Hepatic Metallothionein Concentrations Research Articles

Assessment of Zinc Status

Plasma zinc concentration has been denigrated as a measure of zinc status because it responds to metabolic conditions unrelated to zinc status and because it is insensitive to changes in dietary zinc. The insensitivity of plasma zinc to reductions in dietary zinc reflects the tremendous capacity of the organism to conserve tissue zinc by reductions in zinc excretion and/or reductions in the rate of growth. Changes in plasma zinc concentrations do not seem to occur until the capacity to reestablish homeostasis by reducing excretion and/or growth has been exceeded. Reductions in dietary zinc beyond the capacity to maintain homeostasis lead to utilization of zinc from a small, rapidly turning over pool. This pool is located, at least in part, in the bone, liver, and plasma. Loss of a small, critical amount of zinc from this pool leads to the rapid onset of both biochemical and clinical signs of zinc deficiency. Thus, plasma zinc is a valid, useful indicator of the size of this exchangeable pool of zinc. Plasma metallothionein concentrations may prove useful for identifying poor zinc status. Plasma metallothionine concentrations reflect hepatic concentrations and, therefore, are reduced when the dietary zinc supply is low. However, since hepatic metallothionein concentrations rise in response to stress, plasma metallothionein concentrations are increased in this state when the plasma zinc concentrations are also likely to be low. Thus, measurement of both plasma metallothionein and plasma zinc concentrations could differentiate a low plasma zinc due to dietary zinc deficiency from a low concentration due to stress, infection, or other metabolic conditions.

Hepatic Glutathione, Metallothionein and Zinc in the Rat on Gestational Day 19 during Chronic Ethanol Administration

Ethanol, under certain conditions, alters the metabolism of sulfur amino acids, metallothionein (MT) and zinc. If chronic ethanol administration during pregnancy decreases the availability of sulfur amino acids or Zn, this deficiency could contribute to growth retardation of the fetuses, one of the features of fetal alcohol syndrome. The purpose of this study was to discern whether chronic ethanol administration to pregnant rats alters glutathione (GSH), MT or Zn content of selected tissues of the dams and fetuses. Sprague-Dawley rats were fed from gestational days 5 to 19 either the control diet ad libitum (AF), the ethanol diet ad libitum (EF) or the control diet using the pair-feeding technique (PF). On the 19th day of gestation, total hepatic GSH was significantly lower for the EF and PF dams than for the AF dams. Hepatic MT contents were similar for the AF and EF dams, and hepatic MT content was significantly greater for the PF dams than the AF and EF dams. The three groups did not differ regarding hepatic Zn content of dams or fetuses. In summary, on the 19th day of gestation, chronic ethanol feeding of pregnant rats did not lower the maternal hepatic GSH level below that of PF dams, did not induce hepatic MT in the dams and did not prevent fetuses from achieving body weights and hepatic Zn concentrations equal to those of controls.

Investigation into the combined effects of ethanol and cadmium on rat liver and kidneys

To examine the combined hepatotoxic and nephrotoxic effects of cadmium and ethanol, rats maintained on an ethanol containing liquid diet (5% w/w) were given cadmium either acutely (3 x 1 mg/kg IP) or subacutely (about 14 mg/kg/day PO for 6 weeks). Parameters tested were cadmium, zinc and copper contents of blood and various organs, metallothionein (MT) contents, polysome profile of liver and kidneys, serum SDH and GPT levels and creatinine clearance. Ethanol reduced the hepatic MT contents without altering the polysome profile and the zinc and copper contents. Cadmium on the other hand raised the MT contents in liver and kidneys. This effect of cadmium predominated in the combined treatment. Morphological examination and functional tests (SDH, GPT, creatinine clearance) indicate that cadmium does not enhance the toxic effects of ethanol, and vice versa.

