Metallothionein Synthesis Research Articles

Enhanced synthesis of metallothionein as a possible cause of abnormal copper accumulation in LEC rats

Long-Evans rats with a cinnamon-like coat color (LEC) is an inbred strain accumulating copper (Cu) in the liver abnormally and showing spontaneous hepatitis and hepatoma. The present study was intended to clarify how Cu accumulates in the LEC rat liver. For this purpose, the distribution profiles of Cu and zinc (Zn) and the inducibility of metallothionein (MT) synthesis were examined in the liver between Cu-loaded Long Evans agouti (LEA, the original strain of LEC) rats and were compared with those in control LEC rats. LEA rats (female, five weeks old) were injected subcutaneously with CuCl 2 daily at a dose of 3 mg Cu/kg body weight for 2, 4, 6, and 9 days. The concentration of Cu (124 μg/g) accumulated in the LEA rat liver after four injections was comparable to that in control LEC rats. Only 20% of Cu in the liver of LEA rats was recovered in the supernatant fraction in the form of MT, while Cu in the LEC rat liver (113 μg/g) was recovered mostly in the supernatant fraction, and was bound to MT. Although the increased concentration of Cu in the LEA rat liver was further elevated after additional injections of Cu, the amount of MT did not increase further. The MT mRNA content in the LEA rat liver remained lower than that of LEC rats even after further injections of Cu. Therefore, the present results suggest that LEC rats can accumulate Cu at a high concentration in the liver because of their extremely high inducibility of MT.

Involvement of metallothionein in cadmium accumulation and elimination in the clam Ruditapes decussata

Cadmium is one of the most toxic pollutants in seawater because of its persistence, toxicity and potential for bioaccumulation. It is included on the [open quotes]black list[close quotes] of several international agreements established to regulate the input of pollutants into the marine environment. The deleterious effects of cadmium contamination in marine organisms result from its accumulation within specific tissues. However, most of these organisms have developed subcellular detoxification processes, including the synthesis of metallothioneins, low-molecular weight, metal-binding proteins. Bivalves have the ability to accumulate and concentrate cadmium to levels several orders of magnitude above those found in their environment. The present study was designed to examine the involvement of metallothionein synthesis in cadmium accumulation and elimination in the bivalve Ruditapes decussata when exposed to a sublethal cadmium concentration (100 [mu]g/l) and to a mixture of cadmium (100 [mu]g/1), copper (50 [mu]g/l) and zinc (50 [mu]g/1). 15 refs., 2 figs., 1 tab.

Role of cytosolic copper, metallothionein and glutathione in copper toxicity in rat hepatoma tissue culture cells

Effects of metallothionein (MT) synthesis inhibiting compounds (actinomycin D, cycloheximide), MT synthesis stimulating compounds (dexamethasone, dibu-cAMP) and interfering metals (Cd, Zn) on copper accumulation were investigated in rat hepatoma tissue culture cells. Copper-metallothionein (Cu-MT) and Mt-associated copper levels were determined to find a possible correlation between cytosolic copper concentrations and MT as a Cudetoxifying protein. Further, intracellular non-MT associated copper levels and levels of GSH and SOD were determined. Cell viability was tested under all experimental conditions by measuring LDH-release, K + uptake and total cell protein. Administration of dexamethasone and dibu-cAMP showed no effect on MT levels (compared with controls), and only a marginal effect on 64Cu and total Cu accumulation. Administration of actinomycin D resulted in increased copper accumulation in the particulate fraction, possibly due to inhibition of copper secretion processes and/or protein synthesis. Presence of zinc had no effect on MT levels nor on total Cu and 64Cu levels, in contrast with cadmium which drastically enhanced copper accumulation and MT levels in the cells. Cu/MT ratios varied from 1.0 ± 0.3 to 1.2 ± 1.2, which is far below the assumed maximum molar ratio of 8–12 mol Cu per mol MT. SOD levels appeared to be enhanced up to 2- or 3-fold in the presence of Cd 2+, relative to control values. The role of GSH as Cu-intermediate in intracellular Cu distribution plus its role in copper defence mechanism(s) was tested by application of BSO, an inhibitor of GSH synthesis. It was found that BSO had no effect on intracellular MT level; it was found however that MT-bound copper levels were markedly decreased. The results presented support a model for copper metabolism in hepatoma tissue culture (HTC) cells, where Cu(I) is complexed by GSH immediately after entering the cell. GSH is capable of transferring copper to MT where it is stored. Depletion of GSH (by administration of Cd 2+, actinomycin D, cycloheximide) almost instantaneously results in enhanced cellular toxicity. When also MT is depleted (by actinomycin D) non-MT associated, ‘free’ cytosolic Cu 2+ is elevated, and HTC cells rapidly loose their resistance to copper toxicity, as also reflected in loss of cell viability (LDH, K + and total cell protein).

