Intracellular zinc during cell activation and zinc deficiency

IntroductionZinc is an essential trace element having manifold functions within living cells. Zinc deficiency but also zinc excess impairs cell-specific functions whereas a balanced zinc level is required for an adequate cell behavior.Material and methodsThis study deals with the impact of cellular priming due to stimulation with interleukin (IL)-1, IL-2, IL-4, IL-6 or the chemokine CXCL12a and its subsequent influence on the intracellular free zinc concentration. Since cellular priming and activation is essential for proper immunological reactions, and across that highly cell-type specific, we investigated T cells, B cells, and peripheral blood mononuclear cells (PBMCs). Additionally, alterations of the intracellular zinc content was investigated by inducing zinc deficiency using the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) with subsequent re-supplementation of zinc, hence generating an intracellular zinc flux. Evaluation of zinc staining with FluoZin3-AM, Zinpyr-1 and Zinquin was done by flow cytometry or by fluorescence microscopy.ResultsOur results indicate that cellular priming for different periods of time (10 minutes/one hour) causes decreased intracellular free zinc concentrations in the FluoZin3-AM staining and increased zinc concentrations stained with Zinpyr-1. Furthermore, zinc supplementation after induced zinc deficiency leads to a fast and excessive rise of the intracellular free zinc levels in most cellular compartments.ConclusionOur study emphasizes the importance of zinc homeostasis and zinc distribution during cellular priming and for certain signaling cascades especially in T and B cells. Moreover, we demonstrated that zinc re-supplementation of zinc deficient cells results in significantly elevated intracellular free zinc concentrations compared to untreated controls. Hence, this underlines the need of a balanced zinc homeostasis for proper immune cell function.     

Dietary phytate,zinc and hidden zinc deficiency

Epidemiological data suggest at least one in five humans are at risk of zinc deficiency. This is in large part because the phytate in cereals and legumes has not been removed during food preparation. Phytate, a potent indigestible ligand for zinc prevents it's absorption. Without knowledge of the frequency of consumption of foods rich in phytate, and foods rich in bioavailable zinc, the recognition of zinc deficiency early in the illness may be difficult. Plasma zinc is insensitive to early zinc deficiency. Serum ferritin concentration ≤ 20 μg/L is a potential indirect biomarker. Early effects of zinc deficiency are chemical, functional and may be “hidden”. The clinical problem is illustrated by 2 studies that involved US Mexican-American children, and US premenopausal women. The children were consuming home diets that included traditional foods high in phytate. The premenopausal women were not eating red meat on a regular basis, and their consumption of phytate was mainly from bran breakfast cereals. In both studies the presence of zinc deficiency was proven by functional responses to controlled zinc treatment. In the children lean-mass, reasoning, and immunity were significantly affected. In the women memory, reasoning, and eye-hand coordination were significantly affected. A screening self-administered food frequency questionnaire for office might help caregiver's identify patients at risk of zinc deficiency.     

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.

Kinetics of zinc status and zinc deficiency in Berardinelli-Seip syndrome

Berardinelli-Seip syndrome (BSS) is a very rare disorder characterized by near-complete absence of adipose tissue from birth or early infancy, hypoleptinemia, hypertriglyceridemia, insulin resistance, diabetes mellitus, and other clinical signals. It is caused by mutations in AGPAT2 or Gng3lg. We evaluated 10 BSS patients and 10 healthy subjects. A single dose of 382.43 μmol zinc was administered intravenously before and after 3 months of oral zinc supplementation. Blood samples were collected from the contralateral arm at 0, 30, 60, 90, and 120 min after zinc injection. Plasma and serum were obtained to measure hematological and biochemical parameters. Urine was collected to measure creatinine, protein, and zinc. Basal serum zinc levels were similar in controls and BSS patients. However, serum zinc profiles were significant reduced in BSS patients in comparison with controls. The change in total-body zinc clearance was more significant in BSS patients, indicating that these patients had suboptimum zinc deficiency.     

Nuclear zinc in liver of cadmium and zinc treated rats

X-ray microanalysis was performed to detect quantitatively, the variation of the nuclear zinc in the liver cells of rats. The nuclear zinc concentration showed statistical decrease and increase in response to cadmium and zinc treatments, respectively. The results suggest that the liver responds differently to cadmium and zinc treatments. The difference in response to either treatment may reflect different mechanisms of zinc transport and metabolism in the liver. The difference in binding affinity of metallothionein (MT) may suggest the involvement of Mt in the metabolism and transport of zinc, an effect, which may be modified by treatment.     

