Dietary zinc attenuates renal lead deposition but metallothionein is not directly involved

Chronic lead exposure irreversibly damages the kidneys and may be associated with hypertension and renal insufficiency at sub-clinically toxic levels. Zinc supplementation reduces lead absorption and tissue retention in rodent models but the mechanisms are unknown. Metallothionein (MT) may function in lead detoxification. Our objective was to investigate the effects of marginal zinc (MZ) and supplemental zinc (SZ) intakes on renal lead and zinc accumulation, renal MT immunolocalization and levels. Weanling Sprague Dawley rats were assigned to MZ (8 mg Zn/kg diet), zinc-adequate control (CT; 30 mg Zn/kg), zinc-adequate diet-restricted (DR; 30 mg Zn/kg) or SZ (300 mg Zn/kg) groups, with and without lead acetate-containing drinking water (200 mg Pb/L) for 3 weeks. Kidneys were analyzed for lead and zinc by inductively coupled plasma spectroscopy and MT by immunolocalization and Western blotting. MZ had higher renal lead and lower renal zinc concentrations than CT. SZ was more protective than CT against renal lead accumulation. Renal MT levels reflected dietary intake (SZ ≥ DR ≥ CT ≥ MZ) but lead had no effect on MT staining intensity, distribution, or relative protein amounts. In summary, while SZ lowered renal lead concentration, MT did not appear to function in renal lead accumulation. Future studies should explore alternate mechanisms of renal lead detoxification.     

The zinc/thiolate redox biochemistry of metallothionein and the control of zinc ion fluctuations in cell signaling

Free zinc ions are potent effectors of proteins. Their tightly controlled fluctuations ("zinc signals") in the picomolar range of concentrations modulate cellular signaling pathways. Sulfur (cysteine) donors generate redox-active coordination environments in proteins for the redox-inert zinc ion and make it possible for redox signals to induce zinc signals. Amplitudes of zinc signals are determined by the cellular zinc buffering capacity, which itself is redox-sensitive. In part by interfering with zinc and redox buffering, reactive species, drugs, toxins, and metal ions can elicit zinc signals that initiate physiological and pathobiochemical changes or lead to cellular injury when free zinc ions are sustained at higher concentrations. These interactions establish redox-inert zinc as an important factor in redox signaling. At the center of zinc/redox signaling are the zinc/thiolate clusters of metallothionein. They can transduce zinc and redox signals and thereby attenuate or amplify these signals.     

Expression of metallothionein in the liver and kidney of rats is influenced by excess dietary histidine

It is well known that excess dietary histidine induces the metabolic changes in copper and zinc. Therefore, this study was carried out to clarify whether excess dietary histidine alters the gene expressions of metallothionein-1 and metallothionein-2 in the liver and kidney. Male rats were fed the control (ad libitum and pair-fed) or histidine-excess (50 g of L-histidine per kg of diet) diet for 0, 1 and 3 days. The levels of liver metallothionein-1 and metallothionein-2 mRNA were markedly lower in the rats fed the histidine-excess diet as compared to those of the control (ad libitum and pair-fed) diet, when fed for 1 or 3 days. The levels of renal metallothionein-1 and metallothionein-2 mRNA in the rats fed the histidine-excess diet were higher or tended to be higher as compared with the rats fed the control (ad libitum and pair-fed) diet when fed for 1 or 3 days, respectively. At the same time, hepatic copper content was decreased and renal zinc content was increased by dietary histidine. It thus appears, that such a response on the level of liver metallothionein mRNA might be related to the contents of liver copper, but of kidney metallothionein mRNA might be due to the content of zinc.     