Subcellular Targets of Cadmium Nephrotoxicity: Cadmium Binding to Renal Membrane Proteins in Animals with or without Protective Metallothionein Synthesis

Subcellular Targets of Cadmium Nephrotoxicity: Cadmium Binding to Renal Membrane Proteins in Animals with or without Protective Metallothionein Synthesis

Functional integrity of metallothionein genes in testicular cell lines

The presence and inducibility of the major cadmium (Cd) chelating protein metallothionein (MT) in testicular cells has been controversial. In this study, the induction and production of MT in testicular cells were studied using mouse Leydig and Sertoli cell lines. Metal accumulation was studied by subjecting the cells to increasing levels of Cd. The presence of transcription factors for MT synthesis was analyzed by transfecting the cells with a reporter gene under the control of the MT promoter. The dose- and time-dependent induction of MT were conducted by Northern analyses. Expression of MT genes occurred in both Leydig and Sertoli cells. To avoid cross hybridization of the MT probe with mRNAs encoding testicular metal binding proteins and to investigate the integrity of MT mRNA, isoMT mRNA identification and primer extension experiments were performed. Those studies show that the induced mRNA indeed encodes MT. The biosynthesis of MT was confirmed by following 35S-cysteine incorporation into the protein. Finally, cadmium tolerance of testicular cells is compared with that of fibroblast cells. By these studies, we conclude that the MT genes are functional and inducible in testicular cells.

Characterisation of six additional human metallothionein genes

Human metallothionein (MT) genes are clustered in a locus on chromosome 16, and this report presents the characterisation of the remaining six uninvestigated members of the family. Nucleotide sequencing in whole or part suggested that four of these genes, MT1I, MT1J, MT1K and MT1L do not encode expressed MT proteins, based on the presence of structural faults or atypical amino acid assignments. On the other hand, the structures of MT1H and MT1X are consistent with these genes being functional and encoding unique type 1 isoforms. The promoters of both genes conferred activity to CAT expression constructs when transfected into HeLa cells, and showed differential responses to inducers of MT synthesis. Endogenous MT1H and MT1X genes were expressed at the mRNA level in HeLa cells following cadmium treatment. This work brings the number of functional class 1 and 2 MT genes in the human to eight, and confirms that each encodes structurally unique proteins.

Invertebrate organisms as biological indicators of heavy metal pollution

Some species of invertebrate animals are known to be efficient accumulators of trace elements. Generally, metal accumulation by such organisms is based on efficient detoxification mechanisms, such as intracellular compartmentalization, or metal inactivation by binding to metallothioneins. Metal accumulators have often been used as accumulation indicators of environmental metal pollution. This means that, ideally, metal concentrations in the animal's body reflect quantitatively or semiquantitatively environmental pollution levels. In reality, however, many factors, such as the animal's weight and age, can disturb such quantitative relationships. These factors have, therefore, to be considered carefully before an invertebrate is utilized as accumulation indicator for metal pollution. Apart from accumulation, many invertebrates exposed to elevated metal concentrations respond to this stress by metal-induced synthesis of metallothioneins. Additionally, metallothionein in metal-loaded organisms can be present in different isoforms that are specifically synthesized in response to different metals. These facts make metallothionein a potential biomarker for metal stress in invertebrates. One possibility may be to assess parameters of metallothionein synthesis at the molecular or biochemical level. Moreover, metallothionein isoform patterns could provide information on different isoforms synthesized in response to different metals or chemicals. In any case, however, care must be taken to consider intrinsic physiological parameters, such as nutritional or developmental factors, which could also interfere with metallothionein synthesis.

Influence of dietary iron deficiency on nickel, lead and cadmium intoxication

The influence of dietary iron deficiency on acute nickel, lead or cadmium toxicity as reflected by the induction of hepatic, renal, and intestinal metallothionein (MT), disposition of the metals and alterations in hematological parameters, was investigated in young rats to ascertain whether the toxic effects of these metals modify under anemic conditions. The administration of Cd induced hepatic, renal and intestinal MT while that of Ni or Pb induced hepatic MT only. While dietary Fe deficiency did not affect MT induction by Cd, it enhanced the synthesis of renal and intestinal MT by Ni and Pb. The accumulation of Pb in liver and kidney and that of Cd in liver only, were enhanced by Fe deficiency; the tissue deposition of Ni remained unaffected by Fe deficiency. The induction of hepatic MT by Ni, Pb or Cd appears to be related to the concomitant rise in the hepatic Zn, Ca and Fe levels in normal rats. However, dietary Fe deficiency increased the hepatic Zn in response to Ni or Cd and the hepatic Ca in response to Pb administration.

