Zinc availability regulates exit from meiosis in maturing mammalian oocytes.

Cellular metal ion fluxes are known in alkali and alkaline earth metals but are not well documented in transition metals. Here we describe major changes in the zinc physiology of the mammalian oocyte as it matures and initiates embryonic development. Single-cell elemental analysis of mouse oocytes by synchrotron-based X-ray fluorescence microscopy (XFM) revealed a 50% increase in total zinc content within the 12-14-h period of meiotic maturation. Perturbation of zinc homeostasis with a cell-permeable small-molecule chelator blocked meiotic progression past telophase I. Zinc supplementation rescued this phenotype when administered before this meiotic block. However, after telophase arrest, zinc triggered parthenogenesis, suggesting that exit from this meiotic step is tightly regulated by the availability of a zinc-dependent signal. These results implicate the zinc bolus acquired during meiotic maturation as an important part of the maternal legacy to the embryo.

Characterization of lymphocyte transformation induced by zinc ions.

Lymphocyte cultures from all normal human adults are stimulated by zinc ions to increase DNA and RNA synthesis and undergo blast transformation. Optimal stimulation occurs at 0.1 mM Zn(++). Examination of the effects of other divalent cations reveals that 0.01 mM Hg(++) also stimulates lymphocyte DNA synthesis. Ca(++) and Mg(++) do not affect DNA synthesis in this culture system, while Mn(++), Co(++), Cd(++), Cu(++), and Ni(++) at concentrations of 10(-7)-10(-3) M are inhibitory. DNA and RNA synthesis and blast transformation begin to increase after cultures are incubated for 2-3 days with Zn(++) and these processes reach a maximum rate after 6 days. The increase in Zn(++)-stimulated lymphocyte DNA synthesis is prevented by rendering cells incapable of DNA-dependent RNA synthesis with actinomycin D or by blocking protein synthesis with cycloheximide or puromycin. Zn(++)-stimulated DNA synthesis is also partially inhibited by 5'-AMP and chloramphenicol. Zn(++) must be present for the entire 6-day culture period to produce maximum stimulation of DNA synthesis. In contrast to its ability to independently stimulate DNA synthesis, 0.1 mM Zn(++) inhibits DNA synthesis in phytohemagglutinin-stimulated lymphocytes and L1210 lymphoblasts.

Zinc induces expression of the BH3-only protein PUMA through p53 and ERK pathways in SH-SY5Y neuroblastoma cells.

Accumulating evidence suggests that zinc (Zn2+) contributes to neuronal death in pathologic states such as ischemia. p53-upregulated modulator of apoptosis (PUMA), which is a BH3-only protein, is known to promote apoptosis through a tumor suppressor p53-dependent and -independent mechanism. In this study, we examined the effect of Zn2+ on the induction of the PUMA gene in human neuroblastoma SH-SY5Y cells. The expression of PUMA was induced by Zn2+ in a dose- and time-dependent manner. A reporter assay revealed that Zn2+ activated the PUMA promoter. In addition, the mutation of the p53 binding site in the PUMA promoter region reduced promoter activation by Zn2+. These findings suggest that p53 participates in Zn2+-induced PUMA expression. Furthermore, we also demonstrated here that Zn2+ stimulates the phosphorylation of ERK and that the MEK-ERK pathway inhibitor, U0126, suppressed Zn2+-induced PUMA expression. Taken together, these results indicate that Zn2+ regulates the induction of PUMA through p53 and ERK pathways.

Identification and mechanism of action of two histidine residues underlying high-affinity Zn2+ inhibition of the NMDA receptor.

Zinc (Zn2+) inhibition of N-methyl-D-aspartate receptor (NMDAR) activity involves both voltage-independent and voltage-dependent components. Recombinant NR1/NR2A and NR1/NR2B receptors exhibit similar voltage-dependent block, but voltage-independent Zn2+ inhibition occurs with much higher affinity for NR1/NR2A than NR1/NR2B receptors (nanomolar versus micromolar IC50, respectively). Here, we show that two neighboring histidine residues on NR2A represent the critical determinant (termed the "short spacer") for high-affinity, voltage-independent Zn2+ inhibition using the Xenopus oocyte expression system and site-directed mutagenesis. Mutation of either one of these two histidine residues (H42 and H44) in the extracellular N-terminal domain of NR2A shifted the IC50 for high-affinity Zn2+ inhibition approximately 200-fold without affecting the EC50 of the coagonists NMDA and glycine. We suggest that the mechanism of high-affinity Zn2+ inhibition on the NMDAR involves enhancement of proton inhibition.

Chelation of neurotoxic zinc levels does not improve neurobehavioral outcome after traumatic brain injury.

