Differential transition metal uptake and fluorescent probe localization in hippocampal slices

Metals are taken up by the combined action of metal transporters and ion channels. In this communication we have measured the uptake of the biologically important transition metals Mn, Fe, Co, Ni, Cu, Zn and Cd by rat and mouse hippocampal slices using the fluorescent probes FluoZin-3 (FZ3) and Newport Green (NPG), introduced by acetoxymethyl ester (AM) loading. The combination of metals and probes is also used to attempt to localize cellular sites into which metals translocate. We show that FZ3 and NPG partition into different cellular compartments; FZ3 into neuropil, whereas NPG localizes in neuropil and compartments within the cell bodies of neurons. Ni, Zn and Cd pass across the plasma membrane and then accumulate in intracellular vesicles and within intracellular membranes of cell bodies. The latter accumulate Cd, while synaptic vesicles take up Co. The passage of Mn, Cu and Fe into cells can be detected but there is some uncertainty about their disposition within the cell. All of our experiments are consistent with metals accumulating in intracellular compartments rather than the cytoplasm. Whether and to what extent there are transient elevations of free zinc levels in the cytoplasm remains unclear.     

Detection of heavy metals in bacterial biofilms and microbial flocs with the fluorescent complexing agent Newport Green

The complexing agent Newport Green fluoresces upon binding of nickel, zinc or cobalt. It was used to detect nickel or zinc in MOPS buffer, in gel-like matrices, and in natural biofilms and microbial flocs cultivated in the laboratory. The response curves for increasing nickel concentrations indicated an equimolar binding capacity of Newport Green for nickel in MOPS buffer, whereas zinc fluorescence reached saturation in the presence of a 10-fold excess of zinc ions relative to Newport Green molecules. The maximum fluorescence intensity as determined by luminometry was 8-fold and 4-fold above background for nickel and zinc, respectively. The response of Newport Green to either nickel or zinc in the presence of the other metal is consistent with a different binding affinity of Newport Green for the two metals. Zinc binds more strongly to the complexing agent than nickel but it leads to a weaker fluorescent signal which was detectable by luminometry but not by confocal laser scanning microscopy (CLSM). Newport Green was able to complex nickel in the presence of 1% gelatin or agarose as determined by CLSM and image processing. Its application to fully hydrated bacterial biofilms or microbial flocs revealed the presence of nickel outside of cells. The results suggest that in addition to cellular sorption, metals are bound extracellularly by extracellular polymeric substances in intact and undisturbed microbial aggregates. Journal of Industrial Microbiology & Biotechnology (2000) 24, 116–123. Received 11 June 1999/ Accepted in revised form 04 November 1999     

The zinc indicator FluoZin-3 is not perturbed significantly by physiological levels of calcium or magnesium

There has been some dispute in the literature as to the sensitivity of the zinc indicator FluoZin-3 to calcium, with suggestions that physiological levels of calcium and magnesium effectively occlude the response of the probe to zinc. In this communication we demonstrate that calcium concentrations as high as 10mM do not prevent FluoZin-3 from detecting zinc elevations as low as 100pM. Moreover, the inclusion of a few muM Ca-EDTA does not prevent FluoZin-3 from responding to increases in zinc concentration but does extend the dynamic range of the probe by reducing contaminating zinc levels and allowing the probe to respond to multiple zinc additions. In addition, we have derived a mathematical model to account for the kinetics of FluoZin-3 response to zinc in the presence of an additional zinc and calcium chelator.     

Growth-modulating tripeptide (glycylhistidyllysine): association with copper and iron in plasma, and stimulation of adhesiveness and growth of hepatoma cells in culture by tripeptide-metal ion complexes

