Intracellular Free Zn Research Articles

The five homologous CiaR-controlled Ccn sRNAs of Streptococcus pneumoniae modulate Zn-resistance.

Zinc is a vital transition metal for all bacteria; however, elevated intracellular free Zn levels can result in mis-metalation of Mn-dependent enzymes. For Mn-centric bacteria such as Streptococcus pneumoniae that primarily use Mn instead of Fe as an enzyme cofactor, Zn is particularly toxic at high concentrations. Here, we report our identification and characterization of the function of the five homologous, CiaRH-regulated Ccn sRNAs in controlling S. pneumoniae virulence and metal homeostasis. We show that deletion of all five ccn genes (ccnA, ccnB, ccnC, ccnD, and ccnE) from S. pneumoniae strains D39 (serotype 2) and TIGR4 (serotype 4) causes Zn hypersensitivity and an attenuation of virulence in a murine invasive pneumonia model. We provide evidence that bioavailable Zn disproportionately increases in S. pneumoniae strains lacking the five ccn genes. Consistent with a response to Zn intoxication or relatively high intracellular free Zn levels, expression of genes encoding the CzcD Zn exporter and the Mn-independent ribonucleotide reductase, NrdD-NrdG, were increased in the ΔccnABCDE mutant relative to its isogenic ccn+ parent strain. The growth inhibition by Zn that occurs as the result of loss of the ccn genes is rescued by supplementation with Mn or Oxyrase, a reagent that removes dissolved oxygen. Lastly, we found that the Zn-dependent growth inhibition of the ΔccnABCDE strain was not altered by deletion of sodA, whereas the ccn+ ΔsodA strain phenocopied the ΔccnABCDE strain. Overall, our results indicate that the Ccn sRNAs have a crucial role in preventing Zn intoxication in S. pneumoniae.

Erucic acid increases the potency of cisplatin-induced colorectal cancer cell death and oxidative stress by upregulating the TRPM2 channel.

Erucic acid (ErA) is a source of omega-9 monounsaturated fatty acids. ErA exhibited antitumor effects by causing apoptosis and oxidative stress in tumor cells, with the exception of the HT-29 human colorectal cancer cell line. The apoptotic and Ca2+ signaling pathways in tumor cells are triggered when mitochondrial Ca2+ and Zn2+ accumulation produce reactive free oxygen species (ROS), which in turn activate TRPM2. ErA-induced ROS and TRPM2 stimulation may augment the anticancer action of cisplatin (CSP). We aimed to study the effects of ErA and CSP incubations on ROS, apoptosis, and cell death in the HT-29 cells by activating TRPM2. The cells were divided into five groups: control, ErA (200 µM for 48 h), CSP (25 µM for 24 h), and ErA + CSP + TRPM2 antagonists (200 µM carvacrol and 25 µM N-(p-amylcinnamoyl)anthranilic acid for 24 h). The TRPM2 antagonists reduced ErA plus CSP-induced increases in H2O2-induced intracellular free Ca2+ concentration ([Ca2+]c) and adenosine diphosphate-ribose-caused TRPM2 currents. ErA and CSP were found to cause apoptosis and cell death by raising the intracellular free Zn2+ concentration (Zn2+]c), caspase-3, -8, and -9, mitochondrial membrane dysfunction, and ROS, while lowering reduced glutathione, cell viability, and cell number. The oxidative, apoptotic, and tumor cell death effects of CSP in the cells were enhanced by the increase of ErA-mediated [Ca2+]c and Zn2+]c entering mitochondria through the activation of TRPM2. In conclusion, we observed that the combination of ErA and CSP was synergistic via TRPM2 activation for the treatment of HT-29 tumor cells.

Effect of estrogen and progesterone on intracellular free zinc and zinc transporter expression in bovine oviduct epithelial cells

Zinc (Zn) plays essential roles in numerous cellular processes. However, there is limited understanding of Zn homeostasis within the bovine reproductive system. This study investigated the influence of estradiol (E2) and progesterone (P4) on Zn transporter expression and intracellular free Zn levels in bovine oviduct epithelial cells (BOEC). For this purpose, cells were harvested from slaughtered cows and cultured in vitro. Intracellular Zn concentrations were measured using FluoZin-3AM staining, while real-time polymerase chain reaction assessed Zn transporter gene expression and quantification. Overall, our results confirmed the gene expression of all the evaluated Zn transporters (ZIP6, ZIP8, ZIP14, ZnT3, ZnT7 and ZnT9), denoted and the active role of E2 and P4 in intracellular Zn regulation. Our findings suggest an interaction between Zn, E2 and P4.