In vivo and ex vivo displacement of zinc from metallothionein by cadmium and by mercury

Divalent cadmium and mercury ions are capable in vitro of displacement of zinc from metallothionein. This process has now been studied in vivo and ex vivo, using the isolated perfused rat liver system, in order to determine if this process can occur in the intact cell. Rats with normal and elevated (via preinduction with zinc) levels of hepatic zinc thionein were studied. Cd(II) completely displaces zinc from normal levels of metallothionein and on a one-to-one basis from elevated levels of metallothionein, both in vivo and ex vivo. Hg(II) displaces zinc from metallothionein (normal or elevated) rather poorly, as compared with Cd(II), in vivo, probably due to the kidneys preference for absorbing this metal. Ex vivo Hg(II) displaces zinc from metallothionein (normal or elevated) on a one-to-one basis, with considerably more mercury being incorporated into the protein than in vivo. The results of double-label ex vivo experiments using metal and [35S]cysteine (+/- cycloheximide) were consistent with the above experiments, indicating that de novo thionein synthesis was not required for short term incorporation of cadmium and mercury into metallothionein. These data are supportive of the hypothesis that cadmium and mercury incorporation into rat hepatic metallothionein during the first few hours after exposure to these metals can occur primarily by displacement of zinc from preexisting zinc thionein by a process which does not require new protein synthesis.     

Metallothionein, zinc, and mercury levels in tissues of young rats exposed to zinc and subsequently to mercury

Several studies have described mercury toxicity and the role of metallothioneins (MT) in the detoxification and regulation of metal homeostasis. However, little data exist on this topic during the specific post-natal developmental phase in young mammals. This developmental phase is particularly important since young animals are more sensitive to toxicants than adults. The objective of this work was to investigate whether MT participates in the mechanism of protection conferred by zinc pre-treatment on the toxic effects induced by mercury in neonate rats. Pups were exposed to ZnCl(2) (5 doses of 27 mg/kg/day, s.c.) and subsequently to HgCl(2) (5 doses of 5 mg/kg/day, s.c.); metal (Zn and Hg) and MT contents were analyzed in the liver, kidney, and blood. MT was induced in the liver and kidney of pups of both Zn-sal and Zn-Hg groups, although the greatest increase was in neonates exposed to Zn only. A direct relationship exists between MT and metals for both hepatic and renal tissues, which indicates that the increase in metal levels occurs in parallel to the increase in MT content. Although the heat-treated cytosolic fraction is rich in MT and metals, higher Zn and Hg contents were detected in the insoluble fraction of all tissues. These results suggest that MT is, at least in part, responsible for preventing Hg accumulation in the liver and blood and decreasing renal toxicity.     

Prion protein protects against zinc-mediated cytotoxicity by modifying intracellular exchangeable zinc and inducing metallothionein expression

PrP^C contains several octapeptide repeats sequences toward the N-terminus which have binding affinity for divalent metals such as copper, zinc, nickel and manganese. However, the link between PrP^C expression and zinc metabolism remains elusive. Here we studied the relationship between PrP^C and zinc ions intracellular homeostasis using a cell line expressing a doxycycline-inducible PrP^C gene. No significant difference in ⁶⁵Zn²⁺ uptake was observed in cells expressing PrP^C when compared with control cells. However, PrP^C-expressing cells were more resistant to zinc-induced toxicity, suggesting an adaptative mechanism induced by PrP^C. Using zinquin-ethyl-ester, a specific fluorophore for vesicular free zinc, we observed a significant re-localization of intracellular exchangeable zinc in vesicles after PrP^C expression. Finally, we demonstrated that PrP^C expression induces metallothionein (MT) expression, a zinc-upregulated zinc-binding protein. Taken together, these results suggest that PrP^C modifies the intracellular localization of zinc rather than the cellular content and induces MT upregulation. These findings are of major importance since zinc deregulation is implicated in several neurodegenerative disorders. It is postulated that in prion diseases the conversion of PrP^C to PrP^Sc may deregulate zinc homeostasis mediated by metallothionein.     