The tripeptide H-Gly-His-Lys-OH (GHL) is a human plasma constituent which has been previously shown to modulate the growth and viability of a variety of cell types and organisms. Experimental observations presented herein indicate that GHL is complexed with the transition metal ions Cu++ and Fe++ in vivo and may exert its biological effects as a peptide-metal chelate. At physiological pH in vitro, GHL associates with ionic copper, cobalt, iron, molybdenum, manganese, nickel, and zinc, but has no affinity for calcium, manganese, potassium, and sodium. GHL acts synergistically with copper, iron, cobalt, and zinc to alter patterns of cell growth in monolayer cultures of a tumorigenic hepatoma cell line (HTC4). These transition metals induce cellular flattening and adhesion to support surfaces, and inhibit DNA synthesis and lactic acid production when growth is limited by reduction of serum concentrations in medium. These inhibitory effects are neutralized, and intercellular adhesion and growth are stimulated by GHL in medium at nanomolar concentrations. Cu and Fe are the most active metals when combined with GHL. The results suggest that the inability of HTC4 cultures to replicate without adequate concentrations of serum in medium may reflect deficiency of GHL and transition metals, which appear to form complexes prior to interaction with cells. Chelation of transition metals with GHL and, potentially, with other growth-modulating peptide factors in plasma or medium, may provide a mechanism for expression and regulation of biological activities influenced by transition metals and polypeptide growth factors. The observed effects of GHL-metal complexes, including stimulation of cellular adhesiveness to substratum (flattening) and intercellular attachment (monolayer formation), appear to satisfy requirements for growth of hepatoma cells in monolayer culture.     

Inhibition of iron and copper uptake by iron, copper and zinc

Interactions of micronutrients can affect absorption and bioavailability of other nutrients by a number of mechanisms. In aqueous solutions, and at higher uptake levels, competition between elements with similar chemical characteristics and uptake process can take place. The consequences of these interactions may depend on the relative concentrations of the nutrients. In this work, we measure the effects of increasing concentrations of iron, zinc, and copper on iron and copper uptake in Caco-2 cells. Intracellular Fe or Cu levels were affected by incubating with increased concentrations of metals. However, when the cells already had different intracellular metal concentration, the uptake of Fe or Cu was nor affected. In competition studies, we showed that Cu and Zn inhibited Fe uptake, and while Fe inhibited Cu uptake, Zn did not. When the three metals were given together (1:1:1 ratio), Fe or Cu uptake was inhibited approximately 40%. These results point to a potential risk in the absorption and bioavailability of these minerals by the presence of other minerals in the diet. This aspect must be considered in food supplementation and fortification programs.     

The distribution profile and oxidation states of biometals in APP transgenic mouse brain: dyshomeostasis with age and as a function of the development of Alzheimer's disease

The enrichment of transition metals in the brain and the dyshomeostasis of metals are thought to be important etiological factors for elderly people in a number of neurodegenerative diseases, including Alzheimer's disease (AD). However, the understanding of how biometals dynamically dysregulate in the stages of AD development, such as the exact time-dependent and site-dependent accumulation in the brain with AD progression, is still limited. Herein, by using the APP/V717I transgenic mouse model and age-matched mice as control, we offer distinctive in situ and quantitative images of metals (Cu, Fe, Zn and Ca) in brain sections by synchrotron radiation micro beam X-ray fluorescence (SR-μXRF). The images show that Fe and Ca increased with brain aging in both AD and control (CNT) mice, and Cu, Fe, Zn and Ca appeared significantly elevated in AD mice and showed an obvious age-dependent rise. Fe, Cu and Zn were obviously specifically enriched in the cortex and hippocampus, which were also the plaque-formation sensitive brain regions. Our results demonstrate that the enrichment of transition metals with age and metals' dyshomeostasis in specific regions may contribute together to the etiology and development of AD in elderly people. The XANES measurements of Cu and Fe show evidence that Cu may have redox properties in the AD brain.     

Identification and purification of functional human beta-cells by a new specific zinc-fluorescent probe.

Pancreatic beta-cells contain large amounts of zinc. We took advantage of this to try to localize, quantify, and isolate insulin-producing cells from islet preparations. Our study was designed to identify a non-toxic zinc-sensitive fluorescent probe able to selectively label labile zinc in viable beta-cells and to exhibit excitation and emission wavelengths in the visible spectrum, making this technique exploitable by most instruments. We tested Newport Green, a probe excitable at 485 nm with a dissociation constant in the micromolar range corresponding to a low affinity for zinc. The loading of the lipophilic esterified form of Newport Green was easy, rapid, specific, and non-toxic to cells. Confocal microscopy highlighted an intense fluorescence associated with secretory granules. Regression analyses showed a good relationship between zinc fluorescence and islet number (r = 0.98) and between zinc fluorescence and insulin content (r = 0.81). The determination of Zn fluorescence per DNA enabled us to assess the quality of the different islet preparations intended for islet allografting in terms of both purity and viability. Cell sorting of dissociated Newport Green-labeled cells resulted in a clear separation of beta-cells, as judged by insulin content per DNA and immunocytochemical analysis. This zinc probe, the first able to specifically label living cells in the visible spectrum, appears very promising for beta-cell experimentation, both clinically and for basic research.     

