Induction Of Hepatic Metallothionein Synthesis Research Articles

Induction of hepatic metallothionein synthesis by endoplasmic reticulum stress in mice

Metallothionein (MT) is a small sulfhydryl-rich protein whose levels are elevated by various inducers of organelle stresses, such as nuclear stress (cisplatin), mitochondrial stress (antimycin A, 2,4-dinitrophenol) and lysosomal stress (paraquat). Although abnormal folding of protein in the endoplasmic reticulum (ER) causes ER stress, induction of MT synthesis by ER stress has never been investigated. In this study, we examined the induction of MT by an inducer of ER stress, tunicamycin (Tun), which induces ER stress by inhibiting N-linked glycosylation of protein in the ER. Administration of Tun (0.5–1.5 mg/kg, sc) increased hepatic MT levels in C57BL/6J mice (3.1-fold). The maximal increase in hepatic MT was observed 48–96 h after the administration of Tun (1.0 mg/kg). Expressions of MT-I, II and glucose-regulated protein 78 (Bip/GRP78), which is a molecular chaperone induced by ER stress, mRNA were also detected by administration of Tun. Thapsigargin (Thap), a generator of ER stress by inhibiting ER Ca 2+-ATPase, also increased both hepatic MT levels and expression of MT-I and -II mRNA. The level of expression of Bip/GRP78 mRNA induced by Tun administration in MT-null mice was greater than that in wild-type mice. Taken together, these findings suggest that inhibitors of ER are potent inducers of MT.

Glucocorticoids Suppress the Inflammation-Mediated Tolerance to Acute Toxicity of Cadmium in Mice

Several compounds have been shown to cause acute toxicity to cadmium (Cd). The mechanism of tolerance to Cd toxicity induced by glucocorticoids or by inflammation involves induction of metallothionein (MT) synthesis via glucocorticoid response elements or by inflammatory cytokines. We have demonstrated previously that the synthetic glucocorticoid dexamethasone suppresses inflammation-mediated induction of hepatic MT synthesis. Here we investigated the effect of glucocorticoid on tolerance to Cd induced by inflammation in mice. The LD50 of Cd for mice with induced inflammation by injection with turpentine oil (Tur-mice) was higher than the LD50 in control mice. Pretreatment of Tur-mice with dexamethasone to the Tur-mice (Dex+Tur-mice) resulted in a decrease in LD50 after Cd treatment. A significant increase in plasma alanine aminotransferase and aspartate aminotransferase levels in the Dex+Tur-mice was observed at lower doses of Cd than in the Tur-mice and at higher doses of Cd than in control mice. Dexamethasone did not suppress tolerance to cadmium toxicity in the testes of the Tur-mice. Pretreatment of Tur-mice with dexamethasone resulted in suppression of both plasma interleukin (IL)-6 elevation and in suppression of hepatic MT levels when induced by inflammation but not when induced by Cd. These data suggest that suppression of tolerance to Cd toxicity induced by glucocorticoid may involve hepatic MT synthesis mediated by inflammatory cytokines, such as IL-6. We suggest that the inflammatory response can modulate Cd toxicity by induction of MT by inflammatory cytokines.

Metallothionein synthesis induced by interferon alpha/beta in mice of various zinc status.

We studied the ability of interferon alpha/beta (IFN) to induce metallothionein (MT) synthesis in mice. Male mice were injected intraperitoneally with mouse IFN (5 x 10(5) IU/mouse). Plasma Zn levels were reduced at 4 hr after injection, reached a minimum value at 6 hr, and then returned to the control level at 8 hr. Hepatic MT concentrations began to increase at 4 hr and reached maximum values at 6 hr. Induction of MT gene expression and protein synthesis was confirmed by Northern blot analysis and radioimmunoassay, respectively. The induction of MT synthesis in the liver by IFN was dose-dependent. The data suggest that induction of MT-mRNA and the protein in the liver by IFN occurs rapidly but is rather transient. Furthermore, MT synthesis was not induced by IFN in the liver of mice given a Zn-deficient diet, whereas IFN induced increases in the activity of 2',5'-oligoadenylate synthetase in the spleen were unaffected by Zn status. Thus, induction of hepatic MT synthesis by IFN is influenced by Zn status.

Stress-related induction of hepatic metallothionein synthesis and increase in peripheral polymorphonuclear leukocytes in mice.