Effects of fasting on cadmium toxicity, glutathione metabolism, and metallothionein synthesis in rats

Acute oral toxicity of Cd (as cadmium chloride) was enhanced in rats fasted 24 hr, as shown by a markedly decreased LD50. To examine the relationship among Cd toxicity, hepatic glutathione (GSH), and metallothionein (MT) during fasting, rats were administered 75 mg Cd/kg orally 24 hr after fasting and euthanized after a further 4 or 24 hr for various assays. Serum glutamic-pyruvic transaminase activity 24 hr after Cd treatment was higher in fasted rats than in fed rats. Both total GSH and nonprotein sulfhydryl (NPSH) concentrations in liver decreased to 50% of control levels after 28 hr of fasting and returned to 75% of control values by 48 hr. Total hepatic GSH concentration in fed rats decreased 4 and 24 hr after Cd treatment, whereas that in fasted rats remained unchanged at 4 hr and decreased significantly at 24 hr. Cd uptake by the liver (both concentration and content) 24 hr after Cd treatment was higher in fasted rats than in fed rats. Hepatic MT concentration was markedly increased by Cd treatment and higher in fasted rats than in fed rats. There was no relationship between Cd toxicity and hepatic thiobarbituric acid (TBA) value, an indicator of lipid peroxidation. Fasting had no effect on hepatic GSH peroxidase and GSH reductase activities. These enzymes probably are not involved in Cd toxicity. On histological examination, focal degenerative and necrotic changes were observed from the midlobular to the pericentral region in the livers of fed rats 24 hr after Cd treatment. These changes were enhanced by fasting, diffusing from the pericentral to the periportal region. Histochemical examination revealed a heterogeneous distribution of GSH in the livers of fed rats, with strong staining of GSH in the periportal region. This heterogeneous distribution of GSH in liver was not observed in fed rats 4 hr after Cd treatment or in fasted rats at 24 hr. The present results suggest that hepatic GSH plays an important role in protection against Cd toxicity before the onset of MT synthesis. Animals in bad condition, such as that resulting from interruption of nutrient supply, cannot be protected against Cd toxicity even if the hepatic MT level is high.

Effect of methoxyethanol, cyclophosphamide and cadmium on metallothionein levels during prenatal development in the mouse

Three known teratogenic agents—methoxyethanol (ME), cyclophosphamide (CP) and cadmium (Cd)—are possible inducers of metallothionein (MT) in the embryo and/or in the fetus. Their effect on the MT levels of forelimbs, brain and liver during prenatal development and in dam's liver was studied in the mouse to elucidate whether MT could be used as an early biochemical indicator of teratogenicity. Pregnant mice were injected with 2 doses of each teratogen at different days of the middle gestational phase and their embryos and fetuses were obtained thereafter. Quantitative estimation of MT in the S9 from homogenates of the embryo/fetal tissues and organs and maternal liver showed major alterations in the dam's hepatic MT content but only small changes in prenatal MT levels. These results do not support MT as an early indicator of teratogenicity. However, a causal relationship between the maternal MT changes induced by the tested agents and their teratogenic effect could be possible.

Effect of sulfhydryl-deficient diets on hepatic metallothionein, glutathione, and adenosine 3′-phosphate 5′-phosphosulfate (PAPS) levels in rats

Low dietary concentrations of methionine and cysteine are known to decrease hepatic glutathione content. However, it is not known if restricting the dietary content of these sulfur containing amino acids also affects hepatic levels of adenosine 3′-phosphate 5′-phosphosulfate (PAPS), the cofactor for sulfation, or metallothionein, a protein rich in sulfhydryl groups. Rats were fed diets lacking cysteine and containing various concentrations of methionine (0.15, 0.3, or 0.6%) for 8 days. Control diet contained 0.3% each of methionine and cysteine. Hepatic glutathione levels were decreased approximately 75% in rats fed diets containing 0.15 or 0.3% methionine. In contrast, PAPS and hepatic metallothionein concentrations were not decreased by the low sulfhydryl diets. Additionally, rats on the various diets were challenged by the administration of ZnCl 2 (3 mmol/kg, sc). In both control rats and rats maintained on sulfhydryl-deficient diets, ZnCl 2 increased hepatic metallothionein to the same level. However, significantly lower levels of PAPS were observed after ZnCl 2 in rats receiving sulfhydryl-deficient diets than in controls. In summary, restriction of dietary sulfhydryl markedly decreases the hepatic content of glutathione and has a minor effect on PAPS concentration, but does not decrease the basal hepatic concentration of metallothionein or its induction by ZnCl 2.