A combined biochemical and cellular approach reveals Zn2+-dependent hetero- and homodimeric CD4 and Lck assemblies in T cells

The CD4 or CD8 co-receptors' interaction with the protein-tyrosine kinase Lck initiates the tyrosine phosphorylation cascade leading to Tcell activation. A critical question is: to what extent are co-receptors and Lck coupled? Our contribution concerns Zn2+, indispensable for CD4- and CD8-Lck formation. We combined biochemical and cellular approaches to show that dynamic fluctuations of free Zn2+ in physiological ranges influence Zn(CD4)2 and Zn(CD4)(Lck) species formation and their ratio, although the same Zn(Cys)2(Cys)2 cores. Moreover, we demonstrated that the affinity of Zn2+ to CD4 and CD4-Lck species differs significantly. Increased intracellular free Zn2+ concentration in Tcells causes higher CD4 partitioning in the plasma membrane. We additionally found that CD4 palmitoylation decreases the specificity of CD4-Lck formation in the reconstituted membrane model. Our findings help elucidate co-receptor-Lck coupling stoichiometry and demonstrate that intracellular free Zn2+ has a major role in the interplay between CD4 dimers and CD4-Lck assembly.

An Overexpression of SLC30A6 Gene Contributes to Cardiomyocyte Dysfunction via Affecting Mitochondria and Inducing Activations in K-Acetylation and Epigenetic Proteins.

Intracellular free Zn2+ ([Zn2+]i) is less than 1-nM in cardiomyocytes and its regulation is performed with Zn2+-transporters. However, the roles of Zn2+-transporters in cardiomyocytes are not defined exactly yet. Here, we aimed to examine the role of an overexpression and subcellular localization of a ZnT6 in insulin-resistance mimic H9c2 cardiomyoblasts (IR-cells; 50-μM palmitic acid for 24-h incubation). We used both IR-cells and ZnT6-overexpressed (ZnT6OE) cells in comparison to those of H9c2 cells (CON-cells). The IR-cells have higher ZnT6-protein levels than CON-cells while this level was similar to those of ZnT6OE-cells. The [Zn2+]i in IR-cells was increased significantly and mitochondrial localization of ZnT6 was demonstrated in these cells by using confocal microscopy visualization. Furthermore, electron microscopy analysis demonstrated abnormal morphological appearance in both IR-cells and ZnT6OE-cells characterized by irregular mitochondrion cristae and condensed and dilated cisterna in the sarcoplasmic reticulum. Mitochondria were similarly depolarized in both IR-cells and ZnT6OE-cells. The protein expression level of a mitofusin protein MFN2 in the IR-cells was decreased, significantly, whereas, it was found significantly upregulated in both ZnT6-OE-cells and IR-incubated ZnT6OE-cells, which demonstrates the role of ZnT6-overexpression but not IR. Additionally, the total protein level of a mitochondrial fission protein, dynamin-related protein 1, DRP1 was found to be increased over 1.5-fold in IR-cells while this increase was found to be higher in the ZnT6OE-cells than those of IR-cells, demonstrating an additional effect on IR-increase. ZnT6-overexpression induced also significant increases in K-acetylation, trimethylation of histone H3 lysine27, and mono-methylation of histone H3 lysine36, in a similar manner to those of IR-cells. Overall, our data point out an important contribution of ZnT6-overexpression to IR-induced cellular changes, such as alteration in mitochondria function and activation of epigenetic modifications.

Biological Effects of HDAC Inhibitors Vary with Zinc Binding Group: Differential Effects on Zinc Bioavailability, ROS Production, and R175H p53 Mutant Protein Reactivation.

The coordination of zinc by histone deacetylase inhibitors (HDACi), altering the bioavailability of zinc to histone deacetylases (HDACs), is key to HDAC enzyme inhibition. However, the ability of zinc binding groups (ZBGs) to alter intracellular free Zn+2 levels, which may have far-reaching effects, has not been explored. Using two HDACis with different ZBGs, we documented shifts in intracellular free Zn+2 concentrations that correlate with subsequent ROS production. Next, we assayed refolding and reactivation of the R175H mutant p53 protein in vitro to provide greater biological context as the activity of this mutant depends on cellular zinc concentration. The data presented demonstrates the differential activity of HDACi in promoting R175H response element (RE) binding. After cells are treated with HDACi, there are differences in R175H mutant p53 refolding and reactivation, which may be related to treatments. Collectively, we show that HDACis with distinct ZBGs differentially impact the intracellular free Zn+2 concentration, ROS levels, and activity of R175H; therefore, HDACis may have significant activity independent of their ability to alter acetylation levels. Our results suggest a framework for reevaluating the role of zinc in the variable or off-target effects of HDACi, suggesting that the ZBGs of HDAC inhibitors may provide bioavailable zinc without the toxicity associated with zinc metallochaperones such as ZMC1.