Interferonbeta-induced changes in metallothionein expression and subcellular distribution of zinc in HepG2 cells

We evaluated the changes of metallothionein induction and cellular zinc distribution in HepG2 cells by interferonbeta treatment. Immunohistochemical staining of metallothionein was observed in the cytoplasm and nuclei of hepatocytes; which was observed predominantly in the cells treated with interferon and zinc compared to those with zinc alone, interferon alone or the no-treated control. The cellular zinc level was higher in order of the interferon- and zinc-treated cells, the zinc-alone-treated cells, and the interferon-alone-treated cells. Flow cytometry showed that S-phase population increased in interferon-alone-treated cells and interferon- and zinc-treated cells, but not in zinc-alone-treated ones. Cellular elemental distribution was analyzed using in-air micro-particle induced X-ray emission. In zinc-alone-treated sample, X-ray spectra showed good consistency between the enhanced cellular zinc distribution and the phosphorous map. Localizations of bromine followed by interferon treatment were found accompanying a spatial correlation with the phosphorous map. The samples treated with interferon and zinc showed the marked accumulation of zinc and bromine. Discrete bromine accumulation sites were clearly visible with a strong spatial correlation followed by zinc accumulation. These findings suggest that interferonbeta in combination with zinc predominantly induces metallothionein expression in HepG2 cells. In addition, interferonbeta may promote the translocation of metallothionein-bound zinc from cytoplasm to S-phase nuclei.     

Alterations of tissue glutathione levels and metallothionein mRNA in rainbow trout during single and combined exposure to cadmium and zinc

The objective of this study was to assess the effects of Cd and Zn exposure of rainbow trout (Oncorhynchus mykiss) on (a) hepatic glutathione (GSH) levels; and (b) hepatic and branchial metallothionein (MT) mRNA expression. Juvenile rainbow trout were exposed to waterborne Cd (nominal concentrations: 1.5 or 10 microg Cd l(-1)), Zn (150 or 1000 microg Zn l(-1)) or Cd/Zn mixtures (1.5 microg Cd l(-1) with 200 microg Zn l(-1) or 10 microg Cd l(-1) with 1000 microg Zn l(-1)). After 14 and 28 days of treatment, hepatic concentrations of total glutathione, oxidized glutathione (GSSG) and cysteine were determined by means of fluorometric high performance liquid chromatography (HPLC). Branchial and hepatic expression of MT mRNA was measured by means of semi-quantitative RT-PCR. Exposure of trout to Zn did not result in significantly elevated tissue levels of Zn, whereas Cd accumulation factors changed significantly with time and concentration. Despite of the absence of Zn accumulation, hepatic GSH but not MT mRNA levels were significantly altered in Zn-exposed fish. Cd, on the contrary, affected mainly the MT response but not GSH. Also tissue specific differences in the regulation of the two thiol pools were expressed. The thiol response after exposure to metal mixtures could not be explained by simple addition of the effects of the individual metals. The results indicate that cellular thiol pools show different reaction patterns with respect to specific metals and metal mixtures. Under conditions of long-term, low dose metal exposure, the function of GSH appears to go beyond that of a transitory, first line defense.     

Role of zinc in cellular zinc trafficking and mineralization in a murine osteoblast-like cell line

Zinc (Zn) supplementation stimulates bone growth in Zn-deficient humans and animals. A biphasic pattern of mineralization has been observed in cultured osteoblasts; an initiation phase and a progression phase. We used MC3T3-E1, a murine osteoblastic cell line, to elucidate the physiological role of Zn in osteoblast mineralization and cellular Zn trafficking during the mineralization event. Cells were cultured in media containing Chelex-treated fetal bovine serum and 1, 4, 10 and 20 μM Zn as ZnSO₄ for 14 days (early phase of mineralization) or 21 days (mid-to-late phase of mineralization). During the early phase of mineralization, Alizarin Red staining indicated that mineralization was increased by Zn in a dose-dependent manner. Although Zn exposure did not affect monolayer Zn concentration, metallothionein (MT) mRNA expression increased dose-dependently as assessed by real-time PCR. During the late phase of mineralization, mineralization was maximal at 1 μM Zn and monolayer Zn concentration reflected Zn exposure. The increase in MT mRNA expression during the late phase was similar to that during the early phase, but the difference in expression between culture Zn concentrations tended to be smaller. ZnT-2 mRNA expression decreased significantly with increasing zinc concentrations in the culture medium during the early phase, but increased significantly during the late phase. Osteocalcin mRNA levels were positively correlated to Zn exposure at both time points. Taken together, we propose that Zn may play an important role in osteoblast mineralization through Zn trafficking involving Zn storage proteins and Zn transporters.     

