METALS AND NEURODEGENERATION

Roberto Lucchini, Institute of Occupational Health of the University of Brescia, Italy
Paolo Zatta, Center on Metalloproteins, Department of Biology of the University of Padova, Italy
Susan J Van Rensburg, Department of Chemical Pathology, Tygerberg Hospital and Stellenbosch University, South Africa
Andrew Taylor, Consultant Clinical Biochemist, Royal Surrey County Hospital, Guildford, Surrey, UK

      Until the last decade, little attention has been given by the neuroscience community to the neurometabolism of metals, however, the neurobiology of heavy metals is now of growing interest, since they have been linked to major neurodegenerative diseases. This chapter is a review of some metals that could be possibly involved in neurodegeneration. Two of them are essential, manganese (Mn) and zinc (Zn) and one non-essential, aluminum (Al). Occupational and environmental exposure to these metals has been suggested as a possible cause of neurodegenerative disorders, therefore they deserve more attention from a multi-disciplinary approach that should merge the fields of neurosciences with those more specific for metallochemistry and oxidation chemistry.

1. MANGANISM AND PARKINSON'S DISEASE (PD)

1.1. Occurrence of Manganism
     Neurotoxicity of Mn has been well known since the last century. In 1837 Couper (¹) described already "Manganism" as characterized by extrapyramidal dysfunction and neuropsychiatric symptomatology. Since then this syndrome has been observed in hundreds of cases among miners and industrial workers throughout the world who were exposed to high levels of manganese (^{2, 3, 4, 5, 6, 7, 8}). Manganism occurred also in agricultural workers through Mn based pestcides such as fungicide Maneb (Mn ethylene-bis-dithiocarbamate) and Mancozeb (Mn copper zinc ethylene-bis-dithiocarbamate) (^{9, 10}). Cases of overt Mn poisoning were also reported due to environmental exposure in the drinking water in Japan (¹¹), Australia (¹²), Greece (¹³), or after drinking water low in magnesium while on a diet of foods with high Mn content (¹⁴).
Another possible source of Mn accumulation and clinical toxicity has been identified in excessive Mn levels in total parenteral solution, given intravenously to patients with chronic gastrointestinal illnesses (¹⁵). In addition, since Mn is excreted almost completely via the biliaric tract, individuals with chronic liver failure are at risk of developing hepatic encephalopaty, that is likely to be due to Mn accumulation in the brain (^{16, 17}). More subtle, preclinical effects have been documented in recent literature also for occupational end environmental exposure levels much lower than those at which clinical manganism has been observed (^{18, 19, 20, 21, 22}). Since Mn elimination from the central nervous system is very long, neurotoxic effects may occur later in life, increasing the frequency of parkinsonian disturbances in the geriatric age. Therefore, the problem of manganese neurotoxicity is becoming of great concern because of several factors. Its industrial use will further increase in view of new technological applications both in the metallurgic, as well as the chemical sectors. The use of Mn-based pesticides is widespread, both in industrialized and in developing countries. In addition, the adoption of an organometallic compound such as methyl-cyclopentadienyl manganese tricarbonyl (CH₃C₅H₄Mn(CO)₃), better known as MMT, C1-2 or Ak-33X (antiknock 33X) as a lead substitute in gasoline could become an additional important source of environmental exposure.

1.2. Clinical features of Mn neurotoxicity
     The clinical manifestations of manganism are well documented and detailed due to the excellent work of Huang et al. (^{23, 24, 25}) who followed up a number of workers with Mn intoxication over many years. Three differene stages can be differentiated: (i) behavioral changes; (ii) parkinsonian features; (iii) dystonia and gait disturbances. The early phase includes symptoms of fatigue, headache, muscle cramps, loss of appetite, apathy, insomnia, dimished libido, which are associated with psychotic reactions known as "locura manganica". This Mn madness is a marked aggressive manifestation with emotional liability. The extrapyramidal signs iclude monotone speech, expressionless face, impaired writing and dexterity, and antero- and retro-pulsion. In the established phase, dystonia is evident with a characteristic "cock-walk" gait. Tremor is not frequent and is a low amplitude postural tremor of upper extremities.
The disorder is progressive once established in the most severe stage, and even after removal from exposure. The L-DOPA treatment can improve the symptoms only for an initial short period of time, probably due to a placebo effect.

1.3. Differences and similarity between manganism and PD
    Features of clinical Mn neurotoxicity resemble those of idiopathic parkinsonism. Nevertheless, a careful analysis of cases of Mn poisoning by Calne et al. (²⁶) has revealed clinical, pharmacological and imaging differences. The similarities in the clinical picture include the presence of generalized bradykinesia and widespread rigidity, wherease the dissimilarities are represented, in manganism, by: a) less frequent resting tremor; b) more frequent dystonia; c) a particular propensity to fall backwards d) failure to achieve a sustained therapeutic response to levodopa, and e) failure to detect a reduction in fluorodopa uptake by positron emission tomography (PET).
By the anatomical point of view, the few necropsy studies carried out on Mn-induced parkinsonism have mainly shown degenerative lesions of the globus pallidus and subthalamic nucleus, caudate nucleus, and putamen, with less frequent or less severe lesions of the substantia nigra. This is the basic different picture from the idiopathic PD in which the substantia nigra is typically involved, and the strio-pallidal complex is spared (²⁷). Globus pallidus is known to be sensitive to energy deprivation, and to abnormal excitotoxic injury and contains dopaminergic neurons and receptors.
However, even if manganism and PD appear to be two different entities, several observations have been made in the past years on the possible role of Mn exposure in the etiology of PD. In fact, although PD is one of the most common adult neurologic disorders, its etiology is still unknown and the hypothesis of an interaction between environmental factors and individual genetic susceptibility, both acting on normal aging, has been pointed out (^{28, 29, 30}). Following the discovery that MPTP (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) produces a parkinsonian syndrome and that the MPTP metabolite MPP+ resembles the molecular structure of the herbicide Paraquat, a number of studies have indicated a possible relationship between PD, agricultural work and the use of pesticide chemicals (^{31, 32, 33, 34}). This appears particularly important when considering the widespread use of Mn-based fungicides throughout the world.