Metallothionein as an indicator of heavy-metal exposure in two subtropical fish species

Induction of metallothionein (MT) and levels of cadmium, zinc and copper in liver of the subtropical fish blue-striped grunt Haemulon sciurus were studied after cadmium injection. Hepatic levels of MT, zinc and copper were also analysed in blue-striped grunt and tomtate H. aurolineatum caught at heavy-metal-polluted areas of Hamilton Harbour and Castle Harbour, Bermuda, respectively. In addition, a comparison of the hepatic content of MT, cadmium, zinc and copper was made between blue-striped grunt, tomtate and squirrelfish Holocentrus rufus, caught at unpolluted locations. Hepatic MT was assayed by a radioimmunoassay (RIA), using an antiserum raised against MT from perch Perca fluviatilis and partially purified MT from blue-striped grunt as standard. Hepatic MT was significantly ( P < 0.001) induced in a dose-dependent manner in response to a single i.p. injection of CdCl 2. The doses were 2200, 740, 250, 82, 27 and 0 μg Cd · kg −1 body wt, respectively. Significantly elevated hepatic MT levels ( P < 0.01) were found 2 days after injection of 2200 μg Cd · kg −1 body wt and maximum content of MT in liver was reached after 6 days. MT values in fish 6 days after treatment were increased five-fold compared to the control group. Significantly increased MT levels ( P < 0.05) were also found in livers of blue-striped grunt and tomtate caught at contaminated sites compared to control fish. The increased MT levels were in good accordance with the hepatic zinc and copper levels in these fish. The levels of MT, cadmium, zinc and copper in liver from blue-striped grunt and tomtate, caught at clean sites, were within the range found in other fish species, such as rainbow trout Oncorhynchus mykiss and P. fluviatilis. Squirrelfish, caught at an uncontaminated site, had exceptionally high levels of hepatic MT, zinc and copper.

Combined effects of methylmercury, lead, and cadmium on hepatic metallothionein and metal concentrations in the pekin duck

This study was initiated to determine the effects of combined treatments of methylmercuric chloride (MeHg), lead acetate (Pb) and cadmium chloride (Cd) administered orally on metallothionein (MT) and metal levels in the liver of the pekin duck (Anas platyrynchos). Methylmercury and Pb induced a two-fold increase in MT while Cd caused a seven-fold increase over controls. Concurrent administration of MeHg and Pb caused no increase in MT over single treatments. The hepatic MT content of the MeHg + Cd and MeHg + Pb + Cd groups was not different from the Cdalone group. Hepatic lead levels were lowest followed by Hg and Cd. Lead increased Hg levels when given simultaneously with MeHg, and further addition of Cd increased this effect. In groups given Pb, MeHg increased hepatic levels of this element, but when MeHg was also given with Cd, the combination produced a reduction in levels of Pb. Lead or MeHg had no effect on Cd levels. Methylmercury, Pb, and their combination elevated hepatic Cu over controls. All treatments significantly raised hepatic Zn levels above controls. Combinations of Cd with either MeHg or Pb lowered Zn levels below that caused by Cd alone. The simultaneous administration of all three metals did not produce Zn levels which were additive. The liver content of iron was not significantly altered.