Increases of synaptically released zinc and intracellular accumulation of zinc in hippocampal neurons after traumatic or ischemic brain injury is neurotoxic and chelation of zinc has been shown to reduce neurodegeneration. Although our previous studies showed that zinc chelation in traumatically brain-injured rats correlated with an increase in whole-brain expression of several neuroprotective genes and reduced numbers of apoptotic neurons, the effect on functional outcome has not been determined, and the question of whether this treatment may actually be clinically relevant has not been answered. In the present study, we show that treatment of TBI rats with the zinc chelator calcium EDTA reduces the numbers of injured, Fluoro-Jade-positive neurons in the rat hippocampus 24 h after injury but does not improve neurobehavioral outcome (spatial memory deficits) 2 weeks post-injury. Our data suggest that zinc chelation, despite providing short-term histological neuroprotection, fails to improve long-term functional outcome, perhaps because long-term disruptions in homeostatic levels of zinc adversely influence hippocampus-dependent spatial memory.

D-serine, a selective glycine/N-methyl-D-aspartate receptor agonist, antagonizes the antidepressant-like effects of magnesium and zinc in mice.

Zinc and magnesium are potent inhibitors of the N-methyl-D-aspartate (NMDA) receptor complex. Recent data demonstrate that both zinc and magnesium, like other NMDA receptor antagonists, exhibit antidepressant-like activity in rodent screening tests and depression models. In the present study, we investigated the effect of D-serine (agonist for the glycine(B) site of the NMDA receptor complex; 100 nmol/mouse, icv) on magnesium (30 mg/kg, ip)- and zinc (5 mg/kg, ip)-induced activity during a forced swim test (FST) in mice. The antidepressant-like effect observed during FST for both ions was abolished by D-serine co-treatment. The present study indicates that the NMDA receptor complex, especially the glycine(B) site, plays a role in the antidepressant-like activity of magnesium and zinc in the FST in mice.

Tyrosine kinase potentiates NMDA receptor currents by reducing tonic zinc inhibition.

Activation of the tyrosine kinase Src potentiates NMDA-receptor currents, which is thought to be necessary for induction of hippocampal long-term potentiation. Although the carboxy(C)-terminal domain of the NR2A subunit contains potential tyrosine phosphorylation sites, the mechanisms by which Src modulates synaptic plasticity and NMDA receptor currents is not fully understood. Here we present evidence from NR1 mutants and splice variants that Src potentiates NMDA-receptor currents by reducing the tonic inhibition of receptors composed of NR1 and NR2A subunits by extracellular zinc. Using site-directed mutagenesis, we have identified three C-terminal tyrosine residues of NR2A that are required for Src's modulation of the zinc sensitivity of NMDA receptors. Our data link two modulatory sites of NMDA receptors that were previously thought to be independent.

Absorption of zinc and retention of calcium: dose-dependent inhibition by phytate.

The dose-dependent inhibitory effect of sodium phytate (myo-inositol-hexaphosphate) on absorption of zinc and retention of calcium was studied in man. No systematic study of this dose-response effect has been reported to this time. Forty subjects were served meals containing white wheat rolls without/with additions of phytate. Ten subjects were given test meals containing one or two of the studied levels of phytate and in addition all subjects were served meals to which no phytate was added. The zinc content was 3.1 mg (47 micromol) and the calcium content 266 mg (6.6 mmol). The rolls were labelled extrinsically with radioisotopes, 65Zn and 47Ca, and whole-body retention of both minerals was measured. Totally 105 meals were served, 36 meals in which no phytate was added and 9-10 meals on each level of phytate. The zinc absorption in meals to which either 0, 25, 50, 75, 100, 140, 175 or 250 mg of phytate-P (0, 134, 269, 403, 538, 753, 941 or 1344 micromol phytate) had been added was 22%, 16%, 14%, 11%, 7%, 7%, 7% and 6%, respectively (mean values). The addition of 50 mg phytate-P or more significantly decreased zinc absorption (p=0.01) as compared to absorption from the test meals with no added phytate. The calcium retention at day 7 in the same meals was 31%, 28%, 27%, 26%, 22%, 19%, 14% and 11% (mean values). The addition of 100 mg phytate-P or more significantly decreased calcium retention (p=0.03) compared to the test meals with no added phytate. It was concluded that the inhibitory effect of phytate on the absorption of zinc and the retention of calcium was dose dependent.

Reversible inhibition of the second step of splicing suggests a possible role of zinc in the second step of splicing.