1.4. Epidemiological studies on Mn-associated parkinsonian disturbances
     Therefore, increasing attention is being focused on a possible role of Mn in the etiology of PD (^{35, 36}). An epidemiological study was conducted in Norway on the PD's incidence in the community of Sauda, where the world's largest ferro-manganese plant was active until 1923 (³⁷) . A total number of 13 PD's cases were observed among 5294 inhabitants, which is equivalent to a crude prevalence rate of 245.6/100,000. This rate was higher than most values reported in the literature, for community-based studies, i.e. 70-180 (³⁸), 65-187 (³⁹), or 65-162 (⁴⁰). Preliminary results from another study conducted very recently showed again a frequency of PD cases higher than 200/100.000 among inhabitants in the surroundings of two plants producing ferro-manganese alloys in Northern Italy since the 1970s (⁴¹).
An attempt to evaluate possible differences in Mn concentrations in biological fluids among parkinsonian patients was done by Jimenez-Jimenez FJ et al. (⁴²). Serum levels of Mn and 24-h Mn excretion by urine were compared between 29 PD's patients and 27 matched controls in Spain. No differences were noted between the two groups and the values did not correlate with age, age at onset and duration of the PD, nor the scores of the Unified PD Rating Scale or the Hoehn & Yahr staging among the PD's patients. However, due to the high variability of Mn in serum and urine and the limited number of observation, it is difficult to draw a conclusion from this study.
Zayed et al. (⁴³) found a significant association between PD and self-reported occupational exposure to a combination of Mn, iron and Al, particularly for more than 30 years (O.R. 13.64; p£0.05), but the contribution made by each of this metal to the association with the disease could not be determined. A population-based case-control study by Gorell et al. (⁴⁴) examined the occupational history in 144 PD cases and 464 frequncy-matched for sex, race, and age. More than 20 years exposure to Mn (Odds Ratio = 10.61, 95% C.I. = 1.06-105.83) was found to be associated with PD.

1.5. Mechanism of neurotoxicity
     Neurochemical changes can be observed prior to morphological alterations. After intrastriatal injection of Mn in rodents, Brouillet et al. (⁴⁵) demonstrated that dopamine and GABA concentrations showed a dose-dependent reduction, wherease somatostatin and neuropeptide Y did not change. The striatal lesions were consistent with N-methyl-D-aspartate (NMDA) excitotoxic lesions and could be blocked by prior removal of the corticostriatal glutamatergic input or by prior treatment with the NMDA antagonist MK-801. A severe reduction in dopamine levels in the caudate nucleus, putamen and SN, a distinct reduction of noradrenaline in hypothalamus and normal serotonin in these areas were observed in a patient affected with chronic Mn encephalopathy (⁴⁶). Morphological changes were described in many autopsy studies and animal models and they typically feature the degeneration of the globus pallidus, particularly the medial segment, followed by a degeneration of putamen, caudate nucleus and SNr (⁴⁷). Major histological findings include marked decrease of myelinated fibres and astrocytes proliferation (²⁷), in contrast with those observed in PD: depigmentation and neural loss in SNc, locus ceruleus and dorsal nucleus of vagus, occasional presence of Lewy bodies and neurofibrillary tangles in the cerebral cortex.
The adverse effects on the nervous system probably result from the failure of protective enzymes to detoxify critical amounts of Mn or to alter its oxidation potential. In fact, on one hand the basis of Mn neurotoxicity hinges on the ability of bivalent form Mn²⁺ to oxidize to the trivalent form Mn³⁺, a powerful oxidizing species. On the other hand, Mn shows high affinity to neuromelanin-rich areas, such as the nigrostriatal tract (⁴⁸). Here Mn favours the autoxidation of dopamine by one electron transfer reaction, generating semiquinones and orthoquinones and the production of toxic free radicals (⁴⁹) which can determine substantial lesions of the dopaminergic system. It was presumed by Cotzias (⁵⁰) that in Mn intoxication, brain dopamine is initially elevated and then depleted.
Wolters et al. (⁵¹) examined 4 subjects with clinical features of mild parkinsonism caused by exposure to Mn. The MRI and the 6-fluorodopa positron emission tomography were normal, suggesting that in early manganism, damage may occur from functional disturbances in postsynaptic striatal or even pallidal neurons, rather than from the dopamine depletion that has been documented in postmortem examination of severe manganism cases.
It is misleading to conlcude that a single dysfunction could be a basic mechanism of Mn neurotoxicity. A multifactor hypothesis is more likely to be considered, involving an iron-induced oxidative stress and the direct interaction of Mn with dopaminergic terminal mitochondria leading to selective mitochondrial dysfunction and subsequent excitotoxicity (⁵²). This hypothesis would account for the slow evolution of the pathology, the dopaminergic neuron affinity, and the possible therapeutic advantage of iron-chelation and increased anti-oxidant status.

1.6. Conclusion
     Mn exposure can definitely play an important role in the determination of parkinsonian disturbances. Tehre are several differences between manganism and PD, but it is important to consider that manganism is the result of exposure to very high doses of Mn. Prolonged exposure to lower exposure levels of Mn may act differently, and enhance the onset of parkinsonian disturbances. Therefore, in view of a possible substantial increase of environmental expoure in the general population caused by the adoption of Mn-based gasoline, further epidemiological and neurotoxicological research is needed and should be envisaged.

2. Al IN THE AETIOLOGY OF ALZHEIMER'S DISEASE

     The neurotoxicity of Al has been well known since the last century (⁵³). At present it is clear that Al can exert toxicity on neurological, skeletal and hematological systems in humans with advanced renal failure (⁵⁴). The Al hypothesis in the etiology of Alzheimer's disease (AD) originated when Klatzo et al.(⁵⁵) found that the injection of Al salts into the brains of rabbits produced a neurofibrillary degeneration. Crapper et al. (⁵⁶) repeated the results in cats, and demonstrated increased Al concentrations in the brains of AD patients. However, a controversy developed regarding the role of Al in the etiology of the disease because subsequent experiments produced conflicting results (^{57, 58}). Although Al is one of the most common elements in the biosphere, the amounts taken up into living cells are extremely small and are exceptionally difficult to measure accurately. Hence, some authors regard Al as the root cause of AD, while others believe that the cause lies elsewhere, and that Al is an opportunistic bystander (⁵⁹). The view of the latter group was emphasized in a study in which Landsberg et al. (⁶⁰) stated that they could find no Al in senile plaques from autopsy AD brain material, and hypothesized that Al found previously in plaques (⁶¹), had been caused by contamination from the dyes used to stain the plaques. The Landsberg study was challenged by Good and Perl (⁶²), who stated that the study did not contradict the Al hypothesis, since it was clear from the literature that Al was more often associated with the neurofibrillary tangles than with the plaques. These authors have published the results of a study employing LAMMA technique (laser micoprobe mass analysis), that provides extremely sensitive multi-element detection, with which they demonstrated the presence of significant increases of Al and iron (Fe) in tangles (⁶³).
Despite the criticism, evidence has shown that Al may have a role in the etiology of AD. The metal speciation may also play an important role in the brain metal accumulation and an individual susceptibility to Al uptake is now becoming a relevant etiopathogenic issue. It is becoming more and more evident that the etiology of many diseases, like AD, are found in a combination of several factors, including genetic factors, which render some individuals more vulnerable to adverse environmental factors (⁶⁴).

