Research reportDiverse effects of metal chelating agents on the neuronal cytotoxicity of zinc in the hippocampus
Introduction
Some correlative evidence exists that brain zinc may have a pathological role in the neurological disorders of epilepsy, ischemia and Alzheimer's disease. Exogenous zinc is cytotoxic for neurons and glial cells 8, 9, 11, 15, 31, 32, 34, 35, 36, 61, and can cause epileptic seizures 14, 27, 44, 45. After a period of ischemia or seizure activity in the brain there is increased staining for chelatable zinc within degenerating neuronal perikarya 20, 32, 55. These neurons appear to be dying by an apoptotic process 4, 7, 18, 47, 48. Similarly, cultured lymphocytes undergoing apoptosis also show an increased staining for zinc [63]. In cultures of thymocytes, lymphocytes or splenocytes, exogenous zinc protects, while chelation of zinc induces apoptosis 29, 30, 41, 56, 62. In contrast, Koh et al. [32]recently demonstrated that intraventricular injection of the metal chelate ethylenediamine tetraacetate (EDTA) as the calcium salt, but not as the zinc salt, markedly decreased neuronal loss induced by a 10-min period of global ischemia. In relation to Alzheimer's disease, physiological concentrations of zinc ions, but not of calcium, magnesium or other metal ions, causes the aggregation of human β-amyloid protein 5, 6. The aggregated form of β-amyloid causes neuronal death in cell cultures (possibly via apoptosis) 10, 21, 46.
While these studies are suggestive, only the study of Koh et al. [32]provides direct evidence that zinc is a causative agent for neurodegeneration in neurological disorders. To demonstrate that zinc has a causative action in neurodegeneration requires some means of preventing the actions of zinc. Zinc deficient diets have little effect on brain zinc (30% reduction in hippocampal mossy fibre zinc content after 3 months on a zinc deficient diet) [59]. Metal chelating agents are an alternative means for restricting cellular access to zinc 11, 13, 32, 54. To be used successfully in vivo, the chelator must not be toxic. This is a potential major problem for membrane permeable metal chelators where interference with intracellular processes involving zinc might be expected to have pathological consequences. N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) is membrane permeable [3]and has been shown to stimulate apoptosis in cultured immune cells 29, 30, 56, 63. Yet TPEN, in a dose that blocks all staining for zinc in the hippocampus, does not cause neuronal loss in the adult rat brain [11]. Koh et al. [32]also found that decreasing available zinc with a chelator did not cause toxicity but rather reduced neuronal damage after ischemia.
However, depending on conditions and the concentration reached, release of endogenous zinc could potentially have either neurotoxic or neuroprotective actions. It might also be simply a by-product of neurodegenerative processes. An additional factor to be considered is the source of the zinc found in degenerating cells. In dying thymocytes, the increase in cellular chelatable zinc appears to be by internal mobilization [63]. On the other hand, extracellular zinc would appear to be the source of chelatable zinc in degenerating neurons, as neuronal death was blocked by a membrane impermeable metal chelator (EDTA) [32]. The use of metal chelators with high affinities for zinc and which are either membrane permeant or impermeant is one method for determining the source of chelatable zinc found in cells dying by apoptotic processes.
Techniques for using metal chelating agents require further evaluation before they can be used to determine the physiological functions of chelatable zinc (and other metals) in neurodegeneration. One question to be answered is whether the neuroprotective effect of metal chelators is due to chelation of zinc, or to some other property. If the former is correct, the neuroprotective effects of metal chelators against metal ion toxicity should bear some relationship to their affinities for metal ions. The duration of the action of the chelator in vivo, its long-term toxicity, and its selectivity for zinc compared with other metals normally present in the brain, particularly iron and copper, must also be determined. More generally, there are many endogenous chelators of zinc within neurons such as amino acids. For example, cysteine and histidine complex zinc with affinities of between 1018 M−1 and 1012 M−1 respectively, while proteins generally have affinities of around 107 M−117, 52. Such compounds could act as a reservoir for zinc taken into neurons, allowing for a prolonged release of zinc which might contribute to zinc neurotoxicity. Thus, the ability of metal chelators to permeate cell membranes could be important for the prevention of metal ion neurotoxicity. These issues have been addressed in the current investigation, using the neuronal cytotoxicity of zinc as a model system.
Section snippets
Methods and materials
Adult male Wistar rats (190–210 g) were anaesthetized with 1.5% halothane and placed in a Kopf stereotaxic frame. Due to solubility problems, TPEN (Molecular Probes) was dissolved in dimethylsulfoxide (DMSO) and diluted with 0.9% saline solution to a final concentration of 5 mM in 10% DMSO. Other metal chelators were dissolved in saline and the pH adjusted to pH 7 to 7.4. Zinc chloride was dissolved in either 0.9% saline or 10% DMSO in 0.9% saline to a final concentration of 5 mM (pH 5.5–5.7).
