Rat Liver Metallothionein Research Articles

Type I Interferons Ameliorate Zinc Intoxication of Candida glabrata by Macrophages and Promote Fungal Immune Evasion.

SummaryHost and fungal pathogens compete for metal ion acquisition during infectious processes, but molecular mechanisms remain largely unknown. Here, we show that type I interferons (IFNs-I) dysregulate zinc homeostasis in macrophages, which employ metallothionein-mediated zinc intoxication of pathogens as fungicidal response. However, Candida glabrata can escape immune surveillance by sequestering zinc into vacuoles. Interestingly, zinc-loading is inhibited by IFNs-I, because a Janus kinase 1 (JAK1)-dependent suppression of zinc homeostasis affects zinc distribution in macrophages as well as generation of reactive oxygen species (ROS). In addition, systemic fungal infections elicit IFN-I responses that suppress splenic zinc homeostasis, thereby altering macrophage zinc pools that otherwise exert fungicidal actions. Thus, IFN-I signaling inadvertently increases fungal fitness both in vitro and in vivo during fungal infections. Our data reveal an as yet unrecognized role for zinc intoxication in antifungal immunity and suggest that interfering with host zinc homeostasis may offer therapeutic options to treat invasive fungal infections.

Induction of Metallothionein in Rat Liver by Zinc Exposure: A Dose and Time Dependent Study.

Metallothioneins (MTs) are low molecular weight ubiquitous metalloproteins with high cysteine (thiol) content. The intracellular concentration of zinc (Zn) is tightly regulated and MT plays a crucial role in it. The present study investigates the relationship between the Zn status (as a function of Zn concentration and time) in the rat liver and the occurrence of hepatic MT. For dose dependent study, four experimental groups, one control and three receiving different levels of metal supplementation, were chosen [Group 1 control and Group 2, Group 3, Group 4 receiving subcutaneous dose of 10, 50 and 100mg of Zn/kg body weight (in the form of ZnSO4·7H2O), respectively]. For the time dependent expression of MT, again four experimental groups, i.e. Group 5 control and Group 6, Group 7, Group 8 receiving 50mg of Zn/kg body weight (in the form of ZnSO4·7H2O) subcutaneously and sacrificed at different time intervals after last injection i.e. 6, 18, 48h, respectively were chosen. Isolation of MT was done by using combination of gel filtration and ion exchange chromatography while characterization of MT fraction was carried in the wavelength range 200-400nm. Expression of MT was studied by using Western blot analysis. The results revealed that the MT expression increases with increasing the dose of Zn administered and maximum at 18h after last Zn injection. Accumulation of MT with increase dose would help in maintaining the intracellular Zn concentration by its sequestration which further reduces the possibility of undesirable binding of Zn to other proteins significantly and maintains Zn homeostasis. The maximum expression of MT at 18h is indicative of its half life.

Psychological stress induced zinc accumulation and up-regulation of ZIP14 and metallothionein in rat liver

BackgroundZinc is necessary for normal liver function; and vice versa, the liver plays a central role in zinc homeostasis. The aim of present study is to assess the effects of repeated psychological stress (PS) on the zinc metabolism and related mechanism involved in zinc homeostasis in rat liver.MethodsIn present study, we used communication box to create PS model and investigated the serum corticosterone (CORT), zinc level in serum and liver, liver metallothionein (MT) content and ZRT/IRT-like Protein 14 (ZIP14) mRNA expression.ResultsThe results showed that the serum CORT level increased and serum zinc level decreased significantly after 7 d and 14 d PS treatment. Meanwhile, zinc and MT contents in liver were elevated after 14 d PS exposure, while those in 7 d PS exposure group did not change. ZIP14 mRNA was expressed markedly at 7 d after the onset of PS, while Zip14 mRNA expression in the liver after 14 d PS exposure reached normal level compared with control group.ConclusionsThe results suggest that PS exposure could induce hypozincemia, which might be related to liver zinc accumulation because of high level of MT through glucocorticoid-mediated MT synthesis and ZIP14 expression induced by interleukin-6.

