Hepatic Metallothionein Synthesis Research Articles

Induction of Metallothionein by Manganese Is Completely Dependent on Interleukin-6 Production

Metallothionein (MT) is a cysteine-rich protein that binds to and is inducible by heavy metals such as cadmium and zinc. However, the precise mechanism of MT induction by other metals remains unclear. In the present study, we investigated the mechanism of MT induction by manganese, focusing on the involvement of cytokine production. Administration of MnCl(2) to mice resulted in the induction of MT dose-dependently in the liver with little accumulation of manganese. Speciation analysis of metals in the liver cytosol showed that the major metal bound to the induced MT was zinc. Administration of MnCl(2) caused an increase in mRNA levels of interleukin-6 (IL-6) in the liver as well as an increase in serum levels of IL-6 but not those of other inflammatory cytokines. Subsequently, serum levels of serum amyloid A (SAA), an acute-phase protein induced by IL-6, increased with a peak at 24 h. However, no increase in serum alanine aminotransferase activity was observed, suggesting that manganese enhanced the production of IL-6 and SAA without causing liver injury. In response to IL-6, the expression of a zinc transporter, ZIP14, was enhanced in the liver, possibly contributing to the synthesis of hepatic zinc-MT. In IL-6-null mice, the induction of hepatic MT by treatment with MnCl(2) was completely suppressed to the control level. These results suggest that manganese is a unique metal that induces the synthesis of hepatic MT completely depending on the production of IL-6 without accompanying liver injury.

Interferon alpha induction of metallothionein in rat liver is not linked to interleukin-1, interleukin-6, or tumor necrosis factor alpha

Synthesis of metallothionein (MT) is induced by interferon-alpha (IFN-α) in vitro and in vivo. In addition, IFN-α promotes redistribution of zinc (Zn) from the plasma to the liver in mice. However, it is not clear if IFN-α induces hepatic MT synthesis directly or indirectly via liberation of other cytokines. In order to address this issue, we determined hepatic MT levels, Zn concentration in plasma, liver, and urine, and plasma levels interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNFα) in rats following intramuscular injection of human IFN-α (1.5 × 10 6 UI/m 2). Animals were housed in metabolic cages and sacrificed at various times after IFN-α administration. Zn concentrations in serum, urine, and hepatic tissue were determined by atomic absorption spectrophotometry. MT protein was measured using the MT silver saturation method and expression of MT-1 and MT-2 mRNA was measured by RT-PCR. Plasma levels of rat IL-1, IL-6, and TNFα were determined using an ELISA method. Hepatic MT levels began to increase at 2 h following IFN-α administration and reached maximum levels at 12 h post-treatment. Induction of MT gene expression was confirmed by increases in MT-1 and MT-2 mRNA levels 6, 12, and 18 h after IFN-α administration. IFN-α treatment also resulted in biphasic increases in hepatic Zn, with levels peaking at 2 h, the time-point when MT levels are first increased, and again at 18 h. Concurrently, there were decreases in serum Zn levels at these time points, suggesting IFN-α induced movement of Zn from the blood to hepatic tissue. The decrease in serum Zn was not due to increased excretion since urinary Zn levels were unaffected following IFN-α treatment. IFN-α administration had no effect on plasma IL-1, IL-6, and TNFα levels. These results show that IFN-α promotes the increase of hepatic MT levels and plasma/liver redistribution directly, without IL-1, IL-6, or TNFα participation.

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.

Classical Conditioning of Metallothionein Synthesis in Mice

To clarify the role of metallothionein (MT), an anti-stress protein, induced by psychological stress, we carried out an experiment to determine whether hepatic MT synthesis induced by restraint stress can be classically conditioned by an olfactory odor cue of camphor. Mice were conditioned by exposure to the odor of camphor 1 hr before restraint stress and partly during restraint stress. Although exposure to the odor of camphor did not elevate hepatic MT levels in itself, the odor stimuli significantly elevated hepatic MT levels in mice conditioned for 3 days. The results indicate that MT synthesis is induced by an association of the central nervous system with psychological stress.

Site-Specific Induction of Metallothionein in N-Nitrosodimethylamine-Treated Rat Liver.