Ratiometric Bioluminescent Zinc Sensor Proteins to Quantify Serum and Intracellular Free Zn2.

Fluorescent Zn2+ sensors play a pivotal role in zinc biology, but their application in complex media such as blood serum or plate reader-based cellular assays is hampered by autofluorescence and light scattering. Bioluminescent sensor proteins provide an attractive alternative to fluorescent sensors for these applications, but the only bioluminescent sensor protein developed so far, BLZinCh, has a limited sensor response and a suboptimal Zn2+ affinity. In this work, we expanded the toolbox of bioluminescent Zn2+ sensors by developing two new sensor families that show a large change in the emission ratio and cover a range of physiologically relevant Zn2+ affinities. The LuZi platform relies on competitive complementation of split NanoLuc luciferase and displays a robust, 2-fold change in red-to-blue emission, allowing quantification of free Zn2+ between 2 pM and 1 nM. The second platform was developed by replacing the long flexible GGS linker in the original BLZinCh sensor by rigid polyproline linkers, yielding a series of BLZinCh-Pro sensors with a 3–4-fold improved ratiometric response and physiologically relevant Zn2+ affinities between 0.5 and 1 nM. Both the LuZi and BLZinCh-Pro sensors allowed the direct determination of low nM concentrations of free Zn2+ in serum, providing an attractive alternative to more laborious and/or indirect approaches to measure serum zinc levels. Furthermore, the genetic encoding of the BLZinCh-Pro sensors allowed their use as intracellular sensors, where the sensor occupancy of 40–50% makes them ideally suited to monitor both increases and decreases in intracellular free Zn2+ concentration in simple, plate reader-based measurements, without the need for fluorescence microscopy.

HMA4 and IRT3 as indicators accounting for different responses to Cd and Zn by hyperaccumulator Arabidopsis halleri ssp. gemmifera

Using plants as bioremediation agents are rising worldwide as an environmentally friendly tool to remove contaminants such as heavy metals. Arabidopsis halleri ssp. gemmifera is able to hyperaccumulate Cd/Zn, however, the commonality between Cd/Zn accumulation pathways are still vague. This study focused on HMA4 and IRT3 as indicators and investigated their transcription in A. halleri ssp. gemmifera under Cd/Zn treatments. In addition to the known Cd/Zn xylem loading functions in roots, HMA4 showed a potential role in Cd/Zn redistribution or metal efflux from the vascular bundle in leaves due to increased transcription by 3-fold after 7 d under all treatments. On the other hand, IRT3 as a Zn homeostasis gene was expressed continuously in both roots and leaves under single Cd or Zn treatment. However, IRT3 transcription in leaves plunged to 50-fold lower under combined Cd and Zn treatment. The result indicated an increase of intracellular free Zn ion, implying a new unreported mechanism of Zn homeostasis in leaves under the presence of both metals. Furthermore, no competition between Cd/Zn uptake and accumulation suggested the existence of independent transport system for Cd and Zn in A. halleri ssp. gemmifera, which is differed from what have been reported until now.

Melatonin and Selenium Suppress Docetaxel-Induced TRPV1 Activation, Neuropathic Pain andOxidativeNeurotoxicity in Mice.

Docetaxel (DT) has been reported to positive therapeutic actions in the treatment of glioblastoma, breast tumors, and prostate cancers. However, it can also induce peripheral neuropathic pain and neurotoxicity as adverse effects. Expression level of TRPV1 cation channel is high in dorsal root ganglion (DRG), and its activation via capsaicin and reactive oxygen species (ROS) mediates peripheral neuropathic pain in mice. As cancer is known to increase the levels of ROS, the protective roles of melatonin (MT) and selenium (Se) were evaluated on the TRPV1-mediated neurotoxicity and pain in the DT-treated mice. Mice and TRPV1 expressing SH-SY5Y cells were equally divided into control, MT, Se, DT, DT+MT, and DT+Se groups. In the results of pain tests in the mice, we observed a decrease in DT-mediated mechanical and heat neuropathic pain by MT and Se. The results of plate reader assay and laser confocal microscopy image analyses indicated a protective role of MT and Se on the DT-induced increase of mitochondrial ROS, cytosolic ROS, apoptosis, lipid peroxidation, intracellular free Zn2+, Ca2+, and caspase-3 and -9 levels in the DRG and SH-SY5Y cells. MT and Se modulated DT-induced decreases of total antioxidant status, reducedglutathione and glutathione peroxidase in the DRG. However, the effects of DT were not observed in the non-TRPV1 expressing SH-SY5Y cells. Hence, MT and Se mediated protective effects against DT-induced adverse peripheral oxidative neurotoxicity and peripheral pain. These effects may be attributed to potent antioxidant properties of MT and Se.