Serum zinc levels and zinc binding capacity in thalassemia

Recently, it has been reported that serum zinc binding capacity (ZnBC) is a very important criterion to evaluate body zinc (Zn) status. It has been shown that chronic Zn deficiency occur in the patients with thalassemia major (TM). Zn deficiency in TM may cause hyperzincuria, high ferritin levels, hepatic iron load, hepatic dysfunction. This study was undertaken to determine serum Zn levels and ZnBC in different thalassemia forms and sickle cell disease (SCD). The study has been carried out on 30 Thalassemia Major (TM), 34 Thalassemia Intermedia (TI), 31 Thalassemia Trait (TT) and 10 SCD. As control group,13 healthy children and 20 adults were included. Serum Zn and ZnBC were determined by atomic absorption, then saturation index (SI%: serum Zn/ZnBC x 100) was calculated. Serum Zn levels in all patients were lower than control (p < 0.01). Serum ZnBC was at a normal level in patients with TT and TI but it was found to be lower in TM and SCD than control (p < 0.01). While serum Zn levels decrease and ZnBC increase in nutritionaL Zn deficiency, serum Zn levels decrease but ZnBC doesn't increase in patients with thalassemia.     

Dietary zinc intake,supplemental zinc intake and serum zinc levels and the prevalence of kidney stones in adults

ObjectiveThe association between zinc intake and the risk of kidney stones remains controversial. We examined the associations between dietary zinc intake, supplemental zinc intake and serum zinc levels and the prevalence of kidney stones in adults.MethodsAdult participants from the 2007–2016 NHANES were included. Restricted cubic splines were adopted to assess the dose-response relationships.ResultsDietary zinc intake was linearly associated with the prevalence of kidney stones (P_{for non-linearity} = 0.50), and the odds ratios (95% confidence intervals) of kidney stones were 0.75 (0.51–1.04) for 10 mg/day, 0.65 (0.39-0.97) for 20 mg/day, 0.53 (0.30-0.94) for 30 mg/day and 0.45 (0.22-0.95) for 40 mg/day. The linear relationship was also observed among women and overweight/obese individuals. No association was found between supplemental zinc intake and the prevalence of kidney stones. A non-linear relationship was found between serum zinc levels and the prevalence of kidney stones (P_{for non-linearity} = 0.02), and the odds ratios (95% confidence intervals) of kidney stones were 0.52 (0.33-0.82) for 70 ug/dL, 0.43 (0.24-0.77) for 90 ug/dL, 0.56 (0.32-0.98) for 110 ug/dL and 0.77 (0.37–1.62) for 130 ug/dL. The non-linear relationship was also observed among men and overweight/obese individuals.ConclusionsDietary zinc intake and serum zinc levels were inversely associated with the prevalence of kidney stones in adults, and there may be effect modification by participant sex and body mass index. The present analysis is limited in its ability to establish causality.     

Influence of DNA-methylation on zinc homeostasis in myeloid cells: Regulation of zinc transporters and zinc binding proteins

The distribution of intracellular zinc, predominantly regulated through zinc transporters and zinc binding proteins, is required to support an efficient immune response. Epigenetic mechanisms such as DNA methylation are involved in the expression of these genes. In demethylation experiments using 5-Aza-2′-deoxycytidine (AZA) increased intracellular (after 24 and 48 h) and total cellular zinc levels (after 48 h) were observed in the myeloid cell line HL-60. To uncover the mechanisms that cause the disturbed zinc homeostasis after DNA demethylation, the expression of human zinc transporters and zinc binding proteins were investigated. Real time PCR analyses of 14 ZIP (solute-linked carrier (SLC) SLC39A; Zrt/IRT-like protein), and 9 ZnT (SLC30A) zinc transporters revealed significantly enhanced mRNA expression of the zinc importer ZIP1 after AZA treatment. Because ZIP1 protein was also enhanced after AZA treatment, ZIP1 up-regulation might be the mediator of enhanced intracellular zinc levels. The mRNA expression of ZIP14 was decreased, whereas zinc exporter ZnT3 mRNA was also significantly increased; which might be a cellular reaction to compensate elevated zinc levels. An enhanced but not significant chromatin accessibility of ZIP1 promoter region I was detected by chromatin accessibility by real-time PCR (CHART) assays after demethylation. Additionally, DNA demethylation resulted in increased mRNA accumulation of zinc binding proteins metallothionein (MT) and S100A8/S100A9 after 48 h. MT mRNA was significantly enhanced after 24 h of AZA treatment also suggesting a reaction of the cell to restore zinc homeostasis. These data indicate that DNA methylation is an important epigenetic mechanism affecting zinc binding proteins and transporters, and, therefore, regulating zinc homeostasis in myeloid cells.     