2.1. Evidence for the Al hypothesis

Candy et al. (⁶¹) using energy dispersive X-ray microanalysis, demonstrated the presence of miniscule insoluble Al silicate granules, like tiny grains of sand, in the brains of patients with AD. The granules were surrounded by equally insoluble amyloid protein plaques, leading these authors to hypothesize that the granules may be an early or initiating factor in plaque formation. The presence of a higher concentrations of Al in certain brain regions of autopsy samples from patients with AD was confirmed by Ward and Mason (⁶⁵), although Bjertness et al. (⁶⁶) found no increase in bulk Al concentration in two cortical brain regions of AD patients. Beal et al. (⁶⁷) found that Al-induced neurofibrillary degeneration in rabbits was accompanied by reductions in choline acetyltransferase activity of up to 40% in the entorhinal cortex and hippocampus, as well as reductions in serotonin and noradrenalin, which suggested that some neuronal populations preferentially affected in AD were also affected by Al-induced neurofibrillary degeneration. The binding of Al to paired helical filament tau in AD neurofibrillary degeneration was also demonstrated by Murayama et al. (⁶⁸).

2.2. Barriers against Al uptake

Even though Al is the third most ubiquitous element on earth, very little Al (only about 0.06-0.1% of ingested Al) is absorbed across the GI tract (⁶⁹). In fact, the body has two barriers designed to rigorously exclude Al:

q The gut barrier: The amount of Al absorbed from the gastrointestional tract depends on the presence of other dietary agents such as citrate, which form complexes with the metal (⁷⁰), since most Al compounds are known to be largely insoluble at normal pH (⁷¹). Al uptake is inhibited by calcium (Ca), magnesium (Mg) and silicon (Si) (⁷²). Noteworthy, Taylor et al. (⁷³) elegantly demonstrated that younger people absorbed much less Al from an Al citrate drink than older people taking the same amount of Al (serum concentrations increased by 30 µg/l vs 94.5 µg/l measured after 1 hour), indicating that in younger people the gut barrier is less penetrable by Al than in older people. Interestingly, younger patients with AD had the same Al uptake as older AD patients and older non-demented controls, suggesting that the gut barrier had been overruled in the younger AD patients. Recently, this research group (⁶⁹) demonstrated that the uptake of 26Al by AD patients exceeded controls by a factor of 1.64. Although the exact method of Al uptake from the gut has not been fully elucidated, it is postulated that the iron (Fe) carrier protein transferring (Tf) plays a relevant role in the uptake of both Al and Fe (74). A number of other factors such as vitamin D, parathyroid hormone, and the status of the body iron stores have been suggested to effect Al absorption.

q The blood brain barrier: The blood-brain barrier (BBB) is highly selective as to which substances (including metals) are allowed into the brain. Evidence suggests that the effect of Al on BBB permeability is rapid in onset and quickly reversible and largely dependent on the metal speciation that is expressed by the different physico-chemical properties of the various metal ligands used in experimental toxicology (⁷⁵). Changes in the structure and function of the BBB have been proposed to underlie the findings in Al toxicity and AD (review see ⁷⁶). Tf has been demonstrated to carry Al across the BBB (⁷⁷).

Since the concentration of Al in brain tissue is normally low, the question arises why greater concentrations of Al are found in the brains of AD patients. The answer may be found in a combination of genetic and environmental factors.

q Genetic factors: A genetic factor has been identified which may be involved in an aberrant transport of Al. A higher frequency of a genetic variant of Tf, TfC2, has been found in patients with AD compared to non-demented controls in populations in South Africa (⁷⁸), Japan (79), Sweden (⁸⁰), Ireland (⁸¹) and Italy (⁸²). In addition, an earlier age of onset of AD was demonstrated in patients who had both the ApoE-e4 and the TfC2 alleles (⁸³). Morris et al. (⁸⁴) observed focal accumulation of Al in neurons with high densities of Tf receptors, indicating Tf-mediated uptake, in brain regions such as cortex and hippocampus which are selectively vulnerable in AD. Wong and Saha (⁸⁵) found that TfC2 had a lower Fe-binding capacity than TfC1.

q Epidemiological factors: Al is commonly added to water at municipal water treatment facilities to flocculate and remove turbidity present in water. This can leave Al residues as high as 4 mg/L (⁸⁶). Therefore, under certain circumstances Al ingestion can be considerably greater than the Al normally present as a contaminant in food and beverages. Thirteen epidemiological studies have to date been done world-wide to investigate the hypothesis that there may be a correlation between AD and increased concentrations of Al in drinking water, and nine of these studies have found a positive association (⁸⁷). It would hence seem that a higher concentration of Al in the environment may overcome the gut and brain barriers.

Rogers and Simon (⁸⁸) demonstrated that past consumption of foods containing large amounts of Al additives differed between people with Alzheimer's disease and controls, suggesting that dietary intake of Al may affect the risk of developing this disease. Intake of pancakes, waffles, biscuits, muffins, cornbread and/or corn tortillas differed significantly (P=0.025) between cases and controls. Adjusted odds ratios were also elevated for grain product desserts, American cheese, chocolate pudding or beverages, salt and chewing gum. However, the odds ratio was not elevated for tea consumption.

2.3. The role of al in the expression of AD

In pure phenomenological terms, the interaction of Al with biological systems is documented by an impressive number of papers as reported in a special issue of Coordination Chemistry Reviews in 1996. The literature is particularly rich and a considerable body of information is also available on the interaction between Al and enzymatic systems. No physiological role has so far been found for Al in the body, but the metal has negative effects on several important biochemical reactions involving the binding of other metals such as Mg and Ca (⁸⁹) with proteins. For example, Al is a "dead end" inhibitor of the Mg-ATP-dependent enzyme hexokinase, which has a vital phosphate-transfer function (⁹⁰). Al may also compromise energy production via the Krebs cycle by activating alpha-ketoglutarate dehydrogenase and succinate dehydrogenase, while inhibiting aconitase (⁹¹). At slightly acidic pH, phosphate ligands are prime binding sites for Al, especially when several phosphate groups are geometrically arranged so that cooperative strong binding occurs, as is the case with inositol 1,4,5-triphosphate (⁷¹). The inositol pathway has been found to be involved with long-term potentiation, i.e. with memory formation (⁹²). Al also increases monoamine oxidase type B (MAO-B) (⁹³) and acetylcholinesterase enzymatic activity in a dose dependent manner, which is interesting since both enzymes are significantly alterated in AD (⁹⁴). Alternatively, Al strongly inhibits protease activity of trypsin and chymotrypsin (⁹⁵). Exley (⁹⁶) proposed that Al may potentiate the activities of neurotransmitters by the action of Al-ATP at ATP receptors in the brain.

2.4. Free radical damage

Cell membranes are particularly vulnerable to free radical damage. Although Al cannot change its redox state, it has been shown to exacerbate free radical damage initiated by Fe (⁹⁷). This fact makes Al a very toxic element in the body (⁹⁸). The body has several defense systems which constantly eliminate free radicals and potential free radical initiators in order to protect cells against damage (⁸³). These defense systems include anti-oxidants such as vitamin E, while Fe and Al are removed by Tf, although this function of Tf may be compromised in the mutant TfC2.