A multicomponent complex of proteins and RNA is assembled on the newly synthesized pre-mRNA to form the spliceosome. This complex catalyzes a two-step transesterification reaction required to remove the introns and ligate the exons. To date, only six proteins have been found necessary for the second step of splicing in yeast, and their human homologs have been identified. We demonstrate that the addition of the selective chelator of zinc, 1,10-phenanthroline, to an in vitro mRNA splicing reaction causes a dose-dependent inhibition of the second step of splicing. This inhibition is accompanied by the accumulation of spliceosomes paused before completion of step two of the splicing reaction. The inhibition effect on the second step is due neither to snRNA degradation nor to direct binding to the mRNA, and is reversible by dialysis or add-back of zinc, but not of other divalent metals, at the beginning of the reaction. These findings suggest that the activity of a putative zinc-dependent metalloprotein(s) involved in the second step of splicing is affected. This study outlines a new method for specific reversible inhibition of the second step of splicing using external reagents, and suggests a possible role of divalent cations in the second step of mRNA splicing, most likely zinc.

Effect of transition metals on binding of p53 protein to supercoiled DNA and to consensus sequence in DNA fragments.

Recently we have shown that wild-type human p53 protein binds preferentially to supercoiled (sc) DNA in vitro in both the presence and absence of the p53 consensus sequence (p53CON). This binding produces a ladder of retarded bands on an agarose gel. Using immunoblotting with the antibody DO-1, we show that the bands obtained correspond to ethidium-stained DNA, suggesting that each band of the ladder contains a DNA-p53 complex. The intensity and the number of these hands are decreased by physiological concentrations of zinc ions. At higher zinc concentrations, binding of p53 to scDNA is completely inhibited. The binding of additional zinc ions to p53 appears much weaker than the binding of the intrinsic zinc ion in the DNA binding site of the core domain. In contrast to previously published data suggesting that 100 microM zinc ions do not influence p53 binding to p53CON in a DNA oligonucleotide, we show that 5-20 microM zinc efficiently inhibits binding of p53 to p53CON in DNA fragments. We also show that relatively low concentrations of dithiothreitol but not of 2-mercaptoethanol decrease the concentration of free zinc ions, thereby preventing their inhibitory effect on binding of p53 to DNA. Nickel and cobalt ions inhibit binding of p53 to scDNA and to its consensus sequence in linear DNA fragments less efficiently than zinc; cobalt ions are least efficient, requiring >100 microM Co2+ for full inhibition of p53 binding. Modulation of binding of p53 to DNA by physiological concentrations of zinc might represent a novel pathway that regulates p53 activity in vivo.

How safe are folic acid supplements?

Periconceptual use of folic acid supplements by women is effective in preventing neural tube defects in the fetus. Folic acid supplements also may prevent atherosclerosis and some malignant neoplasms. Nevertheless, safety concerns have delayed recommendations to increase folic acid consumption by the general population. Among the potential safety issues of folic acid supplementation are (1) difficulty identifying cobalamin deficiency, precipitation of neurologic complications of cobalamin deficiency, and lowering of cobalamin levels; (2) folate neurotoxicity; (3) antagonism of drugs that inhibit folate metabolism; (4) reduced zinc absorption; (5) association with malignant neoplasms; (6) hypersensitivity reactions; and (7) increased susceptibility to malaria. The data that suggest that folic acid supplements are unsafe are weak and consist predominantly of case series and reports. Nevertheless, greater difficulty diagnosing cobalamin deficiency due to "masking" of hematologic abnormalities by folic acid is a potential risk. Strict vegetarians need to be informed that they are at risk of cobalamin deficiency. Physicians need to be aware that routine hematologic indexes have a low sensitivity for cobalamin deficiency, especially in patients who are receiving folic acid supplements. Because no high-quality data exclude specific adverse effects, physicians should be vigilant in identifying detrimental effects when patients increase their consumption of folic acid.

Search for nutritional confounding factors in the relationship between iron deficiency and brain function.

This paper on confounding factors in the relationship between iron deficiency and brain function is mainly limited to nutritional factors, primarily factors that can contribute to the development of iron deficiency and that may have an independent direct action on brain function. Three theoretically possible confounders were found in a systematic search for dietary factors: 1) low intake of ascorbic acid, 2) excess of phytates, and 3) increased absorption of lead. Ascorbic acid has a marked effect on the bioavailability of dietary iron and is also known to directly influence various metabolic processes in the brain. Phytates inhibit the absorption not only of iron but also of zinc. An iron deficiency may thus be accompanied by a zinc deficiency which may affect mental performance. A state of iron deficiency may increase the absorption of lead from the diet, which in turn may affect brain function.