The effects of dietary protein and sulfur-containing amino acids on oxidative responses against N-nitrosodimethylamine (NDMA) treatment were investigated in rat liver, focusing on the mechanism of metallothionein (MT) induction. Rats were fed a 10% soybean protein isolate (SPI) diet or SPI supplemented with 0.3% L-methionine (SPI-Met) for 3 weeks after weaning. Rats fed on SPI showed lower glutathione (GSH) levels and higher MT levels in the liver than those fed on SPI-Met. After treatment with NDMA (20 mg/kg, i.p.), MT levels were elevated significantly in both groups, whereas GSH levels were only elevated in the SPI group. Pathologically, necrosis and hemorrhage were seen in the livers of NDMA-treated rats of both dietary groups, suggesting oxidative damage caused by NDMA treatment. Immunological histochemistry using anti-MT antibody showed that most of the MT was distributed in the cytosol region of all the rat groups. The ratios of the MT-positive cells were correlated to MT levels that were chemically analyzed. It was notable that the localization of MT staining in the hepatic lobules was altered after NDMA injection. The MT staining was principally observed around the central vein before injection, whereas it was more markedly observed at the peripheral region after NDMA treatment. These results indicate that the synthesis of hepatic MT basically occurs around the central vein with oxygen stress caused by insufficient oxygen supplement and/or stimulated oxygen consumption. NDMA could be oxidatively metabolized at the peripheral region with sufficient supplemental oxygen from the artery, resulting in oxidative stress and stimulation of MT synthesis there. Thus, distribution of tissue MT might vary depending on the site of oxidative stress.

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.

Bisphenol A enhances cadmium toxicity through estrogen receptor.

To clarify the action of estrogenic endocrine disruptors on cadmium (Cd)-induced metallothionein (MT) synthesis in the liver, we investigated the effects of bisphenol A (BPA) on hepatic MT-I mRNA expression and MT contents after Cd injection. Liver damage after Cd injection was assessed by measuring glutamic-pyruvic transaminase (GPT) and glutamic-oxaloacetic transaminase (GOT) activities in the serum. It was found that BPA reduced the Cd-induced expression of MT-I mRNA and MT protein in the liver. The administration of tamoxifen, an estrogen receptor antagonist, prevented the reduction of hepatic MT content by PA. Moreover both the GPT and GOT activities of the BPA-treated groups were higher than those of the control groups. These findings suggest that BPA reduced hepatic MT synthesis after Cd injection via the estrogen receptor which resulted in increased damage to the liver.

Metallothionein content increased in the liver of mice exposed to magnetic fields.

Although recent studies have shown that various stress can induce metallothionein (MT) synthesis in animal tissues, the induction of MT synthesis by exposure to static magnetic fields (SMF) has not been reported. We measured MT levels in the liver, kidney and brain of mice exposed to SMF and also evaluated the effect of SMF exposure on the induction of hepatic MT by treatment with carbon tetrachloride (CCl4). The MT content in the liver was significantly increased by exposure to 4.7 T of SMF for 6, 24, or 48 h, whereas that in the kidney or brain was not changed compared to the control. The combination of CCl4 injection and SMF exposure induced elevation of the hepatic MT content exceeding that induced by either treatment alone. These results indicate that exposure to the strong SMF induces MT synthesis in the liver in mice and enhances the hepatic MT synthesis induced by CCl4 administration.

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.

Effect of glutathione depletion on metallothionein synthesis induced by paraquat in mice.

The effect of Glutathione (GSH) depletion on the induction of metallothionein (MT) synthesis by paraquat (PQ) was examined in ICR mice. An increase in hepatic MT level in mice was observed after a single PQ administration. Pretreatment of mice with L-buthionine-SR-sulfoximine (BSO), an inhibitor of GSH synthesis, enhanced the induction of hepatic and renal MT synthesis by PQ depending on the decreased tissue GSH level. A similar result was obtained by pretreatment with diethylmaleate, a GSH depleting agent. The ratio of hepatic MT-I to MT-II induced by PQ was not changed by BSO pretreatment. An increase in the hepatic MT level in GSH depleted mice was observed from 3 hr to 24 hr after PQ administration. An increase in the hepatic MT-I mRNA level after treatment with PQ was observed prior to hepatic MT induction in BSO pretreated mice. Pretreatment with actinomycin D, an inhibitor of mRNA synthesis, inhibits the PQ-induced increase in hepatic MT and MT-I mRNA levels in BSO pretreated mice. Pretreatment with BSO did not affect the induction of MT synthesis by zinc, cadmium or dexamethasone. Pretreatment with dexamethasone, an anti-inflammatory agent, enhanced the hepatic MT induction by PQ treatment in GSH depleted mice, while dexamethasone reduced the MT induction by turpentine oil, which is known to induce inflammation and hepatic MT synthesis. These findings suggest that GSH depletion enhances the induction of MT synthesis by PQ because of an increase in the transcription rate, and this enhancement of MT synthesis is not due to an inflammatory response caused by PQ.