Glutathione Depletion and Parkinsonian Neurotoxin MPP+-Induced TRPM2 Channel Activation Play Central Roles in Oxidative Cytotoxicity and Inflammation in Microglia.

Parkinson's disease (PD) is one of most common neurodegenerative diseases. Environmental stressors such as oxidative stress (OS), calcium ion influx, apoptosis, and inflammation mechanisms are linked to activated microglia in patients with PD. The OS-dependent activated transient receptor potential melastatin 2 (TRPM2) channel is modulated in several neurons by glutathione (GSH). However, the cellular and molecular effects of GSH alteration on TRPM2 activation, OS, apoptosis, and inflammation in the microglia remain elusive. The microglia of TRPM2 wild-type (TRPM2-WT) and knockout (TRPM2-KO) mice were divided into control, PD model (MPP), L-buthionine sulfoximine (BSO), MPP + BSO and MPP + BSO + GSH groups. MPP-induced increases in apoptosis, death, OS, lipid peroxidation, PARP1, caspase-3 and caspase-9, inflammatory cytokines (IL-1β, TNF-α, IL-6), and intracellular free Zn2+ and Ca2+ levels in the microglia of TRPM2-WT mice were further increased by the BSO treatment, although they were diminished by the GSH treatment. Their levels were further reduced by PARP1 inhibitors (PJ34 and DPQ) and TRPM2 blockers (ACA and 2-APB). However, the effects of MPP and BSO were not observed in the microglia of TRPM2-KO mice. Taken together, our data demonstrate that maintaining GSH homeostasis is not only important for quenching OS in the microglia of patients with PD but also equally critical to modulating TRPM2, thus suppressing inflammatory responses elicited by environmental stressors.

Resveratrol attenuates hypoxia-induced neuronal cell death, inflammation and mitochondrial oxidative stress by modulation of TRPM2 channel

Hypoxia (HYPX) induced-overload Ca2+ entry results in increase of mitochondrial oxidative stress, inflammation and apoptosis in several neurons. Ca2+ permeable TRPM2 channel was gated by ADP-ribose (ADPR) and reactive oxygen species (ROS), although its activity was modulated in HYPX-exposed neurons by resveratrol (RSV). The aim of this study was to evaluate if a therapy of RSV can modulate the effect of HYPX in the TRPM2 expressing SH-SY5Y neuronal and HEK293 (no expression of TRPM2) cell lines. The SH-SY5Y and HEK293 cells were divided into four groups as control, RSV (50 μM and 24 hours), and HYPX and RSV + HYPX. For induction of HYPX in the cells, CoCl2 (200 μM and 24 hours) incubation was used. HYPX-induced intracellular Ca2+ responses to TRPM2 activation were increased in the SH-SY5Y cells but not in the HEK293 cells from coming H2O2 and ADPR. RSV treatment improved intracellular Ca2+ responses, mitochondrial function, suppressed the generation of cytokine (IL-1β and TNF-α), cytosolic and mitochondrial ROS in the SH-SY5Y cells. Intracellular free Zn2+, apoptosis, cell death, PARP-1, TRPM2 expression, caspase −3 and −9 levels are increased through activating TRPM2 in the SH-SY5Y cells exposed to the HYPX. However, the values were decreased in the cells by RSV and TRPM2 blockers (ACA and 2-APB). In SH-SY5Y neuronal cells exposed to HYPX conditions, the neuroprotective effects of RSV were shown to be exerted via modulation of oxidative stress, inflammation, apoptosis and death through modulation of TRPM2 channel. RSV could be used as an effective agent in the treatment of neurodegeneration exposure to HYPX.

The Zn2+‐Transporter ZnT6 Plays Important Role on Regulation of Cytosolic Free Zn2+ Through Modulation of Mitochondrial Zn2+ in Hyperglycemic Cardiomyocytes