Metal-protein attenuation with iodochlorhydroxyquin (clioquinol) targeting Abeta amyloid deposition and toxicity in Alzheimer disease: a pilot phase 2 clinical trial.

BACKGROUND: Alzheimer disease (AD) may be caused by the toxic accumulation of beta-amyloid (Abeta). OBJECTIVE: To test this theory, we developed a clinical intervention using clioquinol, a metal-protein-attenuating compound (MPAC) that inhibits zinc and copper ions from binding to Abeta, thereby promoting Abeta dissolution and diminishing its toxic properties. METHODS: A pilot phase 2 clinical trial in patients with moderately severe Alzheimer disease. RESULTS: Thirty-six subjects were randomized. The effect of treatment was significant in the more severely affected group (baseline cognitive subscale score of the Alzheimer's Disease Assessment Scale, >/=25), due to a substantial worsening of scores in those taking placebo compared with minimal deterioration for the clioquinol group. Plasma Abeta42 levels declined in the clioquinol group and increased in the placebo group. Plasma zinc levels rose in the clioquinol-treated group. The drug was well tolerated. CONCLUSION: Subject to the usual caveats inherent in studies with small sample size, this pilot phase 2 study supports further investigation of this novel treatment strategy using a metal-protein-attenuating compound.

Histidine abolishes the inhibition by zinc of naloxone binding to opioid receptors in rat brain.

Zinc ions inhibited the specific binding of [3H]naloxone to opioid receptors in the hippocampus, cortex, midbrain and striatum of the rat in a dose-dependent manner. Scatchard analysis performed by using a concentration of zinc close to its IC50 (about 30 microM) revealed that inhibition was due to a decrease in receptor affinity, without change in the number of binding sites. Of several compounds tested, only histidine was capable of reversing the inhibition by zinc in these areas of the brain so preventing the zinc-induced increase in the KD of opioid receptors for naloxone. Histidine alone did not affect the KD or Bmax of opioid receptors for [3H] naloxone in the areas of brain studied. The fact that histidine prevented the zinc-induced increase in the KD but had no effect on receptor affinity or the number of binding sites for [3H] naloxone suggests that histidine exerts its effects by complexing with zinc ions and acting as a chelator. In addition to Zn2+, Cu2+, Cd2+ and Hg2+ inhibited the binding of [3H] naloxone to opioid receptors in the cerebral cortex of the rat in a dose-dependent manner. However, histidine was not capable of abolishing the inhibition to the same extent as that of zinc. Thus, among all of the metal ions studied, the most dramatic effect of histidine was observed with zinc ions.

Nitric oxide reduces Ca(2+) and Zn(2+) influx through voltage-gated Ca(2+) channels and reduces Zn(2+) neurotoxicity.

The translocation of synaptic Zn(2+) from nerve terminals into selectively vulnerable neurons may contribute to the death of these neurons after global ischemia. We hypothesized that cellular Zn(2+) overload might be lethal for reasons similar to cellular Ca(2+) overload and tested the hypothesis that Zn(2+) neurotoxicity might be mediated by the activation of nitric oxide synthase. Although Zn(2+) (30-300microM) altered nitric oxide synthase activity in cerebellar extracts in solution, it did not affect nitric oxide synthase activity in cultured murine neocortical neurons. Cultured neurons exposed to 300-500microM Zn(2+) for 5min under depolarizing conditions developed widespread degeneration over the next 24h that was unaffected by the concurrent addition of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine. Furthermore, Zn(2+) neurotoxicity was attenuated when nitric oxide synthase activity in the cultures was induced by exposure to cytokines, exogenous nitric oxide was added or nitric oxide production was pharmacologically enhanced. The unexpected protective effect of nitric oxide against Zn(2+) toxicity may be explained, at least in part, by reduction of toxic Zn(2+) entry. Exposure to nitric oxide donors reduced Ba(2+) current through high-voltage activated calcium channels, as well as K(+)-stimulated neuronal uptake of 45Ca(2+) or 65Zn(2+). The oxidizing agents thimerosal and 2,2'-dithiodipyridine also reduced K(+)-stimulated cellular 45Ca(2+) uptake, while akylation of thiols by pretreatment with N-ethylmaleimide blocked the reduction of 45Ca(2+) uptake by a nitric oxide donor.The results suggest that Zn(2+)-induced neuronal death is not mediated by the activation of nitric oxide synthase; rather, available nitric oxide may attenuate Zn(2+) neurotoxicity by reducing Zn(2+) entry through voltage-gated Ca(2+) channels, perhaps by oxidizing key thiol groups.

The femtomolar-acting NAP interacts with microtubules: Novel aspects of astrocyte protection.