Effect of phosphorus and zinc nutrition on soybean seed phytic Acid and zinc

The relationships between nutrient P and Zn levels and the phytic acid, P, and Zn concentrations in soybean (Glycine max L. Merr. cv `Williams 79') seed were studied. Phytic acid increased linearly from 4.2 to 19.2 milligrams per gram as nutrient P treatment was varied from 2.0 to 50 milligrams per liter and Zn was held constant at 0.05 milligrams per liter. Leaf P concentration during seed development was found to be closely related to the concentrations of seed P and phytic acid. Leaf and seed Zn concentrations both responded positively to increasing nutrient Zn treatment. The effects of P treatment on plant and seed P and phytic acid were largely independent of the effects of Zn treatment on leaf and seed Zn. Phytic acid to Zn molar ratios ranging from 3.6 to 33.8 were observed.

The effects of nutrient P treatments on the concentrations of phytic acid, seed P, and leaf P were also studied in the P-sensitive (gene np) cultivars `Harosoy' and `Clark' and their respective P-tolerant (gene Np) near-isogenic lines L66-704 and L63-1677. In general, the positive relationships observed among nutrient P, leaf P, seed P, and phytic acid concentrations were similar to those observed in the studies with Williams 79. When fertilized with low or moderate nutrient P (2.5 and 25.0 milligrams P per liter, respectively) no significant differences in any parameter were observed between Harosoy or Clark and their respective P-tolerant isolines. When fertilized with high nutrient P (100 milligrams P per liter), Harosoy seed had a significantly higher concentration of phytic acid (30 milligrams per gram) than did seed of its P-tolerant near-isogenic line L66-704 (24.2 milligrams per gram phytic acid), whereas no significant difference was observed between Clark and its P-tolerant near-isogenic line L63-1677 (22.8 and 21.6 milligrams per gram, respectively). Variation in the phytic acid concentrations in the mature seed of the cultivars and isolines more closely paralleled leaf P concentrations observed during seed development (49 days after flowering), than those observed at the onset of seed development (14 days after flowering). Electrophoresis and ion-exchange chromatography revealed that partially phosphorylated intermediates do not appear when phytic acid accumulation is greatly reduced by limiting the nutrient P or when accumulation is greatly accelerated by excess P.

     

Saccharomyces cerevisiae vacuole in zinc storage and intracellular zinc distribution

Previous studies of the yeast Saccharomyces cerevisiae indicated that the vacuole is a major site of zinc storage in the cell. However, these studies did not address the absolute level of zinc that was stored in the vacuole nor did they examine the abundances of stored zinc in other compartments of the cell. In this report, we describe an analysis of the cellular distribution of zinc by use of both an organellar fractionation method and an electron probe X-ray microanalysis. With these methods, we determined that zinc levels in the vacuole vary with zinc status and can rise to almost 100 mM zinc (i.e., 7 x 10(8) atoms of vacuolar zinc per cell). Moreover, this zinc can be mobilized effectively to supply the needs of as many as eight generations of progeny cells under zinc starvation conditions. While the Zrc1 and Cot1 zinc transporters are essential for zinc uptake into the vacuole under steady-state growth conditions, additional transporters help mediate zinc uptake into the vacuole during "zinc shock," when zinc-limited cells are resupplied with zinc. In addition, we found that other compartments of the cell do not provide significant stores of zinc. In particular, zinc accumulation in mitochondria is low and is homeostatically regulated independently of vacuolar zinc storage. Finally, we observed a strong correlation between zinc status and the levels of magnesium and phosphorus accumulated in cells. Our results implicate zinc as a major determinant of the ability of the cell to store these other important nutrients.     

