Elsevier

Brain Research

Volume 799, Issue 1, 13 July 1998, Pages 97-107
Brain Research

Research report
Diverse effects of metal chelating agents on the neuronal cytotoxicity of zinc in the hippocampus

https://doi.org/10.1016/S0006-8993(98)00482-XGet rights and content

Abstract

Abnormal metabolism of metal ions such as zinc may contribute to neuropathology. Complexing zinc could reduce this pathology. Thus, to examine the effectiveness of metal chelating agents in vivo, a model system was used. This involved determining the ability of chelating agents to prevent neuronal death caused by zinc chloride injected into the rat hippocampus. Significant protection against zinc toxicity was obtained with pyrithione, inositol hexakisphosphate, ethylenediamine tetraacetate (EDTA) and N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). The affinity of these agents for zinc varied between 106 M−1 and 1018 M−1. Thus, the affinity for zinc within this range does not appear to be a major factor affecting the ability of chelators to provide neuroprotection. While almost complete protection was found with EDTA and TPEN given simultaneously with zinc chloride, poor protection was obtained if TPEN was given before or after zinc chloride. Other agents either did not protect against zinc-induced neuronal death (zincon), or exacerbated zinc toxicity (BTC-5N and about 40% of rats injected with a combination of zinc chloride and diethylenetriamine pentaacetate [DTPA]). Rats showing increased damage after zinc plus BTC-5N or DTPA suffered wet dog-like shakes (WDS), suggesting that these zinc chelate complexes can induce seizures resulting in seizure-related damage. In contrast, in the 60% of rats treated with zinc chloride and DTPA that had no WDS, there was about an 80% reduction in the size of the zinc-induced lesion. The ability of chelators to cross cell membranes was examined by determining whether Timm's staining for vesicular zinc was reduced following the injection of a chelator into the hippocampus. TPEN and pyrithione reduced Timm's staining for zinc. However, cell permeability was not necessary for a chelator to protect against zinc toxicity.

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.

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