N-Hexane-Induced Synthesis of Hepatic Metallothionein Is Mediated by IL-6 in Mouse

The mechanism of metallothionein (MT) synthesis in the liver by n-hexane (HX) was examined. The increased synthesis of MT in the liver by HX was inhibited by dexamethasone pretreatment. Serum IL-6 was increased soon after HX injection, reaching a maximum at 8-16 hr, and then decreased, but neither IL-1 nor TNF was increased. The hepatic MT concentration reached a maximum later than did the serum IL-6 concentration, at 2 days after administration. When the MT synthesis induced by HX was inhibited by dexamethasone pretreatment, the concentration of IL-6 in the serum was suppressed to a very low level. Furthermore, the increase in concentration of hepatic MT and plasma fibrinogen was significantly decreased by the anti-mouse IL-6 monoclonal antibody. The concentration of hepatic MT was higher when the concentration of HX in the olive oil of the solution for injection was higher, even when the amount of HX administered was the same. It is suggested that the cytokine(s) is produced by the macrophage and fibroblast through injury and inflammation at the site of administration. These findings suggest that MT synthesis resulting from HX is induced indirectly through cytokine(s) production, especially IL-6.

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.

Differential stimulation of hepatic and brain metallothioneins by ethanol

Administration of ethanol induces the synthesis of hepatic metallothionein and metallothionein mRNA in the liver but not in the brain. Furthermore, ethyl alcohol, methyl alcohol and isopropyl alcohol enhance the synthesis of metallothionein in Chang cells but not in neuroblastoma IMR-32 cells in culture. The results of this study are interpreted to suggest that the mechanisms of synthesis of metallothionein and the utilization of essential metal nutrients in the brain and peripheral tissues are not identical.

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.

Distribution of cadmium in body organs and hepatic metallothionein content following chelation therapy.

1. Calcium trisodium diethylene triamine penta-acetate (CaNa3DTPA), sodium 1,2 diaminocyclohexane tetra-acetate (Na2CDTA), triethylenetetramine hydrochloride (TETA.HCl) and sodium diethyldithiocarbamate (NaDDC) were investigated for their efficacy to mobilize cadmium (Cd) from various tissues and hepatic metallothionein (MT) in Cd-exposed rats. 2. All chelating agents reduced the hepatic Cd burden but did not elicit any influence on other tissues, except that TETA.HCl lowered pancreas Cd and NaDDC increased brain Cd. 3. Cadmium-induced hepatic MT was lowered upon treatment with CaNa3DTPA while it increased further following treatment with NaDDC. 4. The chelating agents, in split doses, are capable of reducing the hepatic burden of Cd and altering the hepatic MT level. 5. While all the chelating agents decreased Cu content of hepatic MT, only CaNa3DTPA decreased its Zn content and TETA.HCl mobilized MT-bound Cd. 6. The administration of chelating agents alone in normal animals showed that NaDDC induced greater hepatic MT synthesis and increased MT-bound Zn than other chelating agents.

Effect of co-exposure to ethanol and cadmium in rats.

Metabolism and toxicity of heavy metals may be influenced by certain factors such as protein malnutrition, essential element deficiency or alcoholism. Ethanol has been found to enhance the absorption of lead in body and alcoholics have been reported to be more susceptible to lead intoxication. As alcoholism may be common among industry workers and a significant section of population, who may be exposed to cadmium, it was considered of interest to investigate the influence of ethanol-cadmium co-exposure on cadmium sensitive hepatic, renal and serum enzymes, tissue accumulation of cadmium, essential trace element status and cadmium induced hepatic metallothione in synthesis in rats.

The interaction of zinc and cadmium in the synthesis of hepatic metallothionein in rats

The interaction of injected zinc salts (Zn) and cadmium salts (Cd) with regard to the synthesis of metallothionein (MT) in adult rat liver was investigated. Male rats received an i.p. injection of Zn (20 mg/kg) or Cd (0.6 mg/kg) with or without pretreatment with Zn (20 mg/kg 16 h prior to the second injection). It was found that both metals, when administered singly, induced the synthesis of significant levels of hepatic MT, but that, when the Cd injection followed the Zn injection, synthesis of MT was not additive. When Zn pretreatment was followed by a second Zn injection, MT accumulation was additive (approx. 2-fold of that observed after a single Zn injection). Also, a highly significant positive correlation, ( r = 0.97, P < 0.01) was noted between hepatic Zn concentration and hepatic MT concentration, a relationship which was independent of the mode of MT induction. The results of the investigation indicate that: (1) in the presence of pre-existing hepatic Zn-MT, the ability of Cd to induce new MT synthesis is greatly reduced; rather, Cd is sequestered by the pre-existing MT; and (2) Zn may play a major role in the induction of MT synthesis both after Zn administration and after Cd administration.

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.