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

An experiment was conducted to invest effects of chronic cadmium poisoning on Zn, Cu, Fe, Ca, and metallothionein gene expression and protein synthesis in liver and kidney in rats. Forty rats, 6 weeks old, were randomly allocated into two groups. A group was given CdCl(2) (1 mg/KgCd(2+)) by intraperitoneal injection once a day. The other group was treated with normal saline in the same way. Liver and kidney were collected for analysis at the end of the third week. Results showed that Cd exposure increased Cd (P<0.01) and Zn (P<0.01) content both in liver and kidney. Fe and Ca concentration had a considerable increase in kidney (P<0.01), while both had different degree reduction in liver. Discrepancies between MT mRNA and protein were observed in liver and kidney. In liver, both MT mRNA and protein had a significant increase (P<0.01), while in kidney, only MT gene increase was checked. Meanwhile, the expression levels of MT-1 mRNA and MT-2 mRNA were distinct between liver and kidney. The present study indicated that changes in tissue Cd and Zn levels tended to reflect MT mRNA expression, but bear no clear relationship with MT protein. There did not have a strict dose-dependent relationship among Cd content, MT gene expression, and MT protein synthesis. What is more, changes of Zn, Fe, Cu, and Ca had a certain interaction with both MT mRNA and protein.

Alignment of Gold Clusters on DNA via a DNA-Recognizing Zinc Finger-Metallothionein Fusion Protein

The complementary recognition of base pairs (bp) is the major strategy in the "DNA lithography" of gold (Au)clusters or nanoparticles, where single-stranded DNAs sulfurized at their termini are generally used to bind Au clusters or nanoparticles. In this report, we discuss a new material that can be used to locate Au clusters on the desired positions of DNA. For this purpose, we combined a two-domain zinc finger (ZF) and the analogue of R domain of rat's liver metallothionein (MT) to utilize the DNA-recognizing ability of ZF motifs and the heavy metal binding ability of MTs, and prepared an artificial fusion protein, ZFZF-MTalpha (1). Titration experiments monitored by absorption and circular dichroism spectroscopies, as well as an electrophoretic mobility shift assay and quantification using 5,5'-dithiobis(2-nitrobenzoic acid), clarified that (1) the ZF domain traps two divalent metal ions to fold in a ZF structure with M(Cys)2(His)2 (M = Co, Zn, and Cd) coordination units, (2) the MT domain traps metal ions to form clusters (Cd2+ particularly forms a Cd4(Cys)9 cluster) without interfering with the folding of the ZF domain, and (3) 1 recognizes the 5'-GGGGGG-3' (G6) bp sequence in the presence of Zn2+ based on the amino acid sequence encoded in the ZF domain. The titration of Au11(PPh3)8Cl3 (Au11) into the solution of 1 in the presence of Zn2+ revealed that the MT domain strongly binds the Au11 cluster with a 1:1 ratio, and a Au11-containing conjugate, ZF(Zn)ZF(Zn)-MTalpha(Au11) was obtained. Transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy showed that this conjugate maintains an Au11 core in the form of Au11(PPh3)4(S-Cys)6 without disturbing the folding nature of the ZF domain. ZF(Zn)ZF(Zn)-MTR(Au11) recognizes the bp sequence G6 with K(d1) = 450 nM, while simultaneously forming a dimer on the DNA with K(d2) = 200 nM. TEM experiments showed that the conjugates form parallelograms or triangles (defective parallelograms) on a double crossover (dx) DNA, according to the positions of G6 encoded in the dx DNA.

Conjugation of Au11 cluster with Cys-rich peptides containing the α-domain of metallothionein

The stoichiometrically controlled complexation of abiological inorganic clusters with proteins is a challenging task that enables us to combine the functions of inorganic clusters with those of biological systems on the basis of chemical characterisation. In this study, we synthesised a 1:1 conjugate between Au11(PPh3)8Cl3 (Au11) and CGMTIIalpha, where Au11 is the smallest cluster, which is situated on the boundary of molecules and nanoparticles, having discrete electronic states and exhibiting Coulomb blockade; on the other hand, CGMTIIalpha is a twelve-cysteine (Cys)-containing peptide consisting of the alpha-domain of rat liver metallothionein II (MTIIalpha)--a heavy metal-binding protein--and an addendum, cysteine-glycine (CG). The Cys residue of the N-terminus is used as a site for elongation by the native chemical ligation (NCL) method. Titration experiments monitored by circular dichroism spectroscopy indicate that CGMTIIalpha reacts with Au11 in a ratio of 1:1. The product exhibits a negative Cotton effect at 222 nm, indicating the formation of a short a-helix. The composition of this hybrid module is quantified as Au11(PPh3)4(S-Cys)6 by transmission electron microscopy (core size observation), energy-dispersive X-ray spectroscopy (elemental analysis), Ellman test (uncoordinated Cys residues), and high-performance liquid chromatography (quantification of eliminated PPh3). These results, as well as those obtained from the simulations of Au 4f X-ray photoelectron spectra, suggest that the Au cluster of this product is Au11(PPh3)4(S-Cys)6. We also prepared F3F3-CGMTIIalpha, where F3F3 is a zinc finger peptide with two domains, in order to demonstrate the applicability of the NCL method to the elongation of CGMTIIalpha. F3F3-CGMTIIalpha also reacts with Au11 to yield a 1:1 conjugate without interfering in the folding of the zinc finger.