Zinc-induced upregulation of metallothionein (MT)-2A is predicted by gene expression of zinc transporters in healthy adults

The usefulness of zinc transporter and metallothionein (MT) gene expressions to detect changes in zinc intake remains unclear. This pilot study aimed to determine the effects of zinc supplementation on zinc transporter and MT gene expressions in humans. Healthy adults (n = 39) were randomised to zinc treatment (ZT), receiving 22 mg Zn/day (n = 19), or no treatment (NT) (n = 20). Blood samples were collected on Days 0, 2, 7, 14, and 21. Plasma zinc and serum C-reactive protein concentrations were analysed. Gene expression of zinc transporters and MT in peripheral blood mononuclear cells was analysed using real-time PCR. Using repeated-measures ANOVA, MT-2A gene expression and fold change were found to be higher in the ZT group (P = 0.025 and P = 0.016, respectively) compared to the NT group, specifically at Day 2 (40 ± 18 % increase from baseline, P = 0.011), despite no significant increase in plasma zinc concentration. In a multiple regression model exploring the changes in gene expressions between Days 0 and 21, the change in MT-2A gene expression was correlated with changes in all zinc transporter expressions (r² = 0.54, P = 0.029); the change in ZIP1 expression emerged as a univariate predictor (P = 0.003). Dietary zinc intake was predictive of zinc transporter and MT expressions (P = 0.030). Physical activity level was positively correlated with baseline ZIP7 expression (r = 0.36, P = 0.029). The present study shows that MT-2A expression is related to changing expression of zinc transporter genes, specifically ZIP1, in response to zinc supplementation. The current report adds to our understanding of MT in the coordinated nature of cellular zinc homeostasis.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-015-0494-y) contains supplementary material, which is available to authorized users.     

Metallothionein transfers zinc to mitochondrial aconitase through a direct interaction in mouse hearts

Previous studies have shown that in a cell-free system, metallothionein (MT) releases zinc when the environment becomes oxidized and the released zinc is transferred to a zinc-binding protein if such a protein is present. However, it is unknown whether and how zinc transfers from MT to other proteins in vivo. The present study was undertaken to test the hypothesis that if zinc transfer from MT to other proteins occurs in vivo, the transfer would proceed through a direct interaction between MT and a specific group of proteins. The heart extract obtained from MT-null mice was incubated with 65Zn-MT or 65ZnCl2 and the proteins receiving 65Zn were separated by blue-native PAGE (BN-PAGE) or sodium dodecyl sulfate-PAGE (SDS-PAGE), and detected by autoradiography. A unique 65Zn-binding band was observed from the 65Zn-MT-incubated, but not the 65ZnCl2-incubated preparation. The analysis using matrix assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry revealed that mitochondrial aconitase (m-aconitase) was among the proteins accepting Zn directly from Zn-MT. The m-aconitase, not the cytosolic aconitase (c-aconitase), was co-immunoprecipitated with MT. This study demonstrates that MT transfers zinc to m-aconitase through a direct interaction.     

