Induction Of Hepatic Metallothionein Research Articles

Induction of hepatic metallothionein by intraperitoneal metal injection: an associated inflammatory response

The nature of hepatic metallothionein (MT) induction by several metals and its relationship to an inflammatory response was studied in chicks. Intraperitoneal (ip) injection of chromium (Cr), managanese, and iron (Fe) caused a much greater increase in hepatic MT (10.2-, 9.0-, and 6.8-fold) compared with cobalt and nickel (2.5- and 2.9-fold); thus not all transition metals are effective. Cr3+ caused markedly greater hepatic MT accumulation than Cr6+, suggesting that the ionic nature of the metal is an important factor. Small organic complexes of Fe (ferrous gluconate or lactate, 6.2-fold) caused significantly greater accumulation of hepatic MT than ferric dextran (1.4-fold), a large organic aggregate. In vitro data from chick hepatocytes and/or fibroblasts clearly indicated that Fe does not effect the induction of MT directly. The role of inflammation, as measured by recruitment of peritoneal exudate cells (PEC), was examined. Endotoxin (LPS), Sephadex (S), and Fe elicited significant elevations in PEC number at 24 h posttreatment (S), and Fe elicited significant elevations in PEC number at 24 h posttreatment (S = Fe greater than LPS much greater than control). The percentage of heterophils but not macrophages was significantly correlated with the accumulation and induction of hepatic MT. In a similar experiment with Cr, we demonstrated that Cr3+ but not Cr6+ stimulated MT messenger RNA accumulation and concomitant hetereophil infiltration at 3 h after injection. Our results indicate that the induction of hepatic MT by the parenteral administration of a number of metals is dependent on the chemical nature of the metal and is associated with an inflammatory response.

Induction of hepatic metallothionein in male B6C3F1 mice exposed to hepatic tumor promoters: Effects of phenobarbital, acetaminophen, sodium barbital, and di(2-ethylhexyl) phthalate

The effects of various compounds known to be hepatic tumor promoters and toxins in the male B6C3F1 mouse liver, including di(2-ethylhexyl)phthalate (DEHP), acetaminophen (ACT), barbital (BB), and phenobarbital (PB) on hepatic metallothionein (MT) concentrations were assessed after chronic exposure. From 6 weeks of age, male mice were maintained on diets containing DEHP at 12,000 or 6000 ppm, ACT at 10,000 or 5000 ppm, BB at 1,000 ppm, or drinking water with PB at 500 ppm for up to 24 weeks. MT was measured in hepatic cytosol at 0, 2, 8, and 24 weeks of exposure. DEHP proved a very effective inducer, producing elevations of MT as high as 11-fold. The increases in hepatic MT with DEHP were both dose- and time-related. ACT was likewise effective in producing hepatic MT elevations (maximum 6.7-fold) in a dose- and time-related fashion. BB and PB, however, had no effect on hepatic MT levels at any time point. While DEHP, BB, and PB treatments produced hepatomegaly, histopathological analysis at 24 weeks revealed that in both DEHP- and ACT-treated livers hepatocellular proliferation was prominent while livers exposed to BB or PB showed predominantly hepatocellular hypertrophy. Gel-filtration of DEHP-treated liver cytosol revealed that zinc was associated with the MT peak. This peak also bound cadmium in vitro and could be extracted by heat treatment and selective acetone precipitation, both typical characteristics of MT. Further confirmation of the presence of MT after DEHP treatment was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10 to 20% acrylamide). Results indicate that some, but not all, tumor promoters can induce target organ MT and that such an induction appears associated with those promoters inducing persistent cellular hyperplasia but not those inducing cellular hypertrophy.

Influence of Diethyldithiocarbamate on Nickel-Induced Hepatic Metallothionein in Rats

The influence of sodium diethyldithiocarbamate (DDC) on 63nickel chloride-induced metallothionein (MT) and alterations in essential metal levels in the liver of rats was investigated. The induction of MT, Zn and Cu levels of the hepatic cytosolic "heat stable fraction" (HSF) by DDC increased with time up to 24 hr. Although MT, Zn and Cu were significantly greater at 17 hr than those at 6.5 hr after 63Ni administration, the Ni level decreased. The treatment with DDC at 6 hr, but not at 10 min, prior to 63Ni administration increased significantly the MT, Zn and Cu contents 17 hr after 63Ni administration over that caused by 63Ni alone at 17 hr., showing a synergistic effect. The induction of hepatic MT by 63Ni or DDC alone was prevented by cycloheximide but not by actinomycin D and seemed to be regulated at the protein synthesis level.