Production of Metallothionein Polyclonal Antibodies Using Chickens as Model

The production of polyclonal antibodies (pAbs) against metallothioneins (MT) has been done in mammals. In this work, we describe a model where pAbs against rat liver MT were produced in chickens. Liver MT-1 and MT-2 isoforms isolated from rats were used as immunogens. MT was purified by exclusion chromatography and MT isoforms isolated by ionic exchange chromatography. Chickens were immunized with each isoform emulsified with Freund adjuvant over 6 weeks. MT-pAbs obtained from egg yolk were purified by ammonium sulfate precipitation followed by thiophilic interaction chromatography. MT-pAbs were characterized by ELISA, SDS-PAGE electrophoresis, and Western blot assays. Results showed significant titers (1:1,000) of MT-1 and MT-2 IgY in the eggs collected 30 days after the first immunization as determined by a direct ELISA assay; results also show a cross-reaction between MT-1 and MT-2 isoforms: however, the Abs obtained did not react with other non-MT proteins in hepatic homogenates. Sensitivity assays showed that MT-pAbs detected MT-1 and MT-2 at nanogram levels. These data suggest that chickens are an alternative model for producing pAbs against mammal high-homology proteins such as MT.

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.

Metal release in metallothioneins induced by nitric oxide: X-ray absorption spectroscopy study.

Metallothioneins (MTs) are low molecular weight proteins that include metal ions in thiolate clusters. The capability of metallothioneins to bind different metals has suggested their use as biosensors for different elements. We study here the interaction of nitric oxide with rat liver MTs by using in situ X-ray absorption spectroscopy techniques. We univocally show that the presence of NO induces the release of Zn atoms from the MT structure to the solution. Zn ions transform in the presence of NO from a tetrahedral four-fold coordinated environment in the MT into a regular octahedral six-fold coordinated state, with interatomic distances compatible with those of Zn solvated in water.

Investigation of metal complexes with metallothionein in rat tissues by hyphenated techniques

The potential of hyphenated techniques based on a combination of microbore reversed-phase (RP) HPLC or capillary zone electrophoresis (CZE) with inductively coupled plasma (ICP) or electrospray (ES) mass spectrometry (MS) was demonstrated for the characterization of metal complexes with metallothionein in rat liver and kidney. The mixture of MT complexes was isolated from the tissues by size-exclusion LC and further characterized in neutral pH conditions (pH 6.8–7.2) by RP-HPLC or CZE. The metal stoichiometry and the molar mass of the eluted complexes was measured by ICP-MS and ES-MS, respectively. An additional dimension to the analysis was achieved by post-column acidification of the chromatographic eluent that allowed the determination of the molecular weight of the demetallated complexes with 10-fold higher sensitivity. The approach allowed the detection of two major metallothionein (MT) isoforms (MT-1 and MT-2) in liver and one MT isoform in kidney. The actual number of peaks in chromatograms and electropherograms was bigger because of the formation of mixed Cd–Cu complexes of the same MT isoform that showed different hydrophobicities.

Analysis for metal complexes with metallothionein in rat liver by capillary zone electrophoresis using ICP double-focussing sector-field isotope dilution MS and electrospray MS detection

The complementarity of ICP sector-field double-focussing (SF-DF) MS and electrospray MS detection in capillary zone electrophoresis (CZE) was studied for the identification and determination of metal complexes with metallothionein (MT) induced in the liver of a rat exposed to intravenously administered Cd2+. The MT fraction investigated was isolated by size-exclusion chromatography, preconcentrated by lyophilization and desalted. CZE-ICP MS allowed the separation of four mixed-metal complexes with one or more MT isoforms. Isotope dilution analysis was developed for the accurate quantification of the MT content in each of the species and the determination of the stoichiometry of the metal complex. A multi-isotope spike (33S,106Cd, 65Cu, 70Zn) was supplied on-line in the makup flow (6 l min−1) of a self-aspirating total consumption micronebulizer that served as the interface. The characterization of the MT fraction was completed by electrospray MS, allowing the determination of the molecular masses of the complexes formed and the identification of the ligands as MT-1 and MT-2 isoforms.

Distribution of zinc-binding metallothionein in cirrhotic liver of rats administered zinc.

