in hepatic Zn levels were observed at 9 h but returned to control values at 12 h. Serum Zn levels increased at 24 h. Cholestasis was confirmed by increased serum alkaline phosphatase (AP) activity. MT increased at 3 h and reached a maximum level at 12 h and remained elevated even at 24 h after the onset of experimental cholestasis. No hepatocellular damage was detected according to the results of alanine aminotransferase (ALT) activities in serum. These results shown that the increases in Zn observed in liver are related to bile stagnation rather than to a hepatocellular damage and that increased MT occurs concurrently with increased hepatic Zn. These observations suggest that the cellular levels of Zn in cholestasis is regulated by homeostatic mechanisms, of which one could be mediated by MT.     

The influence of restraint stress in rats on metallothionein production and corticosterone and glucagon secretion

Earlier studies on metallothionein (MT) induction by stress used a wide range of stimuli, all of them considered as physical stressors. The present paper reports the effect of a basically psychogenic stress such as restraint on serum and liver MT. Male adult rats were stressed for 1, 12, 24 and 48 hours and then killed. Liver MT increased continuously throughout the experimental period. Rats deprived of water and food for 24 and 48 hours showed higher liver MT levels than control but lower levels than stressed ones. Serum MT was not modified by either restraint or water and food deprivation. The lack of relationship between the two MT pools was corroborated by the absence of a significant correlation between them. Restraint stress increased serum corticosterone but not glucagon levels, suggesting that only glucocorticoids are important in MT induction by stress. However, a strong circadian rhythmicity was observed in serum corticosterone but not in serum or liver MT in non-stressed rats. In addition, preliminary data with adrenalectomized rats indicate that glucocorticoids seem not to be essential in MT induction by stress. Likewise glucagon does not appears to be associated with MT induction by stress since its levels were not modified by restraint.     

The relative importance of glutathione and metallothionein on protection of hepatotoxicity of menadione in rats.

The effects of induction of metallothionein (MT) on the toxicity of menadione were investigated in rat liver slices. The protective role of hepatic glutathione (GSH) was also studied and compared to that of MT. A 3-h incubation of rat liver slices with menadione (100-300 microM) containing medium (37 degrees C, pH 7.4, 95%O2:5%CO2) resulted in cellular toxicity, as shown by changes in cytosolic K, Ca and GSH concentrations and lactate dehydrogenase (LDH) leakage. A dose-dependent decrease in cytosolic K and GSH was observed concomitant with an increase in cytosolic Ca and LDH leakage after incubation with menadione. Pretreatment of rats with zinc sulphate (ZnSO4) (30 mg/kg body wt.) increased MT levels in liver slices and suppressed the toxicity of menadione. Intracellular GSH concentrations in liver slices were either depleted or increased by injection of rats with buthionine sulfoximine (BSO), (4 mmol/kg body wt.) and N-acetyl-L-cysteine (NAC) (1.6 g/kg body wt.), respectively. Intracellular GSH was found to be crucial in protection against menadione toxicity. Menadione toxicity was increased when the rats were injected with sodium phenobarbital (PB) (4 x 80 mg/kg body wt.). Pretreatment with Zn provided partial protection against menadione toxicity in liver slices from both BSO- and PB-injected rats. These findings suggest that induction of MT synthesis does protect against quinone-induced toxicity, but the role may be secondary to that of GSH. The mechanisms by which MT protect against menadione toxicity are still unclear but may involve protection of both redox cycling and sulphydryl arylation.     

On metallothionein,cadmium, copper and zinc relationships in the liver and kidney of adult rats

1. A short-term exposure of adult Wistar rats to Cu (50 μg/ml) and Cd (10.0 μg/ml drinking water) caused significant changes in the subcellular concentrations of Cd, Cu, Zn and metallothionein (MT) in the liver and kidney; the concentrations were close to the physiological values, however.2. To establish a relationship between these changes in the subcellular concentrations of Cd, Cu, Zn and the level of MT in the post-mitochondrial fraction of the liver and kidney, the analytical data (N = 42) were subjected to the multiple regression analysis.3. The analysis showed that MT synthesis in the liver was principally induced by small amounts of Cd (0.32–1.4 μg/g wet wt) whereas in the kidney a level of MT in the post-mitochondrial fraction correlated positively with the renal Cd and Cu, as well as with the level of this protein in the liver.4. The above results together with the positive correlation between the level of MT in the post-mitochondrial fraction and the concentration of Cu in this fraction, as well as the fact that under normal physiological conditions the capacity of MT (β-domain) in the liver and kidney was sufficient to bind 50–100% of the total post-mitochondrial Cu suggest that MT, first induced by small amounts of Cd, may be involved in the metabolism of Cu.