Flow cytometric measurement of labile zinc in peripheral blood mononuclear cells

Labile (i.e., free or loosely bound) zinc has the potential to modulate cellular function. Therefore, a flow cytometric assay for the measurement of labile zinc was developed to facilitate the investigation of the physiological roles of zinc. The zinc-sensitive fluorescent probe FluoZin-3 was used to quantify the amount of labile zinc in peripheral blood mononuclear cells isolated from human blood. Maximal fluorescence and autofluorescence of the probe were measured after the addition of zinc in the presence of the ionophore pyrithione, or the membrane-permeant chelator N,N,N',N'-tetrakis-(2-pyridyl-methyl)ethylenediamine, respectively. In this way, the intracellular concentrations of labile zinc in resting cells were estimated to be 0.17 nM in monocytes and 0.35 nM in lymphocytes. The method was successfully employed to monitor phorbol 12-myristate 13-acetate-induced zinc release, which occurred in monocytes but not lymphocytes, and the displacement of protein-bound zinc by the mercury-containing compounds HgCl(2) and thimerosal. Costaining with dyes that emit at higher wavelengths than FluoZin-3 allows multiparameter measurements. Two combinations with other dyes are shown: loading with propidium iodide to measure cellular viability and labeling with antibodies against the surface antigen CD4. This method allows measurement of the concentration of biologically active labile zinc in distinct cell populations.     

Oxidative damage to human red cells induced by copper and iron complexes in the presence of ascorbate

The role of trace metals in the generation of free radical mediated oxidative stress in normal human red cells was studied. Ascorbate and either soluble complexes of Cu(II) or Fe(III) provoked changes in red cell morphology, alteration in the polypeptide pattern of membrane proteins, and significant increases in methemoglobin. Neither ascorbate nor the metal complexes alone caused significant changes to the cells. The rate of methemoglobin formation was a function of ascorbate and metal concentrations, and the chemical nature of the chelate. Cu(II) was about 10-times more effective than Fe(III) in the formation of methemoglobin. Several metals were tested for their ability to compete with Cu(II) and Fe(III). Only zinc caused a significant inhibition of methemoglobin formation by Fe(III)-fructose. These observations suggest that site-specific as well as general free radical damage is induced by redox metals when the metals are either bound to membrane proteins or to macromolecules in the cytoplasm. The Cu(II) and Fe(III) function in two catalytic capacities: (1) oxidation of ascorbate by O2 to yield H2O2, and (2) generation of hydroxyl radicals from H2O2 in a Fenton reaction. These mechanisms are different from the known damage to red cells caused by the binding of Fe(III) or Cu(II) to the thiol groups of glucose-6-phosphate dehydrogenase. Our system may be a useful model for understanding the mechanisms for oxidative damage associated with thalassemia and other congenital hemolytic anemias.     

Human TRPV5 and TRPV6: Key players in cadmium and zinc toxicity

TRPV5 and TRPV6 are two major calcium transport pathways in the human body maintaining calcium homeostasis. TRPV5 is mainly expressed in the distal convoluted and connecting tubule where it is the major, regulated pathway for calcium reabsorption. TRPV6 serves as an important calcium entry pathway in the duodenum and the placenta. Previously, we showed that human TRPV6 (hTRPV6) transports several heavy metals. In this study we tested whether human TRPV5 (hTRPV5) also transports cadmium and zinc, and whether hTRPV5 together with hTRPV6 are involved in cadmium and zinc toxicity. The hTRPV5 mRNA and protein were expressed in HEK293 cells transiently transfected with pTagRFP-C1-hTRPV5. The overexpression of the hTRPV5 protein at the plasma membrane was revealed by cell surface biotinylation and immunofluorescence techniques. We observed that both cadmium and zinc permeate hTRPV5 in ion imaging experiments using Fura-2 or Newport Green DCF. Our results were further confirmed using whole-cell patch clamp technique. Transient overexpression of hTRPV5 or hTRPV6 sensitized cells to cadmium and zinc. Toxicity curves of cadmium and zinc were also shifted in hTRPV6 expressing HEK293 cells clones. Our results suggest that TRPV5 and TRPV6 are crucial gates controlling cadmium and zinc levels in the human body especially under low calcium dietary conditions, when these channels are maximally upregulated.     