Role of metallothionein in biliary metal excretion

We have studied the effect of acute induction of metallothionein on the biliary excretion of a bolus of metal in the rat. Female Sprague-Dawley rats were administered zinc chloride (6.5 mg/kg) or dexamethasone (2 mg/kg), ip in saline daily for 3 d; control animals received saline alone. Zinc causes a 17-fold induction in hepatic metallothionein levels, while dexamethasone caused a 5-fold induction. A bolus of metal chloride, either zinc, mercury, or cadmium, 1 mg/kg iv, was administered, and bile and plasma samples were collected and analyzed for metal content. At 3 h the rats were killed, livers excised, and both metallothionein and the metals associated with metallothionein estimated. Cadmium was excreted into the bile in inverse proportion to the hepatic metallothionein content, while metallothionein content did not appear to bear any relationship to biliary excretion of mercury or zinc. Metallothionein from rats pretreated with zinc contained twice as much zinc per molecule of metallothionein as that found in control rats. Cadmium, which exhibits a very high affinity for metallothionein, replaced this zinc found in association with metallothionein. Conversely, mercury only partially replaced the zinc associated with metallothionein, and a bolus of zinc was completely unable to bind to the already zinc-saturated metallothionein. Consequently, the bolus zinc was found associated with alternative cytosolic proteins, here termed the high-molecular-weight fraction. These findings support the hypothesis that the inhibitory effect of metallothionein on the biliary excretion of metals is dependent on the ability of the metal in question to replace any existing metals associated with metallothionein. Biliary metal excretion was directly proportional to the free metal content, regardless of the metal studied. Consequently, acute induction of metallothionein was inhibitory to biliary cadmium excretion, slightly inhibitory to biliary mercury excretion, and without effect on biliary zinc excretion.

Effect of Salmonella gallinarum Infection on Zinc Metabolism in Chicks

The effect of Salmonella gallinarum infection in chicks on serum, liver, and kidney zinc concentrations was studied. Within 48 h after intraperitoneal administration of the organism, serum zinc declined to approximately one-half the control value. In one experiment, the serum zinc concentration remained low for the 12 days of the experiment, whereas in a second experiment, the concentration gradually increased after 6 days postinoculation but never returned to the control value. Feeding as much as 500 ppm supplemental zinc did not prevent the infection-induced decline in the serum zinc concentration. The infection resulted in a sequestering of zinc in the liver; the kidney remained relatively unresponsive in this system. Fractionation of liver homogenates by gel filtration column chromatography revealed that the zinc in the livers of the infected animals eluted in a volume characteristic of metallothionein, whereas that of control animals was associated with high molecular weight proteins. Increasing the zinc content of the serum by repeated subcutaneous injections of zinc had no effect on mortality from this infection. Restricting feed consumption of uninfected chicks to that of infected animals did mimic the influence of infection of serum zinc and hepatic metallothionein concentrations.

Influences of compounds on metallothionein concentration in mouse tissues. IV. Increase of hepatic metallothionein concentration by drugs for hepatic diseases.

The concentration of metallothionein in the liver of mice injected with drugs for hepatic diseases was determined, and the following results were obtained. 1. The concentration of metallothionein in the liver was increased by the injection of glutathion, N-(2-mercaptopropionyl) glycine, protoporphyrin or Shosaikoto. 2. Among sulfur compounds, an injection of L-cysteine or L-cysteic acid increased the concentration of metallothionein in the mouse liver. 3. Metallothionein induced by N-(2-mercaptopropionyl) glycine or Shosaikoto was zincthionein. Acute cadmium toxicity was prevented by the pre-injection of these compounds. These results indicate that some drugs for hepatic diseases increase the level of metallothionein in the mouse liver.

Increased levels of hepatic metallothionein in rat and mouse after injection of acetaminophen

Induction of hepatic metallothionein (MT) by acetaminophen was characterized in the rat and mouse. Treatment of rats with the hepatotoxin resulted in increase of liver MT in a dose-dependent manner. MT concentration was elevated by 41%, 140% and 260% following acetaminophen injection at doses of 250, 500 and 1000 mg/kg, respectively. The cadmium-binding protein was identified as MT by Sephadex G-75 gel filtration ( V e/ V o = 2.1). In the mouse the hepatotoxin was more potent i.e. maximal effect (increase of 230%) was achieved at the lowest applied dose (250 mg/kg). In both species maximal induction was observed 24 h post exposure and thereafter the hepatic MT content declined, indicating a relatively short half-life of the protein. The elevation of the intracellular concentration of a sulfhydryl-rich protein such as MT may serve as self protecting mechanism of the hepatocyte against highly reactive metabolites of toxic substances.