This experiment examined whether hepatic metallothionein (MT) synthesis induced by stressful stimuli could reinforce the peripheral leukocyte defense mechanism in mice. A 2 x 2 cm section of dorsal skin was excised from male ICR mice (7 w.o.), then the hepatic MT concentration and superoxide anion production (SOA) in peripheral leukocytes were measured at 6 and 24 hr after the excision. The 6 hr-hepatic MT level was 6 times greater in the skin-excised mice than in the controls. SOA in the skin-excised mice was 2.3 times greater at 6 hr than in the controls, then decreased to the control level by 24 hr. Food deprivation increased the hepatic MT and SOA levels at 24 and 48 hr to a remarkably greater level than in the controls. The increases in SOA, which was measured by chemiluminescence response (CL) were found to be due to an increase in the number of polymorphonuclear leukocytes (PMNs) in the peripheral leukocytes in both the skin excision and food deprivation groups. These results suggest that skin excision causes an inflammatory response in mice that results in an acute increase in the number of PMNs concomitant with the acute activation of hepatic MT synthesis. Food deprivation might result in physiologic stress 24 hr or more after food deprivation and cause "emergency" increases in MT synthesis and PMN defense mechanisms. Thus, some unknown linked mechanisms might exist between hepatic MT synthesis and increased peripheral PMNs.

Glucocorticoid inhibition of inflammation-induced metallothionein synthesis in mouse liver

The effects of glucocorticoid treatment on the induction of hepatic metallothionein (MT) during inflammation initiated by turpentine oil (TUR) or endotoxin (LPS) were studied in mice. The administration of TUR increased concentrations of hepatic MT as well as that of plasma fibrinogen. Although hepatic MT was modestly induced by dexamethasone (DEX) alone, pretreatment with DEX (12.5 to 100 mg/kg, sc) inhibited the increases both in hepatic MT and in plasma fibrinogen from a subsequent dose of TUR 6 hr after DEX administration. The concentration of hepatic MT in the DEX-pretreated (25 mg/kg) group was higher than that in the nonpretreated group 4 hr after administration of TUR, but after 24 hr, the MT concentration in the DEX-pretreated group was inhibited to 20% of that in the nonpretreated group. These inhibitory effects were also observed by prednisolone (PRE) but not by salicylic acid. The inhibitory effect of DEX on the induction of MT synthesis during inflammation was observed after administration of the exudate obtained from inflamed tissue. When inflammation was initiated by an injection of LPS as well as TUR, pretreatment of either DEX or PRE inhibited the increase of hepatic MT. Pretreatment of DEX did not affect the induction of hepatic MT synthesis by cadmium. In contrast to inflammation initiated by TUR or LPS, pretreatment of DEX caused an additive increase of hepatic MT concentration after administration of zinc. These results suggest that glucocorticoids, despite being direct inducers of MT, inhibit the induction of MT synthesis during inflammation by the suppression of cytokine production.

Induction of hepatic metallothionein by nonmetallic compounds associated with acute-phase response in inflammation

Induction of hepatic metallothionein (MT) synthesis by several nonmetallic compounds and its relationship to an acute-phase response in inflammation were studied in mice. Subcutaneous injections of menadione, paraquat, carbon tetrachloride (CCl 4), and several organic solvents caused an increase of hepatic MT concentration. This MT contained only zinc. Menadione and n-hexane caused the greatest accumulation of hepatic MT among these nonmetallic compounds (about 13-fold). The concentration of Zn was significantly decreased in plasma in contrast to liver after an injection of these nonmetallic compounds. When 65ZnCl 2 was injected iv after these injections, uptake of 65Zn to the liver was increased. This effect was not observed after treatment with cycloheximide. The association with inflammation of this induction of MT accumulation was examined by determination of acutephase proteins. The concentration of fibrinogen in the plasma was significantly increased following injection of those nonmetallic compounds which caused marked hepatic MT accumulation. An injection of 1 n NaOH, 1 n HCl, turpentine oil, or endotoxin caused a significant increase in the plasma concentration of fibrinogen and in the hepatic MT concentration. Injections of n-hexane as well as turpentine oil significantly increased hepatic MT concentration and plasma concentration of fibrinogen and ceruloplasmin with time. The concentration of fibrinogen was significantly correlated ( r = 0.789) with the concentration of hepatic MT. Neither adrenalectomy nor pretreatment with dexamethasone prevented hepatic MT accumulation caused by these compounds. These results indicate that induction of hepatic MT synthesis by these nonmetallic compounds is associated with an acute-phase response in inflammation and is independent of glucocorticoids.