Free zinc ion (Zn2+), have physiological importance for mammals and is found to be less than 1‐nM in cytosol. However, intracellular free Zn2+ levels ([Zn2+]i) can increase dramatically under pathological conditions including hyperglycemia and cancer. Any increase in [Zn2+]i can also cause significant changes in several intracellular signaling pathways. However, that [Zn2+]i is well regulated by various ways in physiological conditions and that value can fluctuate dynamically in cardiomyocytes. It is accepted that there are two‐types of Zn2+‐transporters, one group of them is responsible Zn2+‐influx from extracellular space and/or organelles to cytosol (ZIPs) and the other group is responsible Zn2+‐efflux from cytosol (ZnTs). Since it is not known whether the Zn2+‐transporter ZnT6 has a role in the regulation of [Zn2+]i in cardiomyocytes under hyperglycemia, we used control and ZnT6 overexpressed H9c2 cells (rat embryonic ventricular cell lines) to investigate its role. In addition, H9c2 cells are incubated with normal (5.5‐mM; control) and high glucose (25‐mM; hyperglycemic) for 24‐h. Following the treatment, control and ZnT6‐overexpressed cells are transfected with Zn+2‐sensitive as well as cytosole, sarco/endoplasmic reticulum S(ER) or mitochondria targeted Förster resonance energy transfer (FRET) sensors to measure cytosolic, S(ER) and mitochondrial [Zn2+], respectively. Furthermore, we examined the mRNA, protein expression levels and intracellular localizations by using molecular, biochemical and immunofluorescence methods. Our results demonstrated that ZnT6 but not ZnT5 mRNA and protein level is increased under hyperglycemia, while ZnT6 overexpression caused increase of ZnT5 protein level. Our immunofluorescence/precipitation experiments showed that ZnT6 localized on mitochondria together with ZnT5 in H9c2 cells. Furthermore, our [Zn2+] measurements by fluorescence imaging showed that cytosolic and mitochondrial [Zn2+] are increased and S(E)R [Zn2+] is decreased in hyperglycemic H9c2 cells. We also found that, ZnT6‐overexpression caused mitochondrial [Zn2+] increase and decrease in cytosolic [Zn2+], without changing the S(E)R [Zn2+]. To understand the role of excess [Zn2+] on mitochondrial function, we used ZnT6 overexpressed or high Zn2+ incubated H9c2 cell lines to measure mitochondrial‐membrane‐potential (MMP), ROS and ATP production. We found that excess intracellular or mitochondrial [Zn2+] caused depolarization of MMP, increase in ROS and decrease in ATP production. Overall, our results showed that ZnT6 has a significant role on [Zn2+]i by modulating mitochondrial [Zn2+] and function in hyperglycemia.Support or Funding InformationTUBITAK‐SBAG‐117S386TUBITAK‐SBAG‐119S661

Molecular characterization of ten zinc (Zn) transporter genes and their regulation to Zn metabolism in freshwater teleost yellow catfish Pelteobagrus fulvidraco

BackgroundZn is an essential trace element for vertebrates, and Zn uptake and transport is related with the ZIP family of Zn transporters. Meantime, Zn also influenced the expression of ZIP family members. MethodsWe cloned and characterized the full-length cDNA sequences of ten Zn transport-relevant genes (ZIP1, ZIP3, ZIP6, ZIP7, ZIP8, ZIP9, ZIP10, ZIP11, ZIP13 and ZIP14) from yellow catfish Pelteobagrus fulvidraco, investigated their mRNA tissue expression. These ZIP mRNA expression was also assessed in the primary hepatocytes and intestinal epithelial cells of yellow catfish in response to three Zn levels (0, 30 μM and 60 μM, respectively). ResultsAll these genes shared the similar domains with the corresponding members in mammals. The mRNA expression of the ten ZIP genes was detected in nine-tested tissues, but variable among these tissues. Flow cytometry analysis and confocal microscopy observation indicated that intracellular free Zn2+ concentration in hepatocytes and intestinal epithelial cells increased with increasing Zn incubation concentration at both 24 h and 48 h. Zn incubation differentially influenced mRNA levels of ZIP transporters in the hepatocytes and intestinal epithelial cells, in a time- and cells-dependent manners. In the hepatocytes, at 24 h, compared to the control, Zn addition down-regulated mRNA levels of ZIP1, ZIP3, ZIP6, ZIP7, ZIP8, ZIP9, ZIP11 and ZIP14; however, ZIP10 mRNA levels were lower in 60 μM Zn group than those in the control and 30 μM Zn group. At 48 h, mRNA levels of ZIP1, ZIP6, ZIP7, ZIP9, ZIP10 and ZIP14 declined with increasing Zn incubation concentrations; ZIP3 mRNA levels were the lowest in 60 μM Zn group and showed no significant differences between the control and 30 μM Zn group. In the intestinal epithelial cells, at 24 h, Zn addition down-regulated mRNA levels of ZIP1, ZIP6, ZIP7, ZIP8, ZIP9, ZIP10, ZIP11, ZIP13 and ZIP14; ZIP3 mRNA levels were lower in 60 μM Zn group than those in the control and 30 μM Zn group. At 48 h, Zn addition up-regulated mRNA levels of ZIP6 and ZIP9, but down-regulated mRNA levels of ZIP8, ZIP10 and ZIP13. ZIP7, ZIP11 and ZIP14 mRNA abundances were the lowest in 60 μM Zn group and showed no significant differences between the control and 30 μM Zn group. ConclusionFor the first time, our study characterized ten ZIP family members in yellow catfish, explored their mRNA tissue expression. Their regulation to Zn addition were also investigated in the hepatocytes and intestinal epithelial cells of yellow catfish. Our study revealed the mechanism of cells exposed to Zn addition and provided novel insights for the regulatory mechanism of Zn homeostasis.