N-acetyl-L-cysteine in the presence of Cu<Superscript>2+</Superscript> induces oxidative stress and death of granule neurons in dissociated cultures of rat cerebellum

Addition into the culture medium of the antioxidant N-acetylcysteine (NAC, 1 mM) in the presence of Cu²⁺ (0.0005-0.001 mM) induced intensive death of cultured rat cerebellar granule neurons, which was significantly decreased by the zinc ion chelator TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine). However, the combined action of NAC and Zn²⁺ did not induce destruction of the neurons. Measurement of the relative intracellular concentration of Zn²⁺ with the fluorescent probe FluoZin-3 AM or of free radical production using a CellROX Green showed that incubation of the culture for 4 h with Cu²⁺ and NAC induced an intensive increase in the fluorescence of CellROX Green but not of FluoZin-3. Probably, the protective effect of TPEN in this case could be mediated by its ability to chelate Cu²⁺. Incubation of cultures in a balanced salt solution in the presence of 0.01 mM Cu²⁺ caused neuronal death already after 1 h if the NAC concentration in the solution was within 0.005–0.05 mM. NAC at higher concentrations (0.1–1 mM) together with 0.01mM Cu²⁺ did not cause the death of neurons. These data imply that the antioxidant NAC can be potentially harmful to neurons even in the presence of nanomolar concentrations of variable valence metals.     

Effect of transition metals on stress, lipid peroxidation and antioxidant enzyme activities in tobacco cell cultures

to-baccoBright Yellow 2 (BY-2) suspension culture to understand the mechanisms of metal resistance in plant cells.We have analysed superoxide dismutase, catalase, and ascorbate peroxidase enzyme activities and superoxidedismutase-isoforms by isoelectric focusing gels in tobacco cells grown at two different toxic concentrations ofeach of the transition metals: copper, iron, manganese and zinc. Exposure of tobacco cells to these metals causedchanges in total superoxide dismutase activity in a different manner, depending on the metal assayed: after cop-perand manganese treatments, total superoxide dismutase activity was enhanced, while it was reduced after ironand zinc exposure. Superoxide dismutase-isoforms were affected by the metal used, and a Fe-SOD band with thesame isoelectric point as a Cu, Zn-SOD from non-treated cells, was induced after iron and zinc treatments. Cu,Zn-SODs were present in all metal-treatments whereas Mn-SOD was not detected in any case. Concerning otherantioxidant enzymes tested, such as catalase and ascorbate peroxidase, the latter showed a remarkable increase inactivity in response to copper treatments and catalase activity was enhanced after iron and with the lowest man-ganeseconcentration. Lipid peroxidation was increased after each metal treatment, as an indication of the oxi-dativedamage caused by metal concentration assayed in tobacco cells. These results suggest that an activation ofsome antioxidant enzymes in response to oxidative stress induced by transition metals is not enough to confertolerance to metal accumulation.     

Regulatory properties and cellular redistribution of zinc during macrophage differentiation of human leukemia cells

Many proteins require the binding of trace metals such as Ca, Fe, Cu, or Zn, which may modulate their structure, function, or activity. To determine if there were any overall changes in metalloprotein distribution or metal concentration during the process of macrophage differentiation we induced human myeloid HL-60 leukemia cells with phorbol 12-myristate 13-acetate (PMA) and quantitatively mapped their metal content using hard X-ray fluorescence micro-analysis. We found a transient increase in the zinc content of HL-60 cell nuclei during the early stages of differentiation induction. This finding was confirmed by spectrofluorometry in HL-60 cells and extended to U-937 leukemia cells. A role for protein kinase C-beta (PKC-beta) in this process was established by examining zinc content in an HL-60 variant, HL-525, which is PKC-beta deficient, and in HL-525 cells in which PKC-beta was restored by stable overexpression. Chemical chelation of both Cu and Zn served to inhibit macrophage differentiation in HL-60 cells, indicating a requirement for these metals during this process. Finally, we demonstrate that growth of HL-60 cells in a low-zinc environment removes their susceptibility to PMA-induced differentiation, and that this capacity can be partially restored by the addition of exogenous zinc.     