Induction of hepatic metallothionein by salicylate in adult rats

Application of salicylate increased the concentration of metallothionein (MT) in liver of pregnant rats as well as of adult male rats, whereas in fetal liver, MT was reduced by salicylate. Induction of MT synthesis by salicylate is an indirect effect because in cultured hepatocytes salicylate did not induce MT synthesis. Salicylate increased MT also in adrenalectomized rats. Indomethacin induced the same concentration of MT in maternal liver as salicylate. However, indomethacin had no effect on MT in fetal liver. Induction of MT in adult liver by salicylate and indomethacin was independent of zinc.

Enzyme-linked immunosorbent assay for human metallothionein: correlation of induction with infection.

Very little information is available concerning the relationship between metallothionein and disease in humans. Recently, investigators have used the Cd-heme method to measure metallothionein levels in human liver samples obtained from autopsy. This assay, however, is not sensitive enough to measure metallothionein in small tissue biopsy specimens. As an alternative, we report the development of a human metallothionein enzyme-linked immunosorbent assay (ELISA). This assay used high-performance liquid chromatography-purified human metallothionein-1 and purified sheep anti-human metallothionein-1 IgG. A limiting antigen coating concentration of 100 ng/ml and a minimal antibody dilution of 1:4000 were chosen. The sensitivity of the ELISA extended to 300 ng/ml (15 ng). The coefficients of interassay and intraassay variation were 15.4% and 4.2%, respectively. Human livers obtained from autopsy were assayed by this method and the values compared with values obtained by the Cd-heme method. The livers were separated by their autopsy reports into four groups: normal, immunosuppression, cancer, and infection. Livers from the infection group (ELISA 2979 micrograms/gm, Cd-heme 1201 micrograms/gm) contained significantly more metallothionein than those from the normal (ELISA, 1035 micrograms/gm, Cd-heme 245 micrograms/gm) and the immunosuppression (ELISA 1272 micrograms/gm, Cd-heme 221 micrograms/gm) groups (p less than 0.05). The cancer group (ELISA 2415 micrograms/gm) also had significantly elevated liver metallothionein levels. We conclude that this ELISA is sensitive enough for the measurement of tissue samples. Furthermore this assay is comparable to the Cd-heme assay in its ability to reflect metallothionein values among various treatment groups. We postulate that hepatic metallothionein induction is mediated by disease-related mechanisms such as interleukin-1, glucocorticoid secretion, or both.

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.

Strain dependence in mice of resistance and susceptibility to the testicular effects of cadmium: Assessment of the role of testicular cadmium-binding proteins

The nature of the cadmium (Cd)-binding proteins in the mouse testes is unknown, although some studies have implied metallothionein (MT) is responsible for the marked strain dependence of Cd-induced testicular necrosis in mice. This study attempted to define the role of MT in strain-dependent Cd resistance using NFS (susceptible) and BALB/c (resistant) mice. In all cases, testicular proteins were compared to hepatic MT isolated after treatment with zinc (Zn). A low-molecular-weight ( M r) Zn-, Cd-binding protein was detected in testicular and hepatic cytosol from both strains after gel filtration. These proteins were extractable by heat treatment and sequential acetone precipitation. When such extracts were further purified with reversephase HPLC, two forms of authentic MT were shown by amino acid analysis from both NFS and BALB/c livers. However, of two testicular forms separated by HPLC from NFS and BALB/c mice, neither could be classified as MT based on amino acid composition. Methylation of the MT-I gene was also studied in testicular and hepatic DNAs isolated from control mice or from mice made resistant to Cd-induced testicular necrosis by Zn treatment. Major differences in methylation between tissues were seen, as the testicular gene of both NFS and BALB/c mice was highly methylated, a condition often linked to genetic quiescence. Zn treatment had no effect on MT-I gene methylation in testes of either strain, although rendering the NFS strain resistant to Cd. Zn treatment did not alter levels of this testicular protein in either strain while causing a marked induction of hepatic MT in both. These results indicate the low- M r Cd-, Zn-binding proteins present in the testes of both resistant and susceptible mice are not MTs and further that the MT gene may not be expressed in either strain.