Acute exposure to formaldehyde induces hepatic metallothionein synthesis in mice

Humans risk inadvertent intraperitoneal or intravenous exposure to formaldehyde (HCHO), commonly used for disinfection of implanted or extracorporeal medical devices. Various chemical and physical stresses are known to induce hepatic metallothionein. This study examined the effect of acute parenteral administration of HCHO on induction of hepatic metallothionein synthesis. Adult male CF1 mice were administered HCHO ip and hepatic metallothionein was quantified by the cadmium-radioassay method. HCHO (50 mg/kg) increased hepatic metallothionein as early as 8 hr after dosing with maximal levels (27-fold increase) occurring at 72 hr. Metallothionein concentrations were elevated (15-fold) 24 hr after 50 or 100 mg HCHO/kg but not at lower dosages. Concomitant elevations in hepatic zinc and copper content were observed. No increases in metallothionein were observed in kidney, pancreas, or intestine 24 hr after HCHO administration (100 mg/kg, ip). Induction of metallothionein by HCHO may reflect direct de novo synthesis since the response was abolished by pretreatment with the RNA synthesis inhibitor, actinomycin D. HCHO induction of metallothionein also does not appear to be mediated by stress-induced release of corticosteroids or catecholamines from the adrenal since the response was unaltered in adrenalectomized mice. Interference with the glutathione (GSH)-dependent oxidation of HCHO by reducing hepatic GSH concentrations to 40% of control after a 2-hr pretreatment with phorone decreased the metallothionein induction response to HCHO by 33%. This result suggests that the induction may be partially due to a HCHO metabolite, e.g., formate. Confirmation of metallothionein synthesis was obtained following spectral and chromatographic analysis. Thus, HCHO and/or a metabolite produces a marked increase in hepatic metallothionein and alters hepatic zinc and copper homeostasis, all of which are transient responses. Although HCHO was only mildly hepatotoxic at the highest dose (as evidenced by an increase in plasma alanine aminotransferase activity), such changes in metallothionein synthesis and essential metal homeostasis may be part of a cellular repair mechanism operant after acute toxic cell injury.

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.

Induction of metallothionein by zinc in lethal milk mutant mice.

Adult lethal milk (lm/lm) mutant mice display increased induction of hepatic metallothionein synthesis compared to wild-type mice following the subcutaneous injection of 40 mumol ZnCl2/kg mouse. At this zinc dose the rate of incorporation of [35S] cysteine into hepatic metallothionein in adult (100- to 230-day-old) lm/lm mice was approximately 2.4-fold greater than the rate of incorporation of isotope in wild-type animals. At a higher zinc dose (160 mumol ZnCl2/kg) the incorporation of [35S] cysteine into hepatic metallothionein was similar in lm/lm and wild-type mice. The altered dose-response to zinc administration was not due to a change in hepatic zinc, copper, or manganese levels, to a difference in 65Zn uptake, or to an alteration in 65Zn bound to differential centrifugation fractions of adult lm/lm liver. 65Zn bound to hepatic metallothionein was, however, increased in aging lm/lm mice with symptomatic skin lesions.

Effect of adrenalectomy on cadmium- and turpentine-induced hepatic synthesis of metallothionein and alpha 2-macrofetoprotein in the rat.

Recent reports have strongly implicated glucocorticoids in the induction of hepatic metallothionein synthesis and hypozincemia which occurs in certain pathophysiologic conditions. Studies were performed in rats to determine the effect of adrenalectomy and glucocorticoid treatment on the hepatic accumulation of metallothionein subsequent to the administration of cadmium and turpentine, two diverse substances known to induce hypozincemia and hepatic synthesis of metallothionein as well as alpha 2-macrofetoprotein in intact rats. By 24 h, both substances induced significant hypozincemia, hepatic metallothionein accumulation, and a severe tissue inflammatory response in adrenalectomized rats. Adrenalectomy only prevented the increase in plasma alpha 2-macrofetoprotein concentration. Results indicate that hepatic synthesis of alpha 2-macrofetoprotein, but not metallothionein, is mediated by adrenal hormones. Thus, glucocorticoids do not play a "vital" role in hepatic metallothionein accumulation or hypozincemia induced by inflammatory stress, as previously postulated.

Altered zinc homeostasis and hepatic accumulation of metallothionein in indomethacin-induced enteropathy

Altered zinc homeostasis is one of several metabolic sequelae of inflammation wherein zinc is redistributed to the liver from endogenous sources such as plasma and sequestered in hepatic metallothionein. Studies were performed in rats to determine the effect(s) of indomethacin, a potent ulcerogenic agent, on zinc metabolism. A single sc dose of indomethacin given to fed rats in amounts between 1 and 10 mg/100 g body wt produced, by 24 hr, profound hypozincemia, greater intestinal absorption of zinc, induction of hepatic metallothionein synthesis, and gastrointestinal lesions. These responses were significantly muted when the drug was administered to fasted rats. Results indicate that the effects of indomethacin on zinc homeostasis are associated with drug-induced enteropathy. Furthermore, results provide evidence that hepatic metallothionein functions, at least in part to sequester zinc in pathophysiologic conditions.