Metal Acquisition and Homeostasis in Fungi

Transition metals, particularly iron, zinc and copper, have multiple biological roles and are essential elements in biological processes. Among other micronutrients, these metals are frequently available to cells in only limited amounts, thus organisms have evolved highly regulated mechanisms to cope and to compete with their scarcity. The homeostasis of such metals within the animal hosts requires the integration of multiple signals producing depleted environments that restrict the growth of microorganisms, acting as a barrier to infection. As the hosts sequester the necessary transition metals from invading pathogens, some, as is the case of fungi, have evolved elaborate mechanisms to allow their survival and development to establish infection. Metalloregulatory factors allow fungal cells to sense and to adapt to the scarce metal availability in the environment, such as in host tissues. Here we review recent advances in the identification and function of molecules that drive the acquisition and homeostasis of iron, copper and zinc in pathogenic fungi.     

Effect of Zn treatment on wild type and MT-null cell lines in relation to apoptotic and/or necrotic processes and on MT isoform gene expression

It has been shown in various systems that zinc is able to antagonize the catalytic properties of the redox-active transition metals iron and copper, although the process is still unclear. Probably, the protective effect of Zn against oxidative stress is mainly due to the induction of a scavenger metal binding protein such as metallothionein (MT), rather than a direct action. To support this hypothesis, in this study, the effects of Zn, Cu, Fe, Zn + Cu and Zn + Fe treatments were investigated in a fibroblast cell line corresponding to an SV40-transformed MT-1/-2 mutant (MT-/-), and in wild type (MT+/+), by valuing metal concentrations and apoptotic and/or necrotic processes. We also investigated the synthesis of MT and the levels of both MT-1 and MT-2 mRNAs. In MT+/+ cells, co-treatment with Zn + Fe caused a decrease in Fe content compared to treatment with Fe alone. After Zn and Zn + Cu exposure the expression of MT-1 and MT-2 isoforms increased with a concomitant increase in MT synthesis. Annexin V-FITC and propidium iodide staining revealed necrotic or apoptotic cells in terminal stages, especially after Fe treatments. Immunofluorescent staining with an anti-ssDNA Mab and annexin detected a lower signal in co-treated cells compared to the single treatments in both cell lines. The intensity and quantity of fluorescence resulting from anti-ssDNA and Annexin V staining of MT null cells was higher compared to wild type cells. These results suggest that Zn alone does not completely exert an anti-oxidant effect against Cu and Fe toxicity, but that induction of MT is necessary.     

Elesclomol elevates cellular and mitochondrial iron levels by delivering copper to the iron import machinery

Copper (Cu) and iron (Fe) are redox-active metals that serve as cofactors for many essential cellular enzymes. Disruption in the intracellular homeostasis of these metals results in debilitating and frequently fatal human disorders, such as Menkes disease and Friedreich’s ataxia. Recently, we reported that an investigational anticancer drug, elesclomol (ES), can deliver Cu to critical mitochondrial cuproenzymes and has the potential to be repurposed for the treatment of Cu deficiency disorders. Here, we sought to determine the specificity of ES and the ES-Cu complex in delivering Cu to cuproenzymes in different intracellular compartments. Using a combination of yeast genetics, subcellular fractionation, and inductively coupled plasma-mass spectrometry–based metal measurements, we showed that ES and ES-Cu treatment results in an increase in cellular and mitochondrial Fe content, along with the expected increase in Cu. Using yeast mutants of Cu and Fe transporters, we demonstrate that ES-based elevation in cellular Fe levels is independent of the major cellular Cu importer but is dependent on the Fe importer Ftr1 and its partner Fet3, a multicopper oxidase. As Fet3 is metalated in the Golgi lumen, we sought to uncover the mechanism by which Fet3 receives Cu from ES. Using yeast knockouts of genes involved in Cu delivery to Fet3, we determined that ES can bypass Atx1, a metallochaperone involved in Cu delivery to the Golgi membrane Cu pump, Ccc2, but not Ccc2 itself. Taken together, our study provides a mechanism by which ES distributes Cu in cells and impacts cellular and mitochondrial Fe homeostasis.     