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.

Induction of hepatic metallothionein by alcohols: Evidence for an indirect mechanism

The purpose of the present study was to determine the ability of various short-chain alcohols to induce metallothionein (MT) in the liver and to determine whether the induction results from a direct action of alcohol on liver or an indirect action mediated by zinc, glucocorticoids, or catecholamines. Mice were administered alcohol by gavage and hepatic MT was quantitated by the Cd-hemoglobin radioassay. Ethanol, methanol, isopropanol, and propanol increased MT content to seven to nine times that of control liver. In vitro, ethanol did not increase MT concentrations in rat hepatocyte cultures, indicating that the in vivo induction is not a direct effect of ethanol on the liver. Adrenergic blocking agents did not reduce the MT content of ethanol-treated mice, indicating that catecholamines are probably not involved in the MT induction. Corticosterone and zinc concentrations in plasma were increased in mice 1 hr after ethanol treatment. Corticosterone, give in vivo, was a less effective inducer of MT than was ethanol treatment. In conclusion, (1) hepatic MT was increased by several alcohols, (2) the induction was not due to direct action of alcohol on the liver, and (3) while the mechanism of alcohol induction of MT is unclear, it may be due to an alteration in zinc and glucocorticoid homeostasis.

Induction of hepatic metallothionein in mouse liver following administration of chelating agents

Chelating agents commonly used in therapy of heavy metal intoxication alter the levels of essential metals in liver, kidneys, and serum. Induction of metallothionein synthesis in liver occurs following exposure to a variety of chemical and environmental insults and, in some cases, has been attributed to enhanced hepatic uptake of zinc. Therefore, the effect of acute exposure to seven common metal chelators on the concentration of metallothionein in liver was investigated. Adult male Swiss Webster mice were injected intraperitoneally with the chelators and hepatic metallothionein was quantified by the cadmium radioassay. Ethylenediaminetetraacetic acid (EDTA) produced a 5- to 6-fold increase in hepatic metallothionein 24 hr after injection of 0.75 to 3.0 g/kg. No significant increase in hepatic MT was observed until 12 hr following injection of EDTA (1.5 g/kg, ip). Maximal levels were reached between 12 and 48 hr following EDTA injection. Cadmium, a known inducer of hepatic metallothionein, produced a 15-fold increase in the concentration of MT in liver 24 hr following injection. By comparison, 2,3-dimercaptopropanol and diethyldithiocarbamate produced a 9-fold and 13-fold increase in hepatic metallothionein levels, respectively, 24 hr following injection. A 4- to 6-fold increase in metallothionein was observed 24 hr following injection of 2,3-dimercaptosuccinic acid, d,l-penicillamine, diethylenetriaminepentaacetic acid, and EDTA, while nitrilotriacetic acid elevated hepatic metallothionein levels by 2-fold. Alterations in the concentration of hepatic metallothionein by chelators may have implications for their efficacy in the treatment of cadmium intoxication.

The influence of maternal cadmium exposure or fetal cadmium injection on hepatic metallothionein concentrations in the fetal rat

The ability of Cd to induce the synthesis of fetal hepatic metallothionein (MT) was investigated in rat fetuses exposed to Cd throughout gestation via the mother's drinking water or injected directly with Cd through the uterine wall on Day 18 of gestation. On Day 21 all dams were killed and fetal and maternal tissues were removed. Tissue MT, Zn, Cu, and Cd concentrations were measured. Fetal hepatic Cd concentration was increased only at the high maternal Cd exposure, whereas Zn concentration was significantly reduced by Cd exposure. Both fetal liver and kidney MT were reduced following maternal Cd exposure. Unlike maternal hepatic MT, fetal hepatic MT was not increased after maternal Cd exposure nor did the direct injection of Cd into the 18 days of gestation fetus induce fetal MT synthesis. These data suggest that fetal rat liver is incapable of synthesizing MT in response to Cd, possibly because Cd is not transported to the site of MT synthesis in the fetal system. Furthermore, neither the route of exposure, the duration of prenatal Cd exposure, nor the stage of gestation appear to account for the differences observed between fetal and adult hepatic MT induction by Cd.