Cross-resistance to heavy metals in hydrogen peroxide-resistant CHO cell variants

Hydrogen peroxide-resistant Chinese hamster ovary (CHO) cells displayed cross-resistance to CdCl 2, HgCl 2 and NaAsO 2 but not to Na 2Cr 2O 7, ZnCl 2, NiCL 2 and CuSO 4. Resistance to hydrogen peroxide and to the metals was partially retained by these cells for many generations despite growth in drug-free medium. The loss of resistance was a slow process, and was different for the various metal compounds. Cell variants had a slightly higher content of non-protein intracellular thiols (NPSH) than sensitive cells. This biochemical feature did not seem to be the cause of resistance to CdCl 2 but accounted for at least part of the resistance to HgCl 2 and NaAsO 2. Increased metallothionein synthesis did not seem to be responsible for the metal-resistant phenotype. These results suggest that resistance to specific metal compounds in cultured mammalian cells adapted to hydrogen peroxide is dependent on a number of factors which differ for the various metal compounds and which are characterized by a different stability.

Zinc transport and metallothionein induction in primary cultures of rabbit kidney proximal cells

Primary cultures of isolated rabbit renal proximal cells were grown on collagen-coated permeable supports. The confluent epithelia were polarized, making possible the measurement of uptakes and effluxes across the apical and the basolateral membranes. Uptakes of 65Zn were assessed under initial rate conditions, after 0.5 min incubation. The kinetic parameters of apical uptake were a J max of 25.1 ± 5.3 pmol min −1 (μ DNA) −1, a K m of 43.3 ± 7.3 μ M and an unsaturable constant of 0.105 ± 0.029 ( n = 7) at 37°C. Cadmium competitively inhibited the zinc uptake, with a K i value of 24.5 ± 7.3 μ M . Basolateral uptake was characterized by a high capacity ( J max = 227.9 ± 46.6 pmol min −1 (μg DNA) −1) and an affinity similar to that of the apical uptake ( K m = 35.4 ± 14.2 μ M ). Cadmium had no effect on the basolateral zinc uptake. Effluxes across the basolateral face of the epithelium always exceeded those across the apical face. Excess zinc in the culture medium induced the synthesis of metallothionein in the epithelia, as judged by the rate of [ 35S]cysteine incorporation into a fraction of cytosolic proteins. Metallothionein induction did not appear to modify the kinetic parameters of the apical zinc uptake. These data suggest that separate saturable transport systems are responsible for the apical and basolateral zinc uptakes in proximal renal cells. Induction of metallothioncin had no apparent effect on apical zinc uptake in this system.

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.

Differential induction of metallothionein synthesis by interleukin-6 and tumor necrosis factor-alpha in rat tissues.

Metallothionein (MT) synthesis induced by the inflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF), was studied in vivo. Administration of recombinant human IL-6 or TNF to rats caused the acute phase responses including rapid decreases in plasma zinc (Zn), and increases in plasma copper (Cu) and ceruloplasmin. Hepatic concentration of MT-I, one of MT isoforms, began to increase within 3 h after the injection of IL-6 or TNF. In IL-6-treated rats, MT-I concentration in liver reached a maximum level at 12 h and decreased with a transient rebound, whereas, in TNF-treated rats, a high level of MT-I lasted for about 48 h. MT-II, the other MT isoform, was induced more than MT-I in liver by both cytokines. MT-I was also induced in lung and heart by TNF, but little by IL-6. The data suggest that IL-6 may be responsible for MT synthesis in liver, whereas TNF may be responsible not only in liver but also in lung and heart. Furthermore plasma concentration of MT did not always reflect the enhanced concentration of MT by TNF and IL-6 in liver, suggesting involvement of many factors influencing plasma MT levels. The interrelation between IL-6 and TNF for MT synthesis has also been discussed.

Metallothionein synthesis and degradation in Indian childhood cirrhosis fibroblasts.