Intrinsic neurotoxicity of metal chelating agents
A range of metal-free chelating agents, with dissociation constants (Kds) for zinc between 3×10−6 M and 10−18 M (Table 1), were first examined for their toxicity in vivo. With a single injection of 10 nmol, most complexing agents showed little toxicity towards neurons, with the size of the lesion being similar to that produced by an equal volume of 0.9% saline or 10% DMSO in 0.9% saline (Fig. 1a). Even a 5-fold higher dose of pyrithione (50 nmol) was not toxic. On the other hand, DTPA and InsP6
General considerations
The use of metal chelating agents to determine the role of a metal ion in neuropathology is fraught with difficulties. Whole body administration of chelators such as dithizone and diethyldithiocarbamate (DEDTC) produce side effects not related to metal chelation, and cause general debilitation of the animal [19]. DEDTC inhibits many enzymes requiring sulphydryl groups for activity [see Ref. [42]]. A further problem is the large number of metal-dependent processes that could potentially be
Acknowledgements
We thank NZ Lottery Health, the University of Auckland Staff Research Fund, the Auckland Medical Research Foundation and the Health Research Council of NZ for providing financial support to GJL. MPC is a recipient of a W.B. Miller Post-Graduate Scholarship from the Neurological Foundation of NZ.
References (63)
- et al.
Cytosolic Ca2+ homeostasis in Ehrlich and Yoshida carcinomas
J. Biol. Chem.
(1985) - et al.
Mechanisms of delayed cell death following hypoxic-ischemic injury in the immature rat: evidence for apoptosis during selective neuronal loss
Mol. Brain Res.
(1995) - et al.
Apoptotic features of selective neuronal death in ischemia, epilepsy, and gp120 toxicity
Trends Neurosci.
(1996) - et al.
Zinc neurotoxicity in cortical cell culture
Neuroscience
(1988) - et al.
Zinc and Alzheimer's disease. Is there a direct link?
Brain Res. Rev.
(1997) Neurobiology of zinc and zinc-containing neurons
Int. Rev. Neurobiol.
(1989)- et al.
Translocation of zinc may contribute to seizure-induced death of neurons
Brain Res.
(1989) - et al.
Zinc toxicity on cultured cortical neurons: involvement of N-methyl-d-aspartate receptors
Neuroscience
(1994) - et al.
The neurotoxicity of zinc in the rat hippocampus
Neurosci. Lett.
(1990) - et al.
Effect of metal chelating agents on the direct and seizure-related neuronal death induced by zinc and kainic acid
Brain Res.
(1998)
Brain lesions induced by specific and non-specific inhibitors of sodium–potassium ATPase
Brain Res.
Neuronal cytotoxicity of inositol hexakisphosphate (phytate) in the rat hippocampus
Brain Res.
The interactions of zinc, nickel, and cadmium with Xenopous transcription factor IIIA, assessed by equilibrium dialysis
J. Inorg. Biochem.
Diethyldithiocarbamate and dithizone augment the toxicity of kainic acid
Brain Res.
Kainate-induced apoptotic cell death in hippocampal neurons
Neuroscience
Characterization of metal ion-induced []inositol hexakisphosphate binding to rat cerebellar membranes
J. Biol. Chem.
A simplified Timm stain procedure compatible with formaldehyde fixation and routine paraffin embedding of rat brain
Brain Res. Bull.
Partial depletion and altered distribution of synaptic zinc in the rat hippocampus after treatment with sodium diethyldithiocarbamate
Brain Res.
Possible role of zinc in the selective degeneration of dentate hilar neurons after cerebral ischemia in the adult rat
Neurosci. Lett.
Apoptosis is dependent on intracellular zinc and independent of intracellular calcium in lymphocytes
Exp. Cell Res.
Brief exposure to zinc is toxic to cortical neurons
Neurosci. Lett.
Flux of intracellular labile zinc during apoptosis (gene-directed cell death) revealed by a specific chemical probe, zinquin
Chem. Biol.
Pyridine derivatives as complexing agents: XI. Thermodynamics of metal complex formation with bis-, tris-, and tetrakis[(2-pyridyl)methyl]-amines
Helv. Chim. Acta
The neuropathology of temporal lobe epilepsy
J. Neuropathol. Exp. Neurol.
Rapid induction of Alzheimer Aβ amyloid formation by zinc
Science
Zinc and Alzheimer's disease
Science
Response of olfactory Schwann cells to intranasal zinc sulfate irrigation
J. Neurosci. Res.
Activation of metabotropic glutamate receptors protects cultured neurons against apoptosis induced by β-amyloid peptide
Mol. Pharmacol.
Prevention of zinc neurotoxicity in vivo by N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN)
NeuroReport
Effect of diethyldithiocarbamate (DEDTC) on sulphide stained boutons. Reversible blocking of Timm's sulphide silver stain for `heavy' metals in DEDTC treated rats (light microscopy)
Exp. Brain Res.
Seizures in rats associated with divalent cation inhibition of Na+/K+-ATPase
Can. J. Biochem.
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Present address: Harvard Medical School, Genetics and Aging Unit, Massachusetts General Hospital East, Bldg. 149, 13th Street, Charlestown, MA 021129, USA.