Mitochondria-Targeting Antioxidant Provides Cardioprotection through Regulation of Cytosolic and Mitochondrial Zn2+ Levels with Re-Distribution of Zn2+-Transporters in Aged Rat Cardiomyocytes.

Aging is an important risk factor for cardiac dysfunction. Heart during aging exhibits a depressed mechanical activity, at least, through mitochondria-originated increases in ROS. Previously, we also have shown a close relationship between increased ROS and cellular intracellular free Zn2+ ([Zn2+]i) in cardiomyocytes under pathological conditions as well as the contribution of some re-expressed levels of Zn2+-transporters for redistribution of [Zn2+]i among suborganelles. Therefore, we first examined the cellular (total) [Zn2+] and then determined the protein expression levels of Zn2+-transporters in freshly isolated ventricular cardiomyocytes from 24-month rat heart compared to those of 6-month rats. The [Zn2+]i in the aged-cardiomyocytes was increased, at most, due to increased ZIP7 and ZnT8 with decreased levels of ZIP8 and ZnT7. To examine redistribution of the cellular [Zn2+]i among suborganelles, such as Sarco/endoplasmic reticulum, S(E)R, and mitochondria ([Zn2+]SER and [Zn2+]Mit), a cell model (with galactose) to mimic the aged-cell in rat ventricular cell line H9c2 was used and demonstrated that there were significant increases in [Zn2+]Mit with decreases in [Zn2+]SER. In addition, the re-distribution of these Zn2+-transporters were markedly changed in mitochondria (increases in ZnT7 and ZnT8 with no changes in ZIP7 and ZIP8) and S(E)R (increase in ZIP7 and decrease in ZnT7 with no changes in both ZIP8 and ZnT8) both of them isolated from freshly isolated ventricular cardiomyocytes from aged-rats. Furthermore, we demonstrated that cellular levels of ROS, both total and mitochondrial lysine acetylation (K-Acetylation), and protein-thiol oxidation were significantly high in aged-cardiomyocytes from 24-month old rats. Using a mitochondrial-targeting antioxidant, MitoTEMPO (1 µM, 5-h incubation), we provided an important data associated with the role of mitochondrial-ROS production in the [Zn2+]i-dyshomeostasis of the ventricular cardiomyocytes from 24-month old rats. Overall, our present data, for the first time, demonstrated that a direct mitochondria-targeting antioxidant treatment can be a new therapeutic strategy during aging in the heart through a well-controlled [Zn2+] distribution among cytosol and suborganelles with altered expression levels of the Zn2+-transporters.

Zinc source influences the gene expression of zinc transporters in jejunum and cecal tonsils during broiler challenge with Eimeria maximaand Clostridium perfringens

The objective of this experiment was to study the effects of dietary zinc (Zn) source on gene expression of Zn transporters (metallothionein [MT], ZIP 3, 5, 8, 9, 10, 11, 13, and 14, and ZnT 1, 4, 5, 6, 7, 9, and 10) in the jejunum and cecal tonsils of broilers challenged with coccidia or coccidia plus Clostridium perfringens. A 2 × 2 factorial design was used with 2 Zn sources (90mg Zn/kg from either ZnSO4 or an organic Zn, Bioplex® Zn) and challenged with approximately 5,000 oocysts of Eimeria maxima at 14 d of age with or without C. perfringens (108 CFU/bird) at 18, 19, and 20 d of age (8 pens per treatment and 8 birds per pen) after which 1 bird/pen was sampled at 21 d of age. In the jejunum, co-infection resulted in higher ZnT 5 and 6 gene expression, while organic Zn fed birds had lower ZIP 5 and 11, and higher ZnT1. Additionally, an interaction of challenge by Zn source was noted wherein ZnT10 was unaffected by the C. perfringens in the organic Zn treatment but was 2.7-fold lower in the co-infected ZnSO4 fed birds. S100A9 gene expression, a biomarker of inflammatory response in necrotic enteritis, increased 2 and 2.8-fold in the cecal tonsils and jejunum with the co-infection, respectively. Supplementation with organic Zn lowered S100A9 by 1.9 and 4.4-fold in the cecal tonsils and jejunum, respectively, when birds were supplemented with ZnSO4. Notably, MT, ZIP 3, 8, 9, 10, 13, or 14, and ZnT 4, 7, and 9 were unaffected by Zn source and/or method of challenge. An interaction of challenge by Zn source was also noted for serum Zn concentration, which was reduced when birds were challenged with C. perfringens and fed ZnSO4 but no difference between challenge method when birds were fed organic Zn. Based on the expression of ZnT and ZIP genes, more Zn trafficking due to treatment occured in the jejunum than cecal tonsils, but further studies are needed to ascertain how Zn source regulates intracellular free Zn concentrations and whole-body Zn status during an enteric challenge.