Induction of hepatic metallothionein following 5-azacytidine administration

Exposure of cells in culture to the pyrimidine analog, 5-azacytidine (AZA-C), stimulates the expression of the metallothionein (MT) gene. Therefore this study was performed in an attempt to extend this observation to a whole animal system. Young Wistar rats (approximately 30 to 35 days old) were administered AZA-C 24 and 16 hr (50 mg AZA-C/kg, ip, each time) prior to the determination of hepatic MT by the Cd-Hem method. Such treatment resulted in an approximately fivefold increase in the concentrations of hepatic MT in cytosol obtained from both male and female rats. Gel-filtration and anion-exchange chromatography, as well as uv spectral analysis, confirmed the presence of MT in the livers of AZA-C-treated amimals. A single dose of AZA-C (65 mg/kg, ip) produced increases in hepatic MT concentrations 8 hr after dosing that were still elevated at 48 hr. Dose-response studies indicated hepatic MT concentrations were increased 24 hr after the administration of 50, 60, and 65 mg AZA-C/kg but were not altered by doses of 30 or 40 mg/kg. Actinomycin-D pretreatment (1.25 mg/kg, ip) 30 min prior to AZA-C (80 mg/kg, ip) prevented the subsequent increases in hepatic MT concentrations. These data indicate that treatment with AZA-C produces an in vivo induction of hepatic MT synthesis that appears to the more persistent in nature than other nonmetallic inducing agents.

Effects of subcutaneous and oral cadmium on iron metabolism: role of ceruloplasmin and metallothionein.

Male ICR mice were either given water containing Cd at a level of 192-200 ppm for 45 days (ingestion group), or were injected subcutaneously once a week with Cd (1 mg/kg) as CdCl2 for 7 weeks (injection group). The control group was given Cd-free water. In both Cd groups, the hematocrit and hemoglobin values did not change markedly. In the ingestion group, the Fe concentration decreased greatly in the liver, kidney, spleen, and duodenum. These decreases may be due to depression of Fe absorption from the intestine. In the injection group, Fe increased in the liver, spleen, and duodenum, although it decreased in the kidney. By Sephadex G-200 gel filtration, Fe-proteins in the hepatic supernatants were located in the void volume region of this gel column in both Cd groups. Apparently, Fe was not a component of metallothionein (MT) protein. The hepatic MT induction by Cd resulted in an increase in hepatic supernatant Cu. Serum Cu and ceruloplasmin (Cp) activity were stimulated only in the injection group. The enhancement of Cp activity may possibly be due to the increase in hepatic Cu which was accompanied by an increase in hepatic Fe, rather than a decrease. Our observations suggest that Fe metabolism is influenced differentially by the administration route of Cd.

Induction of hepatic metallothionein in the rabbit fetus following maternal cadmium exposure

Timed-pregnant rabbits were administered a single, sc injection of cadmium chloride (0, 0.125, 0.25, or 0.50 mg cadmium/kg body wt) 48 hr prior to being killed at term. Although the doses chosen were below that shown to produce significant fetal lethality, the 0.50 mg/kg dose did produce a significant reduction in fetal body weight, liver weight, kidney weight, and crown-rump length while having no effect on placental weight. Fetal hepatic metallothionein levels were significantly increased at all three doses; however, a significant increase in metallothionein zinc content was seen only at the two higher doses. Metal distribution studies indicated that the increase in fetal metallothionein and the accompanying increase in cytosolic zinc coincided with an apparent redistribution of the metal from extrahepatic sites to the liver. The whole fetal content of zinc was not significantly altered by the maternal administration of cadmium.

Induction of hepatic metallothionein by feeding zinc to rats of depleted zinc status.