Early effects of surgery on zinc and metallothionein levels in female rats

Time-response effects of experimental surgery on zinc (Zn) and metallothionein (MT) homeostasis were investigated in female rats up to 24 h. Hepatic Zn content increased at 20 and 24 h postsurgery, whereas serum Zn levels decreased. Hepatic MT increased significantly by 9 h postsurgery and peaked at up to twofold of control at 12 h after surgery. Following the peak at 12 h, hepatic MT content decreased with time but did not reach control levels at the end of this study. When MT isoforms were evaluated, MT-II levels were elevated to the highest extent by 12 h after surgery, whereas MT-I levels started to decrease after 3 h postsurgery but then increased by 20 h. The early increases in MT content are probably mediated by nonmetallic mediators released during the postsurgical inflammatory process, favoring the plasma/tissue mobilization of Zn. This process might be part of the overall mechanisms occurring in the inflammation.     

Consecutive zinc balance trials in growing rats

Rats were fed a purified egg white-based diet containing 5 ppm Cu and 2, 14, or 57 ppm Zn. Zinc and copper balances were determined for eight consecutive weekly trial periods. The zinc-deficient group almost ceased to gain weight and was in slightly negative zinc balance. Groups of rats fed 14 and 57 ppm Zn gained weight at equal rates. These groups were in strongly positive zinc balance for four weeks; thereafter, they fed 57 ppm Zn retained about two times as much zinc as did the group fed the diet containing 14 ppm Zn. All groups were in null or slightly negative copper balance throughout the trial. These results suggest that zinc accumulation may be homeostatically controlled to a level in excess of that needed for maximum growth.     

Evidence for operation of the direct zinc ligand exchange mechanism for trafficking, transport, and reactivity of zinc in mammalian cells

In addition to its critical role in normal cell function, growth, and metabolism, zinc is implicated as a major factor in the development and progression of many pathological conditions and diseases. Despite this importance of zinc, many important factors, processes, and mechanisms of the physiology, biochemistry, and molecular biology of zinc remain unknown. Especially important is the unresolved issue regarding the mechanism and process of the trafficking, transport, and reactivity of zinc in cells; especially in mammalian cells. This presentation focuses on the concept that, due to the existence of a negligible pool of free Zn²⁺ ions in the mammalian cell environment, the trafficking, transport and reactivity of zinc occurs via a direct exchange of zinc from donor Zn-ligands to acceptor ligands. This Zn exchange process occurs without the requirement for production of free Zn²⁺ ions. The direct evidence from mammalian cell studies is presented in support of the operation of the direct Zn-ligand exchange mechanism. The paper also provides important information and conditions that should be considered and employed in the conduct of studies regarding the role and effects of zinc in biological/biomedical research; and in its clinical interpretation and application.     

Dietary zinc effects on zinc, calcium, and magnesium content in bones of growing rats

The aim of the present study was to assess dietary zinc effects on femur weight and mineral content in growing rats. For this purpose, 70 weanling Sprague-Dawley rats were divided into four groups. Each group was subject to a diet containing 2 (BZ), 5 (DZ), 10 (MZ), and 30 (CZ) ppm zinc. The calcium and magnesium content in all diets was 5 g/kg and 507 mg/kg, respectively. The animals were kept on this regime for 28 d and then sacrificed and their femurs were removed for analysis using atomic absorption spectrophotometry. The weights of the BZ and DZ groups were significantly different from the MZ and CZ groups (38.5±10.5, 89.9±13.7, 118.6±13.6 and 134±19.9 g, p<0.01) respectively. There were no differences between the MZ and CZ groups. Femur weight also varied with dietary zinc, as it was significantly different among all groups (BZ, 265±49 mg; DZ, 380±40 mg; MZ, 452±54 mg; CZ, 735±66 mg; p<0.01). The femur zinc content varied with diets, following a different pattern than the above parameters. Femur zinc from the BZ group (51.5±5.4 ppm) was significantly different from the MZ and CZ groups (115.9±14.2 and 175.0±13.5 ppm, respectively), whereas the DZ group (62.5±11.3 ppm) did not differ from the other three groups. The femur content of calcium (BZ, 83.2±9.8 mg/g; DZ, 88.0±9.2 mg/g; MZ, 90.2±13.6 mg/g; CZ, 83.1±14.7 mg/g) and magnesium (BZ, 1.82±0.13 mg/g; DZ, 1.98±0.09 mg/g; MZ, 1.93±14 mg/g; CZ, 1.83±0.19 mg/g) were not significantly different among the groups, nor was the calcium-magnesium ratio. These results suggest that although dietary zinc deficiency retards growth and causes bone fragility, bone deposition of calcium and magnesium and its ratio are not affected.     