2.5. Membranes and brain aging

Cellular, biochemical and biophysical events occur mainly at the membrane level, where structural and dynamic properties provide the control mechanisms. In addition, cellular membranes structurally mediate/regulate several cell functions like permeability, transport, transduction signals etc.). The membrane hypothesis in the etiopathogenesis of AD is currently one of the most appealing, among various hypotheses of aging, in that the biophysical structure of the plasma membrane can be modified with aging and with pathological events with consequent damage due to various factors like oxidative stress, variation of the chemical composition, modification of homeostasis etc. Alteration in plasma membranes in AD has been reported by several authors (^{99, 82}). Recently a great deal of attention has been paid to membrane fluidity in relation to neuropathological events; in fact it has been found that Al significantly modifies the biophysical properties of membranes (¹⁰⁰). Zubenko (¹⁰¹) found that a subgroup of AD patients exhibited increased platelet membrane fluidity. In addition, Al was shown to increase the fluidity of platelet membranes in vitro, probably by binding to the membrane phospholipids, while Zn reversed the effect of Al (¹⁰²). Furthermore, Zn supplements corrected platelet membrane fluidity and improved cognition in patients with AD (¹⁰³).

2.6. Al absorption in the presence of other elements

The presence of other elements has a direct influence on the uptake of Al from the environment. Investigations into another disease complex in which dementia occurs and neurofibrillary tangles are formed, the Amyotrophic Lateral Sclerosis and Parkinsonian Dementia (ALS-PD) complex of Guam, revealed that these neurodegenerative diseases were caused by chronic nutritional deficiencies of Ca and Mg and relative excesses of trace metals such as Al and Mn (¹⁰⁴). Al was found to accumulate in the tangle-bearing neurons in these patients (¹⁰⁵). Epidemiological evidence from Guam and other foci of parkinson-dementia syndromes in the Western Pacific suggest that chronic nutritional deficiency of Ca and Mg leads to a state of secondary hyperparathyroidism, increases absorption of Al, and deposition of the metal within the central nervous system (¹⁰⁶). The ratio of different, mainly metallic, elements is thus vital and it is important to consider the overall elemental status rather than Al alone (⁵⁹).

2.7. Conclusion

An abundance of research has continued to link Al to AD. Contradictory evidence may be the result of failure to appreciate that increased Al concentrations over a short time period may have different effects than a life-long exposure to increased Al concentrations in the brain, whether these may result from increased Al in the environment, or from genetic susceptibility to higher Al uptake. The detailed neuropathological differences between AD and dialysis encephalopathy could result from the fact that in AD the accumulation of Al occurs over many years and focally in neurofibrillary tangles and plaques, whereas the rate of intoxication is much greater (1 2 years) in dialysis encephalopathy with a general accumulation of the metal in all brain mass. However, the possibility of involvement of environmental factors such as Al in the etiology of AD provides hope for prevention and treatment. Genetic factors cannot always be controlled, but if all the environmental factors are identified, they could be counteracted even if they cannot be eliminated completely.

3. ZN AND NEURODEGENERATION

     Zn is an essential trace element. It's importance to health and well-being is well recognised. It is required for a large number of physiological and biochemical functions and a wide range of symptoms develop as a consequence of a deficiency of the metal. These include effects within the central nervous system such as distorted or absent sensory function involving taste, smell and vision (^{107, 108}). Toxicity associated with excessive exposure to Zn is less well known. Situations in which toxicity has been observed include deliberate ingestion, exposure to contaminated food and/or drinking water and other incidents with unidentified causes. Involvement of the nervous system in some of these cases has been documented. Lethargy, light-headedness and loss of neuromuscular co-ordination were noted in a 16-y-old youth following ingestion of 12 g metallic Zn (¹⁰⁹). Numbness and paraesthesia with other signs of CNS demyelination were seen in a subject who had a high serum Zn concentration (¹¹⁰). These authors referred also to epidemiological studies showing associations between undue Zn exposure and the incidence of demyelinating diseases.

3.1. Zn and normal neurological function

     A number of observations suggest that Zn may have a specific role in normal neurological function, quite separate from that as an enzyme cofactor.

• Significant amounts of Zn, at concentrations around 10 µM, are present in the mammalian brain (¹¹¹). Some is protein bound but a substantial proportion is in a chelatable form (^{112, 113}).

• Chelatable Zn is localised to specific areas of the brain, particularly the hippocampus and neocortex (^{113, 114}).

• Within these areas the stainable Zn is contained in presynaptic vesicles at excitatory glutaminergic nerve endings (^{115, 116}).

• Depolarisation of neuronal cells (electrical, or by addition of K+ or kainate) causes glutamate and Zn to be released. Ca²⁺ promotes this exodus and concentrations of Zn in the synaptic space are estimated to be 10-100 µM. With repeated stimulation, up to 300 µM may be obtained (¹¹⁷).

• Secreted Zn is restored to the pre-synaptic nerve ending (¹¹⁸).

The physiological role of this Zn is not clear but it may be involved with stabilisation of glutamate in the vesicles, as is seen in other secretory cells (^{119, 120}), and it may influence the behaviour of post-synaptic membrane receptors/ion channels. In in-vitro systems, addition of 10-100 µM Zn attenuates the NMDA sub-class of glutamate receptors (^{121, 122}) and potentiates AMPA type glutamate receptors (¹²³). GABA receptors, strychnine-sensitive glycine receptors and other membrane sites are also influenced by Zn at these concentrations (¹¹⁷). Thus, a role for Zn in modulating nerve membrane excitability appears likely.

3.2. Zn and neurotoxicity

     Separate from the effects on membrane function, Zn is neurotoxic when present in increased concentrations. Cultured mouse cortical neurons became swollen and granular within 15 min of exposure to 300-600 µM Zn and the cells were completely destroyed after 24 h (¹²⁴). This necrosis was evident when as little as 100-300 µM Zn was applied, if membrane depolarisation was initiated e.g. by kainate or K⁺, and was inhibited by the presence of chelating agents. The mechanism appears to involve release of glutamate and Zn from the pre-synaptic cell and uptake of exogenous and endogenous Zn into the post-synaptic cell (¹²⁵).
A number of elegant studies have been carried out to determine how Zn may enter the cell to reach toxic concentrations. The results indicate that there are several routes of entry but that influx via calcium ion channels is probably the most important.
Freund and Reddig (¹²⁶), working with rat primary cortical cultures, incubated the cells for 30 min with a solution of 50 µM AMPA + 300 µM Zn²⁺ or with this solution together with a test material. Cell viability was monitored by a calcein-staining procedure, for up to 48 h post exposure. Table I shows that with the 30 min exposure to AMPA/Zn²⁺ there was approximately 50% decrease in neuronal viability. In order to investigate the cellular targets involved in this neurotoxicity they included various test agents in the incubation medium. The effects of these agents on cell viability are given in Table II. From the results it was inferred that:

• the NMDA-receptor and its associated Ca²⁺ ion channels are not involved as the blocker, MK-801, had no influence on the toxicity

• toxicity was reduced by the AMPA receptor antagonist, CNQX, indicating that activation of the AMPA receptor is necessary for induction of toxicity

• it is likely that the events involve membrane depolarisation as use of K⁺ instead of AMPA had the same effect (125)

• inhibition of toxicity by EDTA and EGTA demonstrate the requirement for extracellular Ca²⁺ and Zn²⁺

• diltiazem and nimidopine, which block L-type Ca²⁺ channels, have protective effects suggesting the need for activation of these channels with an influx of Ca²⁺ and Zn²⁺

• the influx of Ca2+and Zn2+ initiate neurodegeneration

A slightly different approach to the investigation of Zn influx was employed by Sensi et al. (¹²⁷). These workers cultured mouse neural cells and measured intracellular Zn2+ using a fluorescent dye, mag-fura-5. Cells were briefly loaded with the probe, washed and then treated with test compounds to investigate the movement of Zn²⁺. Entry of the ion into cells was visualised as an increase in emission of light viewed by fluorescence microscopy. Although they have low affinity for the dye, the culture medium contained no Ca²⁺ or Mg²⁺ to ensure that no response to these ions could interfere. A Zn2+ chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylene- diamine (TPEN), was added after responses had been observed. This abolished the fluorescence emission and confirmed that signals were caused by entry of Zn²⁺. Entry of Zn²⁺ under membrane depolarising conditions (addition of K⁺) was examined. A K+ concentration-dependent increase in intracellular Zn2+ was observed which was blocked by inclusion of Ca²⁺ channel blockers (Table III). This inhibition was partially reversed by increasing the extracellular concentration of calcium.
These results are consistent with those of Freund and Reddig (¹²⁶) in suggesting Ca channel involvement. However, Sensi et al. (¹²⁷) also investigated other possible mechanisms using the mag-furan-5 probe. It was found that Zn²⁺ will enter cells via a reverse of an ion-exchange process similar (or identical) to the Na⁺-Ca²⁺ exchanger. Furthermore, NMDA receptor-gated Ca²⁺ channels may also permit transfer of Zn²⁺ into the cell. This mechanism was not favoured by Freund and Reddig (¹²⁶) in their receptor blocker studies but the visualisation technique of Sensi et al. (¹²⁷) showed a response that was NMDA concentration-dependent and which was sensitive to D-APV, a competitive NMDA antagonist. The same research group had previously proposed that Zn neurotoxicity, demonstrated by loss of lactate dehydrogenase (LDH) into the culture medium, could be reduced or prevented by NMDA receptor antagonists (¹²⁸). However, a number of atypical features relating to their results suggested that Zn2+ permeated through the NMDA receptor-gated channels, rather than following conventional ion influx (¹²⁸). Further work by Choi and his colleagues, in which exposure to 300 µM Zn²⁺ + 300 µM NMDA evoked only minimal increases in [Zn²⁺]i and in cell viability, supports the notion that NMDA receptor-gated channels have a minor role in Zn neurotoxicity (¹²⁹).
Marin et al. (¹³⁰) proposed that Zn²⁺ might enter neural cells even when depolarisation has not occurred. Influx into cultured mouse neurons was monitored using another fluorescent probe, N-(6-methoxy-8-quinoyl)-p-toluene sulfonamide and cell viability was determined by labelling with sytox green and 4',6-diamino-2-phenylindol, and by measuring leakage of LDH. The entry of Zn, which was observed by Marin et al. (¹³⁰), was not modified by Ca²⁺ channel blockers as reported by others and, therefore, they suggested that Zn²⁺ may permeate lipid bilayers or be carried by an unidentified transporter.
Use of selective blocking agents has helped to unravel the complex mechanisms involved in uptake of Zn into vulnerable post-synaptic neurons. Entry via voltage-gated calcium channels is a significant route together with receptor-gated calcium channels associated with AMPA and NMDA. Other mechanisms such as an Na⁺-Zn²⁺ ion exchange also play a role.

3.3. Toxic Mechanisms

    Whatever routes are involved in securing entry of toxic concentrations of Zn into neuronal cells, possible mechanisms leading to cell injury and death also need to be understood. Using cell cultures, Kim et al. (¹³¹) observed Zn-induced swelling of cell bodies and of mitochondria, while Sensi et al. (¹³²) found that rotonene (a mitochondrial blocker) afforded protection against Zn toxicity, in a similar model system. Zn toxicity may, therefore, involve inhibition of energy production. It is known that a number of the enzymes required for mitochondrial respiration and glycolysis are inhibited at nM-mM concentrations of the metal (^{133, 134, 129}) while brief exposure to 100 µM Zn²⁺ caused a 50% loss of intracellular ATP (¹³⁰). Sheline and Choi (¹³⁵) reported that Zn-induced ATP loss and cell death were reduced when 2 mM pyruvate was included in the test media. In addition, from the work presented in these and other publications (^{136, 130}) it was suggested that reactive oxygen species (ROS) are produced as a consequence of mitochondrial dysfunction and that cellular necrosis then follows. Zn-induced generation of ROS was visualised using DCDHF, Newport green and HEt as oxidation-sensitive probes by Kim et al. (137), Sensi et al. (^{132, 136}), respectively. Necrotic changes in the same cells (lipid peroxidation) was quantified by the thiobarbituric acid reactive substances (TBARS) assay (^{131, 137}), Table IV. It should be noted that Zn interacts with many macromolecules (¹²⁹) affording other possible harmful perturbations of cellular function in addition to those involving energy production and formation of ROS.

3.4. Implications of Zn and Neurotoxicity

     Neural cell death may be relevant to situations other than undue exposure to Zn. Choi and Koh (¹¹⁷) present evidence to suggest that Zn translocation is a feature of the selective neuronal cell death, which occurs following transient global ischaemia. Similarly, neuronal injury associated with epilepsy or seizures may be Zn-related (¹³⁶). Agents which reduce the uptake of Zn into neurons in experimental situations, as described above, might prove useful in the treatment of these conditions.

3.5. Conclusions

     The use of various Zn-specific dyes and related approaches demonstrate that the effects of undue exposure to Zn are features associated with that metal and may not be dismissed as secondary to Ca²⁺ overload. The relative importance of calcium and glutamate, either alone or together with Zn remains to be determined.