Sublethal treatment with plasma-activated medium induces senescence-like growth arrest of A549 cells: involvement of intracellular mobile zinc.

Plasma-activated medium (PAM) is a solution produced by exposing a liquid medium to non-thermal atmospheric pressure plasma (NTAPP). A number of reactive molecules, such as reactive oxygen species and reactive nitrogen species, are contained in PAM. Therefore, exposure to high doses of PAM results in cell death. We previously demonstrated that intracellular zinc (Zn2+) serves as an important mediator in PAM-induced cell death; however, the effects of sublethal treatment with PAM on cell functions are not fully understood. In the present study, we found that sublethal PAM treatment suppressed cell proliferation and induced senescence-like changes in lung adenocarcinoma A549 cells. Cell cycle analysis revealed that PAM induced cell cycle arrest at the G2/M phase. PAM increased the level of intracellular free Zn2+ and the Zn2+ chelator TPEN counteracted PAM-induced growth suppression, suggesting that Zn2+ functions in PAM-induced growth suppression. In addition, sublethal treatment with PAM induced phosphorylation of ATM kinase, accumulation of p53 protein, and expression of p21 and GADD45A, which are known p53 target genes, in a Zn2+-dependent manner. These results suggest that the induction of growth arrest and cellular senescence by sublethal PAM treatment is mediated by Zn2+-dependent activation of the ATM/p53 pathway.

Biphenyl-induced cytotoxicity is mediated by an increase in intracellular Zn2+

Biphenyl is found both in natural and anthropogenic sources and is used as a fungistat in the packaging of citrus fruits. Acute exposure to high levels of biphenyl has been observed to cause skin irritation and toxic effects on the liver and kidneys. However, the mechanisms of cytotoxicity induced by biphenyl are not yet well understood. In the present study, the cytotoxicity of biphenyl was studied by flow cytometry with fluorescent probes. Biphenyl at 100 μM significantly increased cell lethality after 3 h in rat thymocytes. In addition, biphenyl at 100 μM or more elevated intracellular Zn2+ levels. N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), an intracellular and extracellular Zn2+ chelator, but not diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid (DTPA), a membrane-impermeable Zn2+ chelator, attenuated the biphenyl-induced increase in intracellular Zn2+ levels and cell death. These results suggested that biphenyl-induced cytotoxicity caused an increase in intracellular Zn2+ levels, which was dependent on internal Zn2+. Moreover, biphenyl led to an increase in sensitivity to oxidative stress, while TPEN inhibited this biphenyl-induced increase. Our findings revealed that biphenyl caused an increase in the intracellular free Zn2+ concentration, inducing cytotoxicity, cell death, and an increase in sensitivity to oxidative stress.

ZnT8 Arg325Trp polymorphism influences zinc transporter expression and cytokine production in PBMCs from patients with diabetes

AimsZnT8 Arg325Trp polymorphism has been associated with type 2 diabetes (T2DM) susceptibility. The Arg-325 risk variant shows accelerated zinc (Zn) transport kinetic and reduced glucose-stimulated insulin secretion in pancreatic cells. However, it remains unexplored the role of Znt8 polymorphism in the regulation of Zn homeostasis and inflammatory response in peripheral blood mononuclear cells (PBMCs) from T2DM patients. Methods and resultsA total of 556 healthy controls and 413 T2DM patients were genotyped for ZnT8 Arg325Trp polymorphism confirming the association of Arg-325 variant with an increased T2DM risk (OR = 1.35 95% C.I: 1.10–1.66; p = 0.0044). Moreover, PBMCs from Arg/Arg T2DM subjects showed increased intracellular free Zn, higher gene expression of Metallothioneins, Znt1, Znt8, Zip2 genes, and reduced Znt4 and Znt7. Higher release of IL-1α, IL-1β, IFN-γ, IL-12p70 and TNF-α and a reduced IL-10 secretion after lipopolysaccharide (LPS) stimulation were observed in PBMCs from Arg/Arg T2DM carriers as compared to subjects with the Trp variant. ConclusionsOur data provide evidence of a substantial different Zn homeostasis regulation between Znt8 Arg-325 and Trp-325 carriers in PBMCs from T2DM patients. Moreover, Znt8 Arg-325 risk variant shows an enhanced inflammatory response upon LPS stimulation that might aggravate insulin resistance and the progression of diabetes cardiovascular complications.