Molecular aspects of human cellular zinc homeostasis: redox control of zinc potentials and zinc signals

Zinc(II) ions are essential for all forms of life. In humans, they have catalytic and structural functions in an estimated 3,000 zinc proteins. In addition, they interact with proteins transiently when they regulate proteins or when proteins regulate cellular zinc re-distribution. As yet, these types of zinc proteins have been explored poorly. Therefore the number of zinc/protein interactions is potentially larger than that given by the above estimate. Confronted with such a wide range of functions, which affect virtually all aspects of cellular physiology, investigators have begun to elucidate the molecular mechanisms of cellular homeostatic control of zinc, especially the functions of transporter, sensor, and trafficking proteins, such as metallothioneins, in providing the correct amounts of zinc ions for the synthesis of zinc metalloproteins. The sulfur-containing amino acid cysteine in proteins has an important role in the cellular mobility of zinc ions. Sulfur-coordination environments provide sufficiently strong interactions with zinc ions; they can undergo fast ligand-exchange; and they can serve as molecular redox switches for zinc binding and release. For the cellular functions of zinc, the free zinc ion concentrations (zinc potentials, pZn = −log[Zn²⁺]) and the zinc buffering capacity are critically important parameters that need to be defined quantitatively. In the cytoplasm, free zinc ions are kept at picomolar concentrations as a minute fraction of the few hundred micromolar concentrations of total cellular zinc. However, zinc ion concentrations can fluctuate under various conditions. Zinc ions released intracellularly from the zinc/thiolate clusters of metallothioneins or secreted from specialized organelles are potent effectors of proteins and are considered zinc signals. The cellular zinc buffering capacity determines the threshold between physiological and pathophysiological actions of zinc ions. When drugs, toxins, other transition metal ions or reactive compounds compromise zinc buffering, large zinc ion fluctuations can injure cells through effects on redox biology and interactions of zinc ions with proteins that are normally not targeted.

A differential assay for the reduced and oxidized states of metallothionein and thionein

In the cellular environment, the sulfur ligands in zinc/thiolate coordination sites of proteins can be oxidized with concomitant mobilization of zinc. The characterization of such "redox zinc switches" requires the determination of three species, i.e., the zinc-containing complex and the zinc-free complex with the thiolate ligands either reduced or oxidized. Differential chemical modification of thiol groups in the presence and absence of either reducing or chelating agents allows the analytical speciation of such systems as demonstrated here for the characterization of the redox and metal-binding states of mammalian metallothionein. Thiol derivatization with 6-iodoacetamidofluorescein in the presence and absence of the reducing agent tris(2-carboxyethyl)phosphine, high-performance liquid chromatographic separation, and photometric detection are employed to determine the reduced and oxidized protein. Because the holoprotein reacts only in the presence of a chelating agent such as ethylenediaminetetraacetate (EDTA) its amount can be determined as the difference between measurements in the presence and the absence of EDTA. This method is applied to the study of the chemical and enzymatic oxidation of metallothionein/thionein. It should also greatly facilitate the characterization of the redox and metal-binding properties of zinc/thiolate coordination environments of other proteins such as zinc finger proteins.     