Functional expression of the human hZIP2 zinc transporter

Zinc is an essential nutrient for humans, yet we know little about how this metal ion is taken up by mammalian cells. In this report, we describe the characterization of hZip2, a human zinc transporter identified by its similarity to zinc transporters recently characterized in fungi and plants. hZip2 is a member of the ZIP family of eukaryotic metal ion transporters that includes two other human genes, hZIP1 and hZIP3, and genes in mice and rats. To test whether hZip2 is a zinc transporter, we examined (65)Zn uptake activity in transfected K562 erythroleukemia cells expressing hZip2 from the CMV promoter. hZip2-expressing cells accumulated more zinc than control cells because of an increased initial zinc uptake rate. This activity was time-, temperature-, and concentration-dependent and saturable with an apparent K(m) of 3 microM. hZip2 zinc uptake activity was inhibited by several other transition metals, suggesting that this protein may transport other substrates as well. hZip2 activity was not energy-dependent, nor did it require K(+) or Na(+) gradients. Zinc uptake by hZip2 was stimulated by HCO(3)(-) treatment, suggesting a Zn(2+)-HCO(3)(-) cotransport mechanism. Finally, hZip2 was exclusively localized in the plasma membrane. These results indicate that hZip2 is a zinc transporter, and its identification provides one of the first molecular tools to study zinc uptake in mammalian cells.     

Molecular aspects of Cu, Fe and Zn homeostasis in plants

Proper metal transport and homeostasis are critical for the growth and development of plants. In order to potentially fortify plants pre-harvest with essential metals in aid of human nutrition, we must understand not only how metals enter the plant but also how metals are then delivered to the edible portions of the plant such as the seed. In this review, we focus on three metals required by both plants and humans: Cu, Fe and Zn. In particular, we present the current understanding of the molecular mechanisms of Cu, Fe and Zn transport, including aspects of uptake, distribution, chelation and/or sequestration.     

Simultaneous detection of intracellular free calcium and zinc using fura-2FF and FluoZin-3

Elevation of intracellular free zinc ([Zn2+]i) probably contributes to cell death in injury paradigms involving calcium deregulation and oxidative stress such as glutamate excitotoxicity. However, it is difficult to monitor both ions simultaneously in live cells. Here we present a new method using fluorescence microscopy and the ion sensitive indicators fura-2FF and FluoZin-3 to monitor both [Ca2+]i and [Zn2+]i in primary cortical neurons. We show that the new single wavelength dye FluoZin-3 responds robustly to small zinc loads, is insensitive to high Ca2+ or Mg2+, and is relatively unaffected by low pH or oxidants. The ratiometric indicator fura-2FF is sensitive to both Ca2+ and Zn2+. However, in conditions analogous to excitotoxic glutamate exposure where [Ca2+]i is high relative to [Zn2+]i, we found that fura-2FF responds mostly to [Ca2+]i but is relatively unaffected by low [Zn2+]i. Moreover, fura-2FF ratio changes caused by high [Ca2+]i or high [Zn2+]i could be distinguished because each ion produces a different spectral response. Finally, dual dye experiments showed that FluoZin-3 and fura-2FF respond robustly to [Zn2+]i and [Ca2+]j, respectively, in the same neurons during intense glutamate exposure. These studies provide a novel method for the simultaneous detection of both calcium and zinc in cells.     

Copper exposure modifies the content and distribution of trace metals in mammalian cultured cells

With this work, we have determined the cellular content of Cu, Fe and Zn in different cell lines, by using total reflection X-ray fluorescence spectrometry (TXRF). In addition, we examined whether cellular exposure to 100 moles l^–1 of Cu-His modifies the intracellular content and distribution of these trace metals. Our results indicate that all the cell lines displayed the same pattern of relative intracellular abundance of trace metals (Cu相似文献