Accumulation of zinc and cadmium and localization of zinc in Picris divaricata Vant.

Picris divaricata Vant., a plant species native to subtropical China, was recently identified as the first Cd/Zn hyperaccumulator from Asteraceae. P. divaricata was grown from wild collected seed for 4 months in a series of pH adjusted test soils with added Zn levels 0–7000 mg kg⁻¹ and Cd levels 0–150 mg kg⁻¹. Plants did not hyperaccumulate Zn (threshold >3000 μg g⁻¹) and weakly hyperaccumulated Cd with little or no dose–response.P. divaricata has multicellular simple trichomes concentrated on the leaf margins and midrib. X-ray analysis showed that Zn was concentrated in larger trichomes and epidermal cells adjacent to the trichome but virtually absent in other leaf tissues. Within the trichomes, Zn was localized in ovate spots around the tips of individual cells. These tips and other locations in the trichome cell contained black electron dense material when examined with transmission electron microscopy, some of which was identified as SiO₂. Silicon and Mn were concentrated in the same areas as Zn. Si has been previously associated with alleviating Zn, Mn and Cd toxicity. Our results support this observation and further investigation is warranted.Calcium and P were concentrated in the distal tips of trichomes, similar to patterns previously observed for calcicole plants grown in elevated Ca soils. Overall, nonsecretory trichomes from many plant families may have a common origin as tissues adapted to handle a variety of environmental metals.     

Interaction of zinc and hemoglobin: binding of zinc and the oxygen affinity.

Stripped human hemoglobin was shown to have a high apparent zinc association constant of 1.3 X 10(7) M-1 with a stoichiometry of one zinc for every two hemes. The saturation of this site produces a dramatic 3.7-fold increase in the oxygen affinity. The effect of zinc on the oxygen affinity is interrelated with the interaction of 2,3-diphosphoglyceric acid (2,3-DPG) and hemoglobin. Thus, a smaller zinc effect is observed in the presence of added 2,3-DPG. Information about the location of the zinc-binding site responsible for the increased oxygen affinity has been obtained by comparing the binding of zinc to various hemoglobins. Blocking the beta93 sulfhydryl group decreases the apparent zinc association constant by an order of magnitude. The substitution of histidine-beta143 in hemoglobin Abruzzo [beta143 (H21) His leads to Arg] and hemoglobin Little Rock [beta143 (H21) His leads to Gln] decreases the apparent zinc association constant by two orders of magnitude. The substitution of histidine-beta143 by other amino acids and the reaction of the beta93 sulfhydryl group are known to produce dramatic increases in the oxygen affinity. The binding of zinc to one or both of these amino acids can, therefore, explain the zinc-induced increase in the oxygen affinity.     

Attenuation of abnormal glutamate release in zinc deficiency by zinc and Yokukansan

The mechanism of the abnormal increase in extracellular glutamate concentration in the hippocampus induced with 100 mM KCl in zinc deficiency is unknown. In the present study, the changes in glutamate release (exocytosis) and GLT-1, a glial glutamate transporter, expression were studied in young rats fed a zinc-deficient diet for 4 weeks. Exocytosis at mossy fiber boutons was enhanced as reported previously and GLT-1 protein was increased in the hippocampus. The enhanced exocytosis is thought to increase extracellular glutamate concentration. However, the basal concentration of extracellular glutamate in the hippocampus was not increased by zinc deficiency, suggesting that GLT-1 protein increased serves to maintain the basal concentration of extracellular glutamate. The enhanced exocytosis was attenuated in the presence of 100 μM ZnCl₂, which attenuated the abnormal increase in extracellular glutamate induced with high K⁺ in zinc deficiency. The present study indicates that zinc attenuates abnormal glutamate release in zinc deficiency. The enhanced exocytosis was also attenuated in slices from zinc-deficient rats administered Yokukansan, a herbal medicine, in which the abnormal increase in extracellular glutamate induced with high K⁺ was attenuated. It is likely that Yokukansan is useful for prevention or cure of abnormal glutamate release. The enhanced exocytosis in zinc deficiency is a possible mechanism on abnormal increase in extracellular glutamate in the hippocampus induced with high K⁺.     