Indian childhood cirrhosis (ICC) is a fatal liver disease characterized by the accumulation of copper-sulfur aggregates. We demonstrated that cultured fibroblasts from a patient with ICC contain vesicular aggregates, fibrillar whorls, and crystalloids along with dilated rough endoplasmic reticulum filled with flocculent material. Although the copper content of the fibroblasts was normal, both basal and metal-induced metallothionein (MT) synthesis was reduced in the ICC cells. The lower MT synthesis in ICC cells was seen at copper concentrations of 100, 200, and 400 microM, zinc concentrations of 50 and 100 microM, and a cadmium concentration of 2 microM. The lower MT synthesis in ICC cells was not due to failure of the cells to take up copper because 67Cu uptake kinetics were normal in the mutant cells. MT degradation was also normal in the ICC cells. The size of human MT IIA mRNA was normal in the ICC cells, but its amount was reduced under both basal and metal-induced conditions. The MT IIA gene, which is the predominant MT gene in human beings, showed no sequence alterations in any of its exons, introns, or promoter region in the ICC cells compared with normal cells. These studies demonstrate that this case of ICC represents a genetic disease with some level of expression in cultured fibroblasts; the basic defect may involve insufficient MT mRNA and protein synthesis for the copper load present. However, it remains to be determined whether reduced MT synthesis is a primary or secondary phenomenon.

Enhanced metallothionein gene expression is associated with protection from cadmium-induced genotoxicity in cultured rat liver cells.

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich proteins that appear to play an important role in the cellular defense system against cadmium toxicity. Although substantial evidence exists demonstrating a reduction in cadmium toxicity concomitant with MT induction, little is known about the possible effects of stimulation of MT synthesis on cadmium-induced genotoxicity. Thus, the alkaline elution technique was used to assess single-strand DNA damage (SSD) in TRL-1215 cells, a liver-derived cell line shown to have inducible MT gene expression. The SSD accumulated over a 2-h time period in a time-dependent manner following exposure to 500 microM CdCl2. Low-concentration cadmium pretreatment (10 microM CdCl2, 24 h) provided protection against the genotoxicity of high-concentration cadmium (500 microM CdCl2, 2 h). A 2-h exposure to 500 microM CdCl2 had no effect on viability, as assessed using a tetrazolium-dye based assay, in cells from either the pretreated or nonpretreated group. Metallothionein was induced in a time-dependent manner by low-concentration cadmium pretreatment: Exposure for 24 and 48 h resulted in 3.3- and 6.4-fold increases, respectively. In addition, a 24-h exposure to low-concentration cadmium resulted in an increase in MT-I gene expression. Cadmium accumulation was 2.6-fold greater in low-concentration cadmium-pretreated cells as compared to nonpretreated cells. These data demonstrate that low-concentration cadmium pretreatment provides protection against cadmium-induced single-strand DNA damage and support the hypothesis that this protection is due to stimulation of MT gene expression.

Purification and characterization of a cadmium-induced metallothionein from the shore crab Carcinus maenas (L.)

Two metallothionein variants were purified from the midgut gland of crabs (Carcinus maenas) exposed to a high cadmium concentration (2 p.p.m.). One of the variants was purified from crabs exposed to a low cadmium concentration (0.5 p.p.m.). The purification method involved acetone precipitation, gel filtration and reversed-phase h.p.l.c. The complete amino acid sequences of both variants have been elucidated by m.s. and automated sequence analysis on S-methylated proteins or fragments produced by cleavage of the S-methylated proteins with Staphylococcus aureus proteinase. The two variants from crabs exposed to the high cadmium concentration differed only by a single residue of methionine at the N-terminus. The single variant isolated from crabs exposed to the low cadmium concentration was the one without the N-terminal methionine, indicating that high cadmium concentrations either inhibit the processing enzymes and/or that the processing enzymes cannot keep pace with the increased metallothionein synthesis when cadmium availability is high. Cadmium-induced metallothionein from C. maenas shows a high degree of structural similarity to metallothioneins from the decapod crustaceans Scylla serrata and Homarus americanus.