Demonstration of subcellular migration of CK2α localization from nucleus to sarco(endo)plasmic reticulum in mammalian cardiomyocytes under hyperglycemia.

The cellular control of glucose uptake and glycogen metabolism in mammalian tissues is in part mediated through the regulation of protein-serine/threonine kinases including CK2. Although it participates to several cellular signaling processes, however, its subcellular localization is not well-defined while some documents mentioned its localization change under pathological conditions. The activation/phosphorylation of some proteins including Zn2+-transporter ZIP7 in cardiomyocytes is controlled with CK2α, thereby, inducing changes in the level of intracellular free Zn2+ ([Zn2+] i ). In this regard, we aimed to examine cellular localization of CK2α in cardiomyocytes and its possible subcellular migration under hyperglycemia. Our confocal imaging together with biochemical analysis in isolated sarco(endo)plasmic reticulum [S(E)R] and nuclear fractions from hearts have shown that CK2α localized highly to S(E)R and Golgi and weakly to nuclear fractions in physiological condition. However, it can migrate from nuclear fractions to S(E)R under hyperglycemia. This migration can further underlie phosphorylation of a target protein ZIP7 as well as some endogenous kinases and phosphatases including PKA, CaMKII, and PP2A. We also have shown that CK2α activation is responsible for hyperglycemia-associated [Zn2+] i increase in diabetic heart. Therefore, our present data demonstrated, for the first time, the physiological relevance of CK2α in cellular control of Zn2+-distribution via inducing ZIP7 phosphorylation and activation of these above endogenous actors in hyperglycemia/diabetes-associated cardiac dysfunction. Moreover, our present data also emphasized the multi-subcellular compartmental localizations of CK2α and a tightly regulation of these localizations in cardiomyocytes. Therefore, taken into consideration of all data, one can emphasize the important role of the subcellular localization of CK2α as a novel target-pathway for understanding of diabetic cardiomyopathy.

Induction of endoplasmic reticulum stress and changes in expression levels of Zn2+-transporters in hypertrophic rat heart.

Clinical and experimental studies have shown an association between intracellular free Zn2+ ([Zn2+]i)-dyshomeostasis and cardiac dysfunction besides [Ca2+]i-dyshomeostasis. Since [Zn2+]i-homeostasis is regulated through Zn2+-transporters depending on their subcellular distributions, one can hypothesize that any imbalance in Zn2+-homeostasis via alteration in Zn2+-transporters may be associated with the induction of ER stress and apoptosis in hypertrophic heart. We used a transverse aortic constriction (TAC) model to induce hypertrophy in young male rat heart. We confirmed the development of hypertrophy with a high ratio of heart to body weight and cardiomyocyte capacitance. The expression levels of ER stress markers GRP78, CHOP/Gadd153, and calnexin are significantly high in TAC-group in comparison to those of controls (SHAM-group). Additionally, we detected high expression levels of apoptotic status marker proteins such as the serine kinase GSK-3β, Bax-to-Bcl-2 ratio, and PUMA in TAC-group in comparison to SHAM-group. The ratios of phospho-Akt to Akt and phospho-NFκB to the NFκB are significantly higher in TAC-group than in SHAM-group. Furthermore, we observed markedly increased phospho-PKCα and PKCα levels in TAC-group. We, also for the first time, determined significantly increased ZIP7, ZIP14, and ZnT8 expressions along with decreased ZIP8 and ZnT7 levels in the heart tissue from TAC-group in comparison to SHAM-group. Furthermore, a roughly calculated total expression level of ZIPs responsible for Zn2+-influx into the cytosol (increased about twofold) can be also responsible for the markedly increased [Zn2+]i detected in hypertrophic cardiomyocytes. Taking into consideration the role of increased [Zn2+]i via decreased ER-[Zn2+] in the induction of ER stress in cardiomyocytes, our present data suggest that differential changes in the expression levels of Zn2+-transporters can underlie mechanical dysfunction, in part due to the induction of ER stress and apoptosis in hypertrophic heart via increased [Zn2+]i- besides [Ca2+]i-dyshomeostasis.