Induction of hepatic metallothionein was investigated in zinc-depleted rats after a single feeding (via stomach tube) of a complete diet with or without supplemental zinc. Subsequent to a maximum elevation in serum zinc (3.25 micrograms/ml) at 3 h, the rate of metallothionein synthesis increased 4.5-fold by 10 h after feeding. Changes in the rate of metallothionein synthesis coincided with similar changes in the level of translatable mRNA coding for metallothionein. Accumulation in liver of Zn2+ as metallothionein rose to a maximum by 12 h after diet administration and thereafter remained constant. Radioactive zinc (65Zn) included in the diet rapidly associated with newly formed metallothionein. Unlike Zn2+, 65Zn exhibited marked fluctuations within the 24-h period following feeding, indicating that zinc associated with metallothionein may be capable of exchange and/or dissociation. Changes in total liver 65Zn were accompanied by concomitant changes in metallothionein-bound 65Zn. This study has related (temporally) the metabolism of dietary zinc to the induction, apparently via transcription of mRNA, of hepatic metallothionein.

Effect of cadmium on hepatic metallothionein level in early development of the rat

The induction of hepatic metallothionein (MT) was investigated 24 hr after an intraperitoneal injection of 0.75 mg/kg Cd as CdCl 2 · H 2O in rats of 7, 14, 21, and 90 days. Metallothionein in liver cytosolic fractions collected on Sephadex G-75 was characterized in terms of sulfhydryl, total protein, Cd, and Zn contents. Most of the cytosolic Cd was associated with MT and the concentration of Cd was equal in the different age groups. The higher contents of sulfhydryl, protein, and Zn both in control as well as in Cd-injected rats of 7 and 14 days than in those of 21 and 90 days indicate the presence of more native Zn-thionein in immature pups. However, the increase in sulfhydryl and protein contents showed more prominent induction of MT in Cd-exposed animals of 21 and 90 days than in those of 7 and 14 days. The concentration of Cd was highest in liver followed by the other tissues. While hepatic accumulation of Cd was similar in all age groups, the renal accumulation increased significantly with age. The intestine and spleen of immature pups concentrated more Cd than those of mature animals. The accumulation of the metal did not differ significantly in heart and brain of the animals among the four groups.

Induction of hepatic metallothionein in the immature rat following administration of cadmium

Hepatic metallothionein (MT) levels, as measured indirectly by total metal-binding capacity, are approximately 8.5-fold higher in the 7-day-old rat than in the 28-day-old rat where levels are barely detectable. To stydy how the presence of MT might influence the toxicity of cadmium, a single subcutaneous injection of cadmium chloride was administered at dose levels of 0, 1, 2, 3, and 6 mg Cd/kg body weight, 48 hr prior to sacrifice on Day 7 or Day 28. In both the 7- and 28-day-old groups, there was a dose-related increase in the amount of cadmium bound to MT. There were no significant age-related differences in the amount of cadmium bound to MT at the various doses, with the exception of the 6 mg/kg dose, where 7 day levels were higher. The 28-day-old rats responded to cadmium exposure with induction of MT and subsequent binding of both cadmium and zinc at all doses. The 7-day-old appeared to have sufficient levels of MT to handle cadmium doses at or below 3 mg/kg without induction; however, at the 6 mg/kg dose induction was observed. Despite the presence and inducibility of hepatic MT at Day 7, 30% of the rats treated with 6 mg Cd/kg died within 48 hr of exposure compared with only 4% at Day 28.

Cadmium resistance and content of cadmium-binding protein in cultured human cells.

INDUCTION of hepatic metallothionein (a cadmium-binding protein) is considered to be a protective mechanism in mammals against the toxic cadmium ion. Pretreatment of rats with low doses of cadmium induces the synthesis of metallothionein and also protects against subsequent exposure to an otherwise lethal dose1. But the role of metallothionein in cadmium resistance is not clear2. Restriction of food intake in the rat increases the level of metallothionein3, but does not alter the LD50 of cadmium2. Also the synthesis of metallothionein seems to continue for a longer time than the protection against cadmium in both rats and mice1,2. Lucis, Shaikh and Embil4 found uptake of Cd in cultured human embryonic fibroblasts, HeLa cells and monkey kidney epithelial cells after 8 d of incubation with Cd. At that time the cells also contained a Cd-binding protein. The authors did not, however, describe growth of the cells in the presence of Cd or development of cell strains resistant to Cd. Webb and Daniel5 recently described the synthesis of a Cd-binding protein in cultures of cells derived from the cortex of the adult pig kidney.