References

1. Couper. Br. Ann. Med. Pharm. Vital. Stat. Gen. Sci.; 1:41-42 (1837)
2. R.H.Flinn, P.A.Neal, W.H.Reinhart, J.M.DallaValle, W.B.Fulton, A.E.Dooly. Publ. Health Bull. 247:77 (1940)
3. J.Ansola, E.Uiberall, E. Escudero. Rev. Med. Chile, 72: 222-228 (in Spanish) (1944)
4. P.Schuler, H.Oyanguren, V.Maturana, A.Valenzuela, E.Cruz, V.Plaza, E.Schmidt, R.Haddad. Ind. Med. Surg., 26: 167-173 (1957)
5. J.Rodier. Brit. J. Ind. Med. 12:21-35 (1955)
6. S.Tanaka, J.Lieben. Arch Environ. Health. 19:674-684 (1969)
7. S.V.Chandra, S.V.Seth, J.K.Mankeshwar. Environ Res. 7:374-380 (1974)
8. J.D.Wang, C.C.Huang, Y.H.Hwang, J.R.Chiang, J.M.Lin, J.S.Chen. Br. J. Ind. Med.; 46:856-859 (1989)
9. H.B.Ferraz, P.H.F.Bertolucci, J.S.Pereira, J.G.C.Lima, L.A.F.Andrade. Neurology. 38:550-553 (1988)
10. G.Meco, V.Bonifati, N.Vanacore, E.Fabrizio. Scand. J. Work Environ Health. 20:301-305 (1994)
11. R.Kawamura, H.Ikuta, S.Fukuzumi, R.Yamada, S.Tsubaki. Kisato Arch. Exp. Med. 18: 145-171 (1941)
12. C.J.Kilburn. Neurotoxicology, 8:421-429 (1987)
13. X.G.Kondakis, N.Makris, M.Leotsinidis, T.Papapetropoulos. Arch. Environ. Health. 44 (3): 175-178 (1989)
14. O.Iwami, T.Watanabe, C.S.Moon, H.Nakatsuka, M.Ikeda. Sci. Total Environ. 149 (1-2): 121-35 (1994)
15. G.Alves, J.Thebot, A.Tracqui, T.Delangre, C.Guedon, E.Lerebours JPEN; 21: 41-45 (1997)
16. D.Krieger, S.Krieger, O.Jansen, P.Gass, L.Theilmann, H.Lichtnecker. Lancet, Jul 29, 346 (8970): 270-274 (1995)
17. G.Pomier--Layrargues, L.Spahr, R.F.Butterworth. [letter]. Lancet, Mar 18, 345 (8951): 735 (1995)
18. L. Alessio. and R. Lucchini. In: Data profiles for selected chemicl series. Argentesi F., Roi R., Sevilla Marcos JM. (eds.) (European Commission Joint Research Project Centre, Luxembourg. SPI 96, 59. 1996)
19. J.Cranmer, D.Mergler, M.Williams-Johnson (eds.) Manganese. Are there effects from long-term, low-level exposure ? Neurotoxicology, volume 20, number 2-3 (1999)
20. R.Lucchini, L.Selis, D.Folli, P.Apostoli, A.Mutti, O.Vanoni, A.Iregren, L.Alessio. Scand. J. Work. Environ. Health. 21:143-149 (1995)
21. R.Lucchini, E.Bergamaschi, A.Smargiassi, P.Apostoli. Environ. Res. 73: 175-180 (1997)
22. R.Lucchini, P.Apostoli, C.Perrone, D.Placidi, E.Albini, P.Migliorati, D.Mergler, M.P.Sassine, S.Palmi, L.Alessio. Neurotoxicol. 20(2-3): 287-298 (1999)
23. C.C.Huang, N.S.Chu, C.S.Lu, J.D.Wang, J.L.Tsai, J.L.Tzeng, E.C.Wolters, D.B.Calne. Arch. Neurol. 46: 1104-1106 (1989)
24. C.C.Huang, C.S.Lu, N.S.Chu, F.Hochberg, D.Lilienfield, W.Olanow, D.B.Calne. Neurology. 43 (8): 1479-1483 (1993)
25. C.C.Huang, N.S.Chu, C.S.Lu, R.S.Chen, D.B.Calne. Neurology. 50:698-700 (1998)
26. D.B.Calne, N.S.Chu, C.C.Huang, C.S.Lu, W.Olanow. Neurology. 44 (9):1583-1586 (1994)
27. M.Yamada, S.Ohno, I.Okayasu, R.Okeda, S.Hatekeyama, H.Watanabe. Acta Neuropathol. (Berlin), 70: 273-278 (1986)
28. D.B.Calne. The Lancet, December 24/31, pp 1457-1459 (1983)
29. M.L.Bleecker. Neurotoxicol and Teratol. 10: 475-478 (1988)
30. M.Zuber and A.Alperovitch. Rev. Epidem. et Sante' Publ., 39: 373-387 (1991)
31. A.Barbeau, M.Roy, G.Bernier, G.Campanella, S. Paris Can. J. Neurol. Sci., 14: 35-41 (1987)
32. C.Hertzman, M.Wiens, D.Bowering, B.Snow, D.B.Calne. Am. J. Ind. Med. 17: 349-355 (1990)
33. J.R.Goldsmith, Y.Herishanu, J.M.Abarbanel, Z.Weinbaum. Arch. Environ. Health. 45 (2): 88-94 (1990)
34. K.M.Semchuk, E.J.Love, R.G.Lee. Neurology, 42: 1328-1335 (1992)
35. R.Feldman. Ann. NY Acad. Sci. 648:266-267 (1992)
36. C.M.Tanner. In: Anderson W., Schoenberg DG, (eds.) Neuroepidemiology: a tribute to Bruce Schoenberg. (CRC Press, Boca Raton 1990), pp: 193-211
37. K.?ygard, T.Riise, B.Moen, B.A.Engelsen. In: Proceedings from the Symposium on Manganese Toxicity. (International Manganese Institute, Paris, 1992), pp:179-182
38. I.Kessler. In: Schoenberg B. (ed.) Neurological epidemiology: principles and clinical applications. (Adv. in Neurology, Vol.19, Raven Press, N.Y.: 355-384. 1978)
39. C.M. Tanner. In: Stern, MB., Koller WC., (eds.) Parkinsonian syndromes. (New York, Marcel Dekker, 1993) pp: 145-154
40. J.De Pedro-Cuesta. Acta Neurol. Scand. 84: 357-365 (1991)
41. R.Lucchini, R.Gasparotti, E.Albini, L.Benedetti, L.Alessio. In: abstracts from the 26th International Congress on Occupational Health. 27 August-1 September 2000, Singapore
42. F.J.Jiménez-Jiménez, J.A.Molina, M.V.Aguilar, F.J.Arrieta, A.Jorge-Santamaria, F.Cabrera-Valdivia, L.Ayuso-Peralta, M.Rabasa, A.Vazquez, E.Garcia-Albea, M.C.Martinex-Para. Acta Neurol. Scand. 91: 317-320 (1995)