This study was undertaken in order to investigate the effect of zinc (Zn) administration on induction of Zn-binding metallothionein in rat liver with thioacetamide-induced cirrhosis, and the localization of metallothionein in the liver. Normal and cirrhotic rats received intraperitoneal injections with or without Zn. Subsequently, metal analyses, purification of metallothionein by gel filtration and immunohistochemical assessments of metallothionein were carried out. Although in Zn-injected cirrhotic rats, the Zn contents in the liver and plasma increased significantly depending upon the dose of Zn, the Zn contents in the liver and plasma of the cirrhotic rats were lower than those of normal rats after the same dose of Zn. The results of gel filtration also showed that the levels of Zn-metallothionein in the cirrhotic liver were reduced in comparison with those of the normal liver. By the immunohistochemical method, the presence of metallothionein in the parenchymal areas but not in the fibrotic areas of the cirrhotic liver was confirmed. These results suggested that the induced metallothionein was only located in the parenchymal areas. The metallothionein induced in the parenchymal areas was considered to play a role in protecting the parenchymal cells against the progression of fibrosis, because metallothionein has been thought to be involved in the cellular defense against oxidative stress.

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

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

Immunohistochemical localization of metallothionein in rat liver, kidney and small intestine after dietary zinc manipulations: [formula omitted]. Foods & Nutrition Dept., Univ. of Manitoba, Winnipeg, MB, R3T 2N2

Whey protein concentrate (WPC) was oxidized by peroxyl radicals derived from 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) and the kinetics of droplet stability in O/W and W/O emulsions stabilized by oxidized WPC were evaluated by studying the micro-rheology. Degrees of protein oxidation were indicated by carbonyl concentration and emulsion types were distinguished by fluorescence microscopy. Oxidation resulted in free sulfhydryl groups degradation and surface hydrophobicity decrease. Moderate protein oxidation promoted to form diminutive droplets, which aggregated quickly to gel-network structure and decreased the motion rate of droplets, leading to the increased elasticity and viscosity, which led to better stability. Over-oxidation underwent severe droplet aggregation and sediment with increased motion rate, which resulted in instability of emulsions. The W/O emulsions of oxidized WPC were more inclined to block the motion of droplets and form a stable structure with higher viscosity, compared with the O/W emulsions.

Conformational changes and surface binding property of rat liver Cd5Zn2‐metallothionein

AbstractA series of conformational changes of native rat liver metallothionein were observed under different ionic strength by 1H NMR. Furthermore, binding of magnetic probes to MT gives us an implication that positively charged groups of lysine are not completely folded back towards the inner core of metallothionein, the negatively charged metal‐sulfur core is slightly exposed to the solvent.

Rat liver metallothioneins are metal dithiolene clusters

Abstract Rat liver metallothioneins (MTs) were studied by means of differential pulse polarography (DPP) and preparative electrolyses in aqueous buffered phosphate solutions (0.05 mol l−1) at micromolar concentrations. DP polarograms of MTs involving cadmium and zinc, (Cd,Zn)MT, showed four peaks at approximately −0.45 (A), −0.55 (C), −0.85 (B) and −1.1V (D) vs. SCE. Remetallation reactions of the apoprotein, thionein, allowed peaks A and B to be attributed to the two-step reduction of cadmium, and peaks C and D to the two-step reduction of zinc centres. Copper in MTs showed two DPP peaks only in the presence of EDTA (at −0.35 and −0.75V). The two-step one-electron reduction of bound metals (proved by coulometry for the case of cadmium thionein) indicated the metal dithiolene character of the metal core in MTs.

Metallothionein: A potential marker for differentiating benign and neoplastic gastrointestinal lymphoid infiltrates

Metallothioneins (MT) are low molecular weight, metal-binding proteins. The induction of MT synthesis by cytokines, hormones and other cytotoxic agents indicates its role in cellular proliferation and differentiation as well as in cellular defense mechanisms. Previous studies have detected expression of MT in various human tumors by immunohistochemical staining. In certain cases the presence of MT in a tumor may be associated with its resistance against radiation and chemotherapeutic agents. Immunohistochemical staining of MT using a rabbit polyclonal anti-rat liver MT antibody was carried out in eight gastric, two small bowel and one large bowel lymphomas, and in ten control gastrointestinal (gastric, colonic and small bowel) biopsies or excised bowel segments with benign lymphoid infiltrates. The primary antibody against rat liver MT readily cross-reacts with human MT. The neoplastic cells in nine of 11 malignant lymphomas showed weak to intense staining for MT, mostly in cytoplasm. In these cells a few nuclei (less than 5% of all tumor cells) were stained positively for MT. The benign lymphocytes in the gastrointestinal excised specimens and biopsies were mostly negative; four cases showed few positive cells. The giant cells were also positive for MT in a Crohn's disease case. We conclude that the presence or absence of MT in lymphocytes, detected by immunohistochemistry, may indicate the growth patterns of these cells. The distinct pattern of MT staining in malignant lymphoma in our study is suggestive of a potential use of MT staining as a discriminator between benign and malignant lymphoid tissues.