Altered Zn status by α-hederin in the pregnant rat and its relationship to adverse developmental outcome

The hypothesis that an acute-phase reaction in the pregnant animal causes a systemic redistribution of Zn, resulting in a transient but developmentally adverse Zn deficiency in the embryo, was tested by treating pregnant rats during organogenesis with α-hederin, an agent reported to induce substantial metallothionein (MT) synthesis in rat liver, and determining hepatic MT concentration, hepatic and plasma Zn concentration, and systemic distribution of a pulse of 65Zn after treatment. Developmental toxicity was assessed by evaluating morphologic development in term fetuses. A single dose of α-hederin, injected sc at dosages of 3 to 300 μmol/kg, caused an acute phase response, indicated by decreased Fe and Zn, and increases Cu, α 1-acid glycoprotein, and ceruloplasmin concentration in plasma, along with a dosage-related increase in maternal hepatic MT concentration. The maximum induction of MT was 11 to 15-fold greater than control and occurred at dosages of 30 μmol/kg and higher, and MT concentration reached its peak 12 to 24 h after treatment. Zn concentration in liver and liver cytosol increased along with MT, reaching a maximum level at dosages of 30 μmol/kg and higher. Plasma Zn concentration decreased after α-hederin treatment to a level approximately 75% of control at a dosage of 30 μmol/kg and 50% of control at 300 μmol/kg. Therefore, hepatic MT induction was associated with most, but not all, of the decrease in plasma Zn concentration. Zn distribution was evaluated by giving an oral pulse of 65Zn 8 h after treatment with 0, 30, or 300 μmol/kg α-hederin on gestation day 11, and measuring 65Zn levels 18 h after treatment. The fraction of 65Zn distributed to the liver of treated rats (either dosage) was twice that of control, but distribution of 65Zn to other maternal tissues was decreased. 65Zn accumulation by conceptuses was significantly decreased, attributable to decreased accumulation in decidua, but not in visceral yolk sacs or embryos; however, at this stage of development the decidua accounts for a greater quantity of Zn than either of the other products of conception and may serve as the Zn-storing tissue for the conceptus. Both 30 and 300 μmol/kg increased resorption incidence, and 300 μmol/kg also decreased fetal weight and increased the incidence of abnormal fetuses. Serum collected from rats two hours after α-hederin treatment (i.e., before the onset of MT synthesis) supported rat embryo development in vitro, whereas serum collected 18 h after treatment did not. Adding Zn to this serum restored normal embryonic development. These data are consistent with the hypothesis that systemic changes in Zn status, brought about by a hepatic acute phase response, including a substantial induction of hepatic MT, may be a mechanism for maternally mediated abnormal development.

Protective effects of metallothionein on streptozotocin-induced diabetes in rats

The protective roles of metallothionein (MT) in streptozotocin (STZ)-induced pancreatic damage were investigated using zinc (Zn) as the inducer of MT synthesis in rats. Diabetes was produced in a group of Sprague Dawley rats by a single injection of STZ. In another group of rats, to induce the synthesis of MT, Zn was injected subcutaneously about 12 h before injection of STZ. Rats were sacrificed at about 30 h, 3 and 6 weeks after administration of STZ. The plasma glucose levels in STZ and Zn+STZ group increased significantly in 30 h, 3 and 6 weeks compared with the control group, but the level of glucose in Zn+ STZ group was lower than that in the STZ group. Both Zn and Mt levels increased in pancreas, liver, and kidney of rats injected with Zn or Zn + STZ. The induction of MT synthesis in pancreas of Zn-injected rats was also demonstrated by Western Blot analysis. Lipid peroxidation induced by STZ was inhibited significantly by pretreatment with Zn. Injection of STZ decreased the activities of superoxide dismutase (SOD) in plasma, liver and pancreas, but injection of Zn had no effect on the activities of SOD. The results support that pretreatment with Zn can partially prevent the development of diabetes induced by STZ injection and it may be related to the effects of MT as a scavenger for the oxygen free radicals.