Prevalence of kidney dysfunction in humans – Relationship to cadmium dose, metallothionein, immunological and metabolic factors

Long term cadmium (Cd) exposure in occupational and general environments may give rise to kidney dysfunction. This effect is usually considered to be the critical effect, i. e. the effect that occurs at relatively low level of exposure. The present review focused on studies of the prevalence of cadmium-related kidney dysfunction among population groups residing in cadmium contaminated areas in China. Dose–response relationships were shown between UCd and the prevalence of increased levels of biomarkers in urine of renal tubular dysfunction such as urinary beta-2-microglobulin or N-acetyl-beta-d-glucosaminidase – NAG or urinary albumin, a biomarker of glomerular kidney dysfunction. Factors that influence these dose–response relationships include: 1) Metallothionein mRNA levels in peripheral blood lymphocytes, used as a biomarker of the ability of each person, to synthesize metallothionein (a protein known to provide intracellular protection against cadmium toxicity). 2) The occurrence of increased levels in blood plasma of autoantibodies against metallothionein. 3) Concomitant changes in glucose metabolism i e Type II diabetes. 4) Concomitant exposure to other nephrotoxic agents such as inorganic arsenic. Increased susceptibility in diabetics has been shown also in population groups in Europe. In persons with type II diabetes and increased levels of autoantibodies against metallothionein in blood plasma or in persons with concomitant exposure to environmental inorganic arsenic, indications of Cd-related kidney dysfunction was observed at UCd levels around 1 μg/g creatinine, levels found among “unexposed” population groups in many countries.     

Zinc binding ligands and cellular zinc trafficking: apo-metallothionein, glutathione, TPEN, proteomic zinc, and Zn-Sp1

Many cell types contain metal-ion unsaturated metallothionein (MT). Considering the Zn(2+) binding affinity of metallothionein, the existence of this species in the intracellular environment constitutes a substantial "thermodynamic sink". Indeed, the mM concentration of glutathione may be thought of in the same way. In order to understand how apo-MT and the rest of the Zn-proteome manage to co-exist, experiments examined the in vitro reactivity of Zn-proteome with apo-MT, glutathione (GSH), and a series of common Zn(2+) chelating agents including N,N,N',N'-(2-pyridylethyl)ethylenediammine (TPEN), EDTA, and [(2,2'-oxyproplylene-dinitrilo]tetraacetic acid (EGTA). Less than 10% of Zn-proteome from U87mg cells reacted with apo-MT or GSH. In contrast, each of the synthetic chelators was 2-3 times more reactive. TPEN, a cell permeant reagent, also reacted rapidly with both Zn-proteome and Zn-MT in LLC-PK(1) cells. Taking a specific zinc finger protein for further study, apo-MT, GSH, and TPEN inhibited the binding of Zn(3)-Sp1 with its cognate DNA site (GC-1) in the sodium-glucose co-transporter promoter of mouse kidney. In contrast, preformation of Zn(3)-Sp1-(GC-1) prevented reaction with apo-MT and GSH; TPEN remained active but at a higher concentration. Whereas, Zn(3)-Sp1 is active in cells containing apo-MT and GSH, exposure of LLC-PK(1) cells to TPEN for 24h largely inactivated its DNA binding activity. The results help to rationalize the steady state presence of cellular apo-MT in the midst of the many, diverse members of the Zn-proteome. They also show that TPEN is a robust intracellular chelator of proteomic Zn(2+).     

Biological function of metallothionein. VI. Metabolic interaction of cadmium and zinc in rats

The accumulation and depletion of cadmium in liver and kidney metallothionein (MT) and the effects of dietary zinc deficiency on cadmium metabolism were studied in rats. The accumulation of cadmium in liver MT started to plateau after 80 days, but there was a linear accumulation of this element in kidney MT over the entire 300-day experiment. Cadmium in MT fractions was depleted very slowly when rats were changed to a diet without cadmium. The accumulation of cadmium in MT also caused zinc to accumulate in this protein, even in rats fed zinc-deficient diets. However, the reverse situation was found not to be true; zinc did not cause cadmium to accumulate in MT. Dietary zinc deficiency limited the binding of injected¹⁰⁹Cd to MT of liver, but not of kidneys or testes. However, zinc-deficient rats fed cadmium in their diets metabolized cadmium similarly to zinc-supplemented rats, suggesting that zinc deficiency does not limit the ability of cadmium to stimulate MT synthesis.