Developmental and sex differences in cadmium distribution and metallothionein induction and localization.

Age- and sex-related differences in hepatic and renal distribution of cadmium (Cd) and the effect of Cd injection (10 mumol/kg) on tissue zinc (Zn), copper (Cu) and metallothionein (MT) levels were investigated in 2- to 84-day old rats. Renal Cd accumulation increased with age of the animal. Sex differences in renal Cd accumulation were noted in young animals where the 2- and 8-day old males had significantly greater concentration than the females. There were no clear effects of Cd on renal Zn. Renal Cu levels, however, were elevated in the adults. The adult females contained about twice as much MT as the adult males. Cd treatment had no effect on renal MT levels of 8- to 84-day old animals but depressed the levels in 2-day old. Age-related increase in hepatic Cd accumulation was also found; the pattern was more clear cut in females than in males. In addition, in the females the hepatic Cd concentration was significantly higher than in the males. Cd-injection significantly increased hepatic Zn and MT concentrations only in weaned animals. While there were no sex differences in MT levels in the young animals, the weaned females had significantly more MT than the corresponding males. Immunohistochemical staining for MT showed positive staining in both cytoplasm and nuclei of the parenchymal cells. The number of MT-positive nuclei was dependent on the relative MT concentration of the liver. In spite of the intense nuclear staining in 2-day old controls and 84-day old Cd-injected rats, less than 1% of the hepatic MT was present in the nuclear fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Metallothionein induction by sodium selenite at two different ambient temperatures in mice.

The induction of metallothionein (MT) synthesis by sodium selenite was investigated in mice with regard to the hypothermic response known to be caused by sodium selenite. Mice received a subcutaneous injection of sodium selenite at two doses (20 and 45 mumol/kg) under two ambient temperature (22 and 33 degrees C) conditions. Hepatic MT concentration was significantly increased by an injection of sodium selenite compared to the control, whereas no significant effect of ambient temperature was observed. The distribution of radiolabeled selenium was examined in vivo and in vitro. When sodium selenite was injected into mice, radiolabeled selenium was mostly eluted in a fraction larger in molecular weight than MT and was not found in a fraction corresponding to MT. When sodium selenite was added to the hepatic supernatant of the mice that had been injected with zinc sulfate, zinc in zinc-thionein was not displaced by radiolabeled selenium.

Physiological role of glucocorticoids on rat serum and liver metallothionein in basal and stress conditions.

Serious contradictions exist at present in our understanding of the physiological role of glucocorticoids on the synthesis of the metal-binding protein, metallothionein (MT). In addressing this problem, we have examined in vivo the role of glucocorticoids on liver and serum MT levels in the rat under a spectrum of experimental conditions. The experiments confirm that stress has a major positive effect on hepatic MT levels. It was found that adrenocorticotropic hormone (ACTH) administration has an inhibitory effect on hepatic MT levels in response to restraint stress and that adrenalectomy (ADX) leads to an increase in basal MT levels and in MT levels in response to acute and chronic immobilization stress. Similar results followed treatment with the glucocorticoid receptor blocker, RU 486. The effect of ADX was abolished by corticosterone replacement. The relations found among hepatic MT, serum MT, and glucocorticoid concentrations indicate that in some circumstances glucocorticoids have a permissive role in mobilizing MT from tissues to serum and that in physiological conditions corticosterone has an inhibitory role in the maintenance of hepatic MT levels.