Synthesis and characterization of nanocrystalline zinc sulfide via zinc thiobenzoate-lutidine single-source precursor

ZnS nanocrystals were prepared both in the form of mesoporous powder and thin films by one step thermal decomposition technique from a single-source procure (SSP) [Zn(SOCPh)₂Lut₂·H₂O]. The final product was characterized by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), N₂ adsorption-desorption isotherm, UV-Vis absorption spectroscopy and photoluminescence (PL) study. Structural analyses of the prepared ZnS revealed the formation of cubic crystallites with diameters around 5 and 10 nm for the thin films and powder materials, respectively. On the other hand, the powder form showed mesoporous nature (type IV isotherm) with an average pore diameter of 37.9 Å and BET specific surface area of 51.73 m²/g.     

Attenuation of abnormal glutamate release in zinc deficiency by zinc and Yokukansan

The mechanism of the abnormal increase in extracellular glutamate concentration in the hippocampus induced with 100 mM KCl in zinc deficiency is unknown. In the present study, the changes in glutamate release (exocytosis) and GLT-1, a glial glutamate transporter, expression were studied in young rats fed a zinc-deficient diet for 4 weeks. Exocytosis at mossy fiber boutons was enhanced as reported previously and GLT-1 protein was increased in the hippocampus. The enhanced exocytosis is thought to increase extracellular glutamate concentration. However, the basal concentration of extracellular glutamate in the hippocampus was not increased by zinc deficiency, suggesting that GLT-1 protein increased serves to maintain the basal concentration of extracellular glutamate. The enhanced exocytosis was attenuated in the presence of 100 μM ZnCl₂, which attenuated the abnormal increase in extracellular glutamate induced with high K⁺ in zinc deficiency. The present study indicates that zinc attenuates abnormal glutamate release in zinc deficiency. The enhanced exocytosis was also attenuated in slices from zinc-deficient rats administered Yokukansan, a herbal medicine, in which the abnormal increase in extracellular glutamate induced with high K⁺ was attenuated. It is likely that Yokukansan is useful for prevention or cure of abnormal glutamate release. The enhanced exocytosis in zinc deficiency is a possible mechanism on abnormal increase in extracellular glutamate in the hippocampus induced with high K⁺.     

The relative efficiency of zinc carriers on growth and zinc nutrition of corn

Summary A comparison of different zinc carriers showed that application of Zn-DTPA, Zn-EDTA, Zn-fulvate and ZnSO₄ significantly increased the dry matter yield and zinc uptake by corn over the control treatment where no zinc was applied. The chelates in particular enhanced to a greater extent the uptake of both native and applied sources than that observed with ZnSO₄ as the zinc carrier. Both the dry matter yield and zinc uptake by corn showed a positive and significant relationship with self-diffusion coefficient of zinc showing thereby that diffusion contributed mainly the supply of Zn from the ambient soil matrix to plant roots. The effectiveness of the chelates varied depending on their capacity to retain Zn in a soluble form in the soil solution.It is evident that zinc nutrition of plants in alkaline and calcareous soils can be more effectively regulated by both synthetic and natural chelates or organic manures which contain substantial amount of complexed zinc.Journal Paper No. 1 from the Department of Soil Science and Agric. Chemistry, Tirhut College of Agriculture, Dholi, Muzaffarpur, Bihar, India.     

Mutagenic and cytotoxic effectiveness of zinc dimethyl and zinc diisononyldithiocarbamate in human lymphocyte cultures

The mutagenic and cytotoxic effectiveness of the vulcanisation accelerators zinc dimethyldithiocarbamate (ZDMC; ziram) and zinc diisononyldithiocarbamate (ZDINDC; arbestab Z) was tested in lymphocyte cultures of five healthy probands. ZDMC and ZDINDC (c=0.1, 1.0 and 10.0microg/ml) were studied in lymphocyte cultures without external metabolic activation. Additionally, incubation of the compounds (c=10.0microg/ml) was performed in the presence of liver microsomes from aroclor-induced rats (1 and 2h, 1 and 2mg microsomal protein). Genotoxicity testing was performed by analysis of chromosomal aberrations (CA), sister chromatid exchanges (SCEs) and micronuclei (MN). For evaluation of antiproliferative effects, mitotic index (MI) and cell cycle kinetics (CCK) were determined. In contrast to earlier investigations we found no significantly increased mutagenic or cytotoxic activity of ZDMC; ZDINDC also was inactive under these conditions.