Cadmium affects the activity of rat liver tyrosine aminotransferase and its induction by dexamethasone.

The effects of cadmium (Cd) administration to intact rats on hepatic glucocorticoid receptor (GR) steroid binding capacity and DNA-binding ability were examined and correlated with the influence of the metal on rat liver tyrosine aminotransferase (TAT) activity and its induction by dexamethasone. It was found that 24 h after i.p. administration of Cd doses ranging from 0.5 to 4 mg/kg, the GR steroid- and DNA-binding activities were significantly reduced in a dose-dependent manner. The same doses of Cd also affected the basal and dexamethasone-induced level of TAT activity, as well as the concentration of metallothionein in rat liver. The decrease in TAT activity and in its induction by dexamethasone observed in response to low Cd doses was proportional to the alterations of the GR functional properties. Higher doses of Cd, which were more effective in reducing both the GR binding of the hormone and to DNA, however, stimulated TAT activity and potentiated dexamethasone induction of the enzyme. The results led to the conclusion that Cd may alter physiological response of rat liver cells to glucocorticoids interfering with the GR-dependent transcriptional regulation of the TAT gene.

Differential sensitivity of metallothionein-1 and -2 in liver of zinc-injected rat toward proteolysis

The characterization of degradation products of metallothionein (MT) in rat liver is reported. Using gel filtration chromatography on Sephadex G-75 an unknown zinc (Zn)-containing peak was found in livers of rats injected with zinc. It contained at least 13 peptide fractions, designated as LPI-1, LPI-2, LPI-3, LPI-4, LPII-1, LPII-2, LPII-3, LPIII-1, LPIII-2, LPIII-3, LPIII-4, LPIII-5 and LPIII-6. While two of these fractions (LPII-1 and LPIII-4) were identified as the carboxyl-terminal half (α-domain) of rat MT-1, LPI-2 and LPI-3 corresponded to the amino-terminal half (β-domain) of rat MT-2. In contrast, neither the β-domain of rat MT-1 nor the α-domain of rat MT-2 could be recovered. The data may indicate that the two metal clusters of Zn-MT-1 and Zn-MT-2 differ in their stabilities resulting in differences in their susceptibility towards proteolytic degradation.

Evidence that nitric oxide enhances cadmium toxicity by displacing the metal from metallothionein.

Cadmium is carcinogenic in humans and rodents. Although extensive evidence indicates that the toxicity and genotoxicity of Cd is ameliorated by binding to cysteine clusters in metallothionein (MT), the factors governing Cd release at intracellular target sites remain unknown. Nitric oxide is a pollutant gas and an important intercellular messenger in the inflammatory immune response. When growing Chinese hamster ovary cells were treated for 24 h with 0.5, 0.75, or 1.0 mM CdCl2 followed by a 1-h exposure to 1.0, 1.5, or 2.0 mM 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO), an NO-generating sodium salt, NO enhanced Cd-induced inhibition of colony forming ability without affecting Cd-induced cytolethality. In experiments designed to determine whether NO acts by displacing Cd from cellular MT, cells treated with 2.0 mM CdCl2 followed by 1.5 or 3.0 mM DEA/NO exhibited 29 and 38% reductions, respectively, in the amount of Cd bound to MT. When purified rat liver MT was used to further characterize NO-induced release of Cd from MT, dose-related increases in Cd displacement were observed at DEA/NO concentrations between 0.1 and 0.5 mM, and a plateau was reached at 3 mol of Cd displaced/mol of MT at higher DEA/NO concentrations. Compared to cells exposed to Cd or DEA/NO alone, cells treated with Cd followed by DEA/NO also exhibited a transient 2-3-fold decrease in c-myc proto-oncogene expression. Taken together, our results support the hypothesis that NO mediates Cd release from MT in vivo and suggest that intracellular generation of free Cd may induce DNA damage and force cells into a period of growth arrest. Such findings may have particular relevance with regard to the etiology of Cd-induced carcinogenesis in human populations.