Activity-dependent neuroprotective protein (ADNP), a gene product essential for brain formation, contains a short octapeptide sequence NAPVSIPQ (NAP) that protects neurons against a wide variety of insults. At the pico-molar concentration range, NAP provides neuroprotection by direct interaction with neurons. At the femtomolar concentration range, NAP requires the presence of glial cells to provide neuroprotection. To further understand the mechanism of neuroprotection afforded by NAP, specific binding proteins were searched for. Tubulin, the major subunit protein of microtubules, was identified as a NAP binding molecule. NAP structure allows membrane penetration, followed by tubulin binding and facilitation of microtubule assembly toward cellular protection in astrocytes. NAP (10(-15) M) promoted microtubule assembly in vitro and protected astrocytes against zinc intoxication which is associated with microtubule disruption. A two hour incubation period of astrocytes with femtomolar concentrations of NAP resulted in microtubule re-organization and transient increases in immunoreactive non-phosphorylated tau. Microtubules are the key component of the neuronal and glial cytoskeleton that regulates cell division, differentiation and protection, while tau pathology is a major contributor to Alzheimer's disease and other dementias. The findings described here may open up new horizons in research and development of neuroprotective compounds.

Corticosterone attenuates zinc-induced neurotoxicity in primary hippocampal cultures.

Primary hippocampal cultures derived from newborn rats were exposed to zinc chloride at 50, 75, 100, 150 and 200 microM concentrations. Neuronal injury was assessed morphologically and by the lactate dehydrogenase (LDH) efflux assay. Zinc exposure increased LDH efflux in a concentration-dependent manner. Exposure to 100 microM zinc for 24 h resulted in beading of neurites and swelling of neuronal soma. When cultures were co-exposed to zinc at 100 microM and corticosterone in the range of 10-5 to 10-7 M, degeneration of neurons caused by zinc was attenuated. Our study suggests that corticosterone can protect neurons from zinc-induced neurotoxicity at low doses.

Involvement of amygdalar extracellular zinc in rat behavior for passive avoidance.

On the basis of the evidence that zinc is released from glutamatergic neuron terminals in the amygdala, the effect of chelation of amygdalar extracellular zinc on glutamate release from the neuron terminals was studied by using in vivo microdialysis. When the amygdala was perfused with 100 microM CaEDTA to chelate extracellular zinc, glutamate concentration in the perfusate was decreased significantly, whereas that tended to be increased by perfusion with 100 microM ZnEDTA as a control. The effect of CaEDTA on extracellular glutamate levels was different between the amygdala and hippocampus, implying that modulation of glutamate signaling by zinc is different between them. To evaluate chelation of zinc in rat behavior, perfusion of the amygdala with CaEDTA was started 40 min before behavioral test for passive avoidance. The behavior for passive avoidance was impaired during perfusion with CaEDTA. On the other hand, the behavior during perfusion with ZnEDTA was more rapidly developed than that with vehicle only. These results suggest that amygdalar extracellular zinc is involved in the behavior for passive avoidance.

Attenuation of zinc-induced neuronal death by the interaction of growth inhibitory factor with Rab3A in rat hippocampal neurons.

We examined the protective effect of growth inhibitory factor (GIF) against zinc-induced neuronal death in rat hippocampal neurons. In an in vitro cell culture system, 300 microM Zn(2+) readily induced death of hippocampal neuronal cells, which was characterized by massive necrosis and a minor degree of apoptosis. Neither the addition of recombinant GIF nor Rab3A alone could rescue these cells from death. However, the combination of GIF with Rab3A could significantly enhance the survival of the hippocampal neurons. This result was supported by both Annexin -V FITC/propidium dual staining and chromosomal DNA analysis. These findings suggest that GIF may inhibit Zn(2+)-induced neuronal death via its interaction with Rab3A.

From soil to brain: zinc deficiency increases the neurotoxicity of Lathyrus sativus and may affect the susceptibility for the motorneurone disease neurolathyrism.

Zinc deficiency and oversupply of iron to the roots of grass pea (Lathyrus sativus) induce increases in the content of the neurotoxin beta-L-ODAP (3-oxalyl-L-2,3-diaminopropanoic acid) in the ripe seeds. The transport of zinc to the shoots is enhanced by the addition of beta-L-ODAP. The neurotoxin of L. sativus is proposed to function as a carrier molecule for zinc ions. Soils, depleted in micronutrients from flooding by monsoon rains (Indian subcontinent) or otherwise poor in available zinc and with high iron content (Ethiopian vertisols), may be responsible for higher incidence of human lathyrism, one of the oldest neurotoxic diseases known to man. A role for brain zinc deficiency in the susceptibility for lathyrism is postulated.