43. J.Zayed, S.Ducic, G.Campanella et al. Can. J. Neurol. Sci. 17:286-291 (1990)
44. J.M. Gorell, C.C. Johnson, B.A. Rybicki, E.L. Peterson, G.X. Kortsha, G.G. Kortsha and R.J. Richardson. Neurotoxicology. 20 (2-3): 239-248 (1999)
45. E.P.Brouillet, L.Shinobu, U.McGarvey, F.Hochberg, M.F.Beal. Exp.Neurol. 120:89-94 (1993)
46. H.Bernheimer, W.Birkmayer, O.Hornykiewicz, K.Jellinger, F.Seitelberger. J.Neurol.Sci. 20:415-455 (1973)
47. H.Shinotoh, B.J.Snow, K.A.Hewitt, B.D.Pate, D.Doudet, R.Nugent, D.P.Perl, W.Olanow, D.B.Calne. Neurology. 45:1199-1204 (1995)
48. A.Lydén, B.S.Larsson, N.G.Lindquist. Acta Pharmacol. Toxicol. (Copenh) 55:133-138 (1984)
49. J.Donaldson, D.McGregor, F.LaBella. Can. J. Physiol. Pharmacol. 60:1398-1405 (1982)
50. G.C. Cotzias, S.T.Miller, P.S.Papavisiliou, L.C.Tang. Med. Clin. North Am. 60:729-738 (1976)
51. E.C.Wolters, C.C.Huang, C.Clark, R.F.Peppard, J.Okada, N.S.Chu, M.J.Adam, T.J.Ruth, D.Li, D.B.Calne. Ann. Neur. 26(5):647-651(1989)
52. A.Verity. Neurotoxicol. 20(2-3):489-498 (1999)
53. Doelken. Nauym-Schmiedelbergs Arch. Exp- Pathol. Pharmak. 40:58-61 (1887)
54. A.Alfrey In: Al in Chemistry, Biology and Medicine. Nicolini M., Zatta P. and Corain B. (eds) (Raven Press, New York. 1991)
55. I.Klatzo, H.Wisniewski and E.Streicher. J. Neuropathol. Exper. Neurol. 24:187-199 (1965)
56. D.R.Crapper, S.S.Krishnan, A.J.Dalton. Science. 180:511-3 (1973)
57. P.Zatta. Trace Elem. Med. 10: 120-128 (1993)
58. D.G.Munoz. Arch Neurol. 55:737-9 (1998)
59. F.C.V.Potocnik. GeriatRx 2:14-18 (1990)
60. J.P.Landsberg, B.McDonald and F.Watt. Nature 360:65-68 (1992)
61. 61) J.M.Candy, A.E.Oakley, J.Klinowski, et al.. Lancet i:354-357 (1986)
62. P.F.Good and D.P.Perl. Nature. 362:418 (1993)
63. P.F.Good, D.P.Perl, L.M.Bierer and J.Schmeidler. Ann Neurol. 31:286-292 (1992)
64. M.J.Kotze, H.J.Davis, S.Bissbort, E.Langenhoven, J.Brusnicky, C.J.J.Oosthuizen. Clin Genet. 43: 295-299 (1993)
65. N.I.Ward and J.A.Mason. Journal of Radioanalytical and Nuclear Chemistry 113:515-526 (1987)
66. E.Bjertness, J.M.Candy, A.Torvik, P.Ince, F.McArthur, G.A.Taylor, S.W.Johansen, J.Alexander, J.K.Gronnesby, L.S.Bakketeig, J.A.Edwardson. Alzheimer Dis Assoc Disord. 10:171-4 (1996)
67. M.F.Beal, M.F.Mazurek, D.W.Ellison, N.W.Kowall, P.R.Solomon and W.W. Pendlebury. Neuroscience 29:339-346 (1989)
68. H.Murayama, R.W.Shin, J.Higuchi, S.Shibuya, T.Muramoto, T.Kitamoto. Am J Pathol 155:877-85 (1999)
69. P.B.Moore, J.P.Day, G.A.Taylor, I.N.Ferrier, L.K.Fifield, J.A.Edwardson. Dement Geriatr Cogn Disord. 11:66-9 (2000)
70. M.W.Whitehead, G.Farrar, G.L.Christie, J.A.Blair, R.P.Thompson, J.J.Powell. Am J Clin Nutr 65:1446-52 (1997)
71. J.D.Birchall and J.S.Chappell. Lancet ii:1008-1010 (1988)
72. R.A.Amstrong, J.Anderson, J.D.Cowburn, J.Cox, J.A.Blair. Biol Chem Hoppe Seyler 373:1075-1078(1992)
73. G.A.Taylor, I.N.Ferrier, I.J.McLoughlin, A.F.Fairbairn, I.G.McKeith, D.Lett and J.A.Edwardson. Age and Ageing 21:81-90 (1992)
74. L.R.Purves, M.Purves, N.Linton, W.Brandt, G.Johnson and P.Jacobs. Biochim. Biophys. Acta 966: 318-327 (1988)
75. M.Favarato, P.Zatta, M.Perazzolo, L.Fontana and M.Nicolini. Brain Res. 569:330-335 (1992)
76. W.A.Banks, A.J.Banks and P.Zatta In: Non-Neuroral cells in Alzheimer's disease. Zatta P. and Nicolini M. (Eds) (World Sci. Publ., Singapore, London, 1995), pp: 1-12
77. A.J.Roskams and J. R.Connor. Proc. Natl. Acad. Sci. USA 87:9024-9027 (1990)
78. S.J.Van Rensburg, M.E.Carstens, F.C.V.Potocnik, A.K.Aucamp and J.J.F.Taljaard. NeuroReport 4:1269-1271 (1993)
79. K.Namekata, Imagawa M., A.Terashi, S.Ohta, F.Oyama, Y.Ihara. Human Genetics 101: 126-129 (1997)
80. G.F.Van Landeghem, C.Sikström, L.E.Beckman, R.Adolfsson, L.Beckman. NeuroReport 9: 177-179 (1998)
81. S.P.McIlroy, M.D.Curran, K.D.Dynan, M.D.Vahidassr, B.M.Gleenon, J.T.Lawson, M.Middleton and A.P.Passmore. J. Alzheimer's Dis. 2: 43 (2001)
82. P.Zatta and M.Suwalsky. In: Al and Alzheimer's disease. Exley C. (ed). (Elsevier, UK, 2001) (in press)
83. S.J.Van Rensburg, F.C.V.Potocnik, J.N.P.de Villiers, M.J.Kotze and J.J.F.Taljaard. Ann NY Acad Sci 903:200-203 (2000)
84. C.M.Morris, J.M.Candy, A.E.Oakley, G.A.Taylor, S.Mountfort, H.Bishop, M.K.Ward, C.A.Bloxham, J.A.Edwardson. J Neurol Sci. 94:295-306 (1989)
85. C.T.Wong, N.Saha. Acta haemat. 75:215-218 (1986)
86. R.G.Miller, F.C.Kopfler, K.C.Kelty et al. Am. Water Works Assoc. 77: 84-89 (1984)
87. T.P.Flaten. Brain Res. Bull (In press) (2001)