Factors affecting hepatic metallothionein levels in marine flatfish

Tissue metallothionein concentrations in marine flatfish have been evaluated as a potential indicator of metal pollution in the sea. The effect on metallothionein in flatfish of agents known to induce its synthesis in mammals was examined. (1) Metals. Intraperitoneal injection of zinc into plaice ( Pleuronectes platessa) gave rise to a rapid and large increase in hepatic metallothionein. This had a half-life of about a month. In contrast, dietary zinc at a concentration of 1000 mg/kg (dry wt) did not significantly increase hepatic nor renal metallothionein levels in the turbot ( Scophthalmus maximus). (2) Stress. There was no effect on hepatic metallothionein in plaice exposed to the stress of capture nor the injection of endotoxin, cortisol, dexamethasone or turpentine. Seasonal variation was found in female plaice. During the breeding season mature fish had occasional very high concentrations of hepatic metallothionein, possibly associated with egg development.

Influences of compounds on metallothionein concentration in mouse tissues. I. Increase of hepatic metallothionein concentration by organic solvents and fatty acids.

Influences of compounds on metallothionein concentration in mouse tissues. I. Increase of hepatic metallothionein concentration by organic solvents and fatty acids.

Influence of chemical and environmental stressors on acute cadmium toxicity.

Previous investigations have demonstrated that the cytosolic protein metallothionein (MT) is induced not only by exposure to certain heavy metals but also by a variety of other factors, including environmental stress. While MT synthesis has been observed with exposure to cold temperatures, there is a paucity of data concerning the influence of cold on heavy-metal toxicity. The present investigation focused on the influence of metal and cold pretreatments on the acute toxicity of cadmium. Mortalities of 80% and 100% were observed for mice orally administered challenge doses of 100 mg Cd/kg and 150 mg Cd/kg, respectively. To determine a protective cadmium pretreatment dose, animals were administered 2.5, 5, 10, 20, 25, and 50 mg Cd/kg 24 h prior to cadmium challenge. In animals pretreated with 10 mg Cd/kg, mortalities of 20% and 70% were observed with the respective challenge doses. Immediately following cold stress (4 degrees C, 12 h), mortalities of 30% and 90% were observed with cadmium challenge doses of 100 and 150 mg Cd/kg, respectively. Significant correlations were demonstrated between induced hepatic MT concentrations and cadmium pretreatment (r = 0.99), as well as cold pretreatment (r = 0.87). Results of this investigation indicate that stressors, such as cold, influence the acute toxicity of cadmium to the same magnitude as metal pretreatment. This induced tolerance to cadmium was attributed, in part, to the induction of MT synthesis. Furthermore, the induced levels of MT resulting from cold stress may confound the simplistic approach of using MT as a biological monitor of occupational exposure to cadmium.

Ontogenically reculated 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.

Induction of hepatic metallothionein following administration of urethane

Induction of hepatic metallothionein (MT) by urethane (ethyl carbamate) was characterized. Male CF-1 mice were treated with urethane (0, 0.5, 1.0, 1.5, and 2 g/kg; ip) and 18 hr later hepatic MT concentrations were determined with the Cd-hemoglobin radioassay. Urethane (1 g/kg and higher) significantly increased hepatic MT levels, resulting in a 14-fold increase after 2 g/kg. Time-course experiments indicated that MT levels were increased significantly at 6 hr after administration of urethane (1.5 g/kg) and reached a maximum between 12 and 24 hr. Gel filtration, anion-exchange chromatography, and ultraviolet spectral analysis were used to characterize the protein induced by urethane. Pretreatment with actinomycin- d prevented induction of MT by urethane. Administration of equimolar dosages (20 mmol/kg) of urethane, N-hydroxyurethane, and methyl carbamate indicated that urethane and N-hydroxyurethane induce MT but that methyl carbamate does not. MT induction was also not observed with other commonly used anesthetics (pentobarbital and phenobarbital). In conclusion, urethane induces hepatic MT but this effect is not related to its anesthetic action, nor is it a common property of all carbamates.