Effects of copper on rat hepatoma HTC cells and primary cultured rat hepatocytes

Cellular responses to copper, applied in concentrations varying from 0.5 to 200 μ.M Cu 2+, were investigated in two different cell types: rat hepatoma cells (HTC) and primary cultured rat hepatocytes. Accumulation of 64Cu, copper (AAS) levels, cellular viability parameters (cell growth and proliferation, LDH leakage, total cell protein, K + uptake, and ATP levels), and cell toxicity parameters (metallothionein (MT), glutathione (GSH) and superoxide dismutase (SOD)) were examined over 24 hr incubation periods. Accumulation of radiolabeled copper (applied copper concentrations: 15–200 μM Cu 2+) showed a four-fold increase in HTC cells (0.88–3.45 nmol Cu/mg cell protein) and a three-fold increase in hepatocytes (4.94–14.66 nmol Cu/mg cell protein), although quantitative uptake in HTC cells was five times lower. Most of the copper accumulated in the hepatoma cells and hepatocytes was found predominantly in the particulate fraction (i.e., cell membranes and organelles), while only a small quantity was present in the soluble fraction (cell cytosol). Metallothionein concentrations in HTC cells were increased from 43 pmol/mg cell protein (0.5 μM Cu 2+ application) up to 223 pmol/mg cell protein (200 μM Cu 2+ application), whereas MT in rat hepatocytes were elevated from 139 pmol/mg cell protein to 546 pmol/mg cell protein over the same range of administered Cu 2+. Metallothionein synthesis rendered both cell types well equipped to deal with increasing intracellular copper levels. In hepatocytes however, MT synthesis resulted in decreasing non-MT-associated copper levels in the cytosol for Cu administrations up to 100 μM. Above that point however, MT failed to stay in line with increasing cytosolic Cu levels, resulting in cytotoxic effects shown by changes in cell viability and GSH/SOD levels. In HTC cells MT synthesis suppressed the free Cu levels in the cytosol to below 0.1 nmol Cu/mg cell protein over the total range of copper concentrations applied. The results presented indicate that hepatoma HTC cells are more capable of dealing with high accumulated Cu levels than the better established rat hepatocytes. Furthermore, it is clear that comparison of these two cell types regarding their ability to respond on (sub)toxic Cu should be discussed with full consideration for the copper applications involved.

成人歯肉由来線維芽細胞のＤＮＡ合成とタンパク質合成におよぼすカドミウムとフッ素の影響

Cadmium (Cd) and fluoride (F) affect the metabolism of hard tissues, but their effect on human beings is not yet known. The purpose of this study was to investigate the effect of Cd and/or F on DNA and protein syntheses of human cultured cells. Four human gingival fibroblast primary cultures (GF-5, 6, 12 and 13 cells) and two human malignant cell lines (HeLa S3cells and KU-2 cells) were used. All cells were grown in Eagle's minimum essential medium supplemented with 10% fetal calf serum. After 24hr incubation, cells were cultured in media containing CdCl2 and/or NaF in order to study their growth, DNA and protein synthesis, and intracellular Cd level. The growth and DNA synthesis of cells were inhibited by 2.0mM NaF or 10-4mM CdCl2. The incorporation of 14C-leucine in cells was increased by 0.5mM NaF or 10-5mM-10-4mM CdCl2. The enhancement of incorporation of 14C-leucine was greater in cells exposed to NaF plus CdCl2 than in cells exposed to CdCl2 alone. Increased incorporation of 35S-cysteine in cells was observed with 10-5mM-10-4mM CdCl2 or NaF plus CdCl2. However, F had no effect on the incorporation of 35S-cysteine. Gel filtration was performed with Sephadex G75. 35S peak was found in an metallothionein (MT) fraction. The intracellular Cd level was lower in gingival fibroblasts than in malignant cells. These results indicate that Cd induced the synthesis of MT in both human gingival fibroblasts and malignant cells. It also seems that F has the ability to stimulate the inducement of MT synthesis by Cd.

Zinc-induced metallothionein synthesis could protect from gentamicin nephrotoxicity in suspended proximal tubules of rats.

We have reported that preinjection of zinc could ameliorate gentamicin-induced nephrotoxicity in vivo via the induction of metallothionein. The present project was designed to study whether preinjection of zinc could protect from the harmful effects of gentamicin on DNA synthesis, Na(+)-K(+)-ATPase activity, and free radical production in rat renal proximal tubules (PT) in vitro. PT were prepared and cultured from normal, saline-preinjected, and Zn-preinjected (Zn 10 mg/k/day s.c. 5 days) Wistar male rats weighing 80-120 g. The results showed that DNA synthesis and Na(+)-K(+)-ATPase activity were significantly suppressed in the normal and saline-preinjected rats' PT by addition of gentamicin to the media in the concentration of 12.4 mg/mL; however, these reactions were not suppressed by gentamicin in Zn-preinjected rats' PT, and malondialdehyde and hydroxyl radical production in Zn-preinjected rats' PT were significantly lower than those in the normal and saline-preinjected rats' PT (p < .01). On the other hand, the metallothionein in Zn-preinjected rats' PT was very significantly higher than that in the normal and saline-preinjected rats' PT (p < .001). Our data indicate that gentamicin-induced suppression of DNA synthesis and Na(+)-K(+)-ATPase activity in rat PT, which lead to renal injury, may be relevant to free radicals generated by gentamicin; and that preinjection of zinc could ameliorate gentamicin-induced nephrotoxicity via the induction of the metallothionein synthesis in rat PT to scavenge free radicals generated by gentamicin.