88. M.A.Rogers, D.G.Simon. Age Ageing 28(2):205-209 (1999)
89. L.Gandolfi, M.P.Stella, P.Zambenedetti, and P.Zatta. Biochim. Biophys. Acta. 1406: 315-320 (1998)
90. G.A.Trapp. Neurotoxicology 1:89-100 (1980)
91. P.Zatta, E.Lain and C.Cagnolini. Eur. J. Biochem. 267: 3049-3055 (2000)
92. M.Berridge. Nature 323:294-295 (1986)
93. P.Zatta, P.Zambenedetti, M.Milanese. NeuroReport 10: 1-4 (1999)
94. P.Zatta, P.Zambenedetti, V.Bruna and B.Filippi. NeuroReport 5: 1777-1780 (1994)
95. P.Zatta, C.Bordin and M.Favarato. Arch. Biochem. Biophys. 303: 407-411 (1993)
96. C.Exley. J Inorg Biochem. 76:133-40 (1999)
97. J.M.C.Gutteridge, G.J.Quinlan, I.Clark and B.Halliwell. Biochim. Biophys. Acta. 835:441-447 (1985)
98. S.J.Van Rensburg, F.C.V.Potocnik and J.J.F.Taljaard. In: Non-neuronal cells in Alzheimer's disease. P. Zatta and M. Nicolini (eds.) (World Scientific, Singapore 1995), pp: 25-37
99. P.Zatta and M.Nicolini (Eds) Non-neuronal cells in Alzheimer's disease (World Sc. Publ., Singapore, London, 1995)
100. P.Zatta, P.Zambenedetti, A.Toffoletti, C.Corvaja, and B.Corain. J. Inorg. Biochem. 65: 109-114 (1997)
101. G.S.Zubenko, B.M.Cohen, C.F.III Reynolds, F.Boller, I.Malinakova and N.Keefe. Am J Psychiatry 144:860-868 (1987)
102. S.J.Van Rensburg, M.E.Carstens, F.C.V.Potocnik, A.K.Aucamp, J.J.F.Taljaard and K.R.Koch. Neurochemical Research 17:825-829 (1992)
103. F.C.V.Potocnik, S.J.Van Rensburg, J.J.F.Taljaard and R.A.Emsley. S Afr Med J 87:1116-1119 (1997)
104. R.M.Garruto, R.Yanagihara and D.C.Gajdusek. Lancet ii:1079 (1988)
105. D.P.Perl, D.C.Gajdusek, R.M.Garruto, R.T.Yanagihara and C.J.Jr.Gibbs. Science 217:1053-1055 (1982)
106. R.Yanagihara, R.M.Garruto, D.C.Gajdusek, A.Tomita, T.Ushikawa, Y.Konagaya, K.Chen, I.Sobue, C.C.Plato and C.J.Gibbs. Ann Neurol 15:42-48 (1984)
107. A.Taylor. Ann Clin Biochem. 33: 486-510 (1996)
108. A.Taylor. Ann Clin Biochem. 34: 142-50 (1997)
109. J.V.Murphy. JAMA. 212: 2119-20 (1970)
110. C.I.Prody, N.R.Holland. Neurology. 54: 1705-6 (2000)
111. C.J.Fredrickson. Int Rev Neurobiol. 31:145-238 (1989)
112. P.Szerdahelyi, P.Kasa. Int Rev Cytol. 89: 1-33 (1984)
113. C.J.Fredrickson, E.J.Kasarskis, D.Ringo, R.E.Fredrickson. J Neurosc Methods. 20:91-103 (1987)
114. G.Danscher, G.Howell, J.Perez-Clausell, N.Hertel. Histochemistry. 83:419-22 (1985)
115. J.Perez-Clausell, G.Danscher. Brain Res. 337: 91-8 (1985)
116. C.Beaulieu, R.Dyck, M.Cynader. NeuroReport. 3:861-4 (1992)
117. D.W.Choi and J.Y.Koh. Annu Rev Neurosc. 21: 347-75 (1998)
118. G.A.Howell, M.G.Welch, C.J.Fredrickson. Nature. 308: 736-8 (1984)
119. S.E.Pattison and M.F.Dunn. Biochemistry. 15: 3691-6 (1976)
120. P.D.Zalewski, S.H.Millard, I.J.Forbes, O.Kapaniris, A.Slavotinet. J Histochem Cytochem. 42: 877-84 (1994)
121. S.Peters, J.Koh, D.W.Choi. Science. 236: 589-93 (1987)
122. G.L.Westbrook, M.L.Mayer. Nature. 328: 640-3 (1987)
123. N.Hori, T.Galeno, D.O. Cell Mol Neurobiol. 7: 73-90 (1987)
124. M.Yokoyama, J.Koh, D.W.Choi. Neurosc Lett. 71: 351-5 (1986)
125. J.H.Weiss, D.M.Hartley, J.Koh, D.W.Choi. Neuron. 10:43-9 (1993)
126. W-D Freund and S.Reddig. Brain Res. 654:257-64 (1994)
127. S.L.Sensi, L.M.T.Canzoniero, S.P.Yu, H.S.Ying, J-Y Koh, G.A.Kerchner, D.W.Choi. J Neurosc. 17: 9554-64 (1997)
128. J-Y Koh, D.W.Choi. Neurosci. 60:1049-57 (1994)
129. L.Canzoniero, D.M.Turetsky, D.W.Choi. J Neurosc. 19: RC31, 1-6 (1999)
130. P.Marin, M.Israel, J.Glowinski, J.Premont. Europ J Neurosci. 12: 8-18 (2000)
131. E.Y.Kim, J.Y.Koh, Y.H.Kim, S.Sohn, E.Joe, J.Gwang. Europ J Neurosc. 11:327-34 (1999)
132. S.L.Sensi, H.Z.Yin, S.G.Carriedo, S.S.Rao, J.H.Weiss. Proc Natl Acad Sci. 96: 2414-9 (1999)
133. B.Krotkiewska, T.Banas. Int J Biochem. 24:1501-5 (1992)
134. H.Manev, E.Kharlamov, T.Uz, R.P.Mason, C.M. Exp Neurol. 146:171-8 (1997)
135. C.T.Sheline, D.W.Choi. Soc Neurosc Abstr. 23: 2255 (1997)
136. S.L.Sensi, H.Z.Yin, J.H.Weiss. NeuroReport. 10: 1723-7 (1999)
137. Y-H Kim, E.Y.Kim, B.J.Gwang, S.Sohn, J-Y Koh. Neuroscience. 89:175-82 (1999)

Table I.

Viability of rat cortical cultures exposed to 50 µM AMPA and/or 300 µM Zn2+. n = 4-6; * = significant difference from control culture - p < 0.05.

Data from Freund and Reddig, 1994

Table II.	Viability of rat cortical cultures exposed to 50 µM AMPA and 300 µM Zn2+, and to test materials as shown. n = 4-6; * = significant difference from AMPA/Zn culture (i.e. protective effect) - p < 0.05.

Data from Freund and Reddig, 1994

Table III.

Mean (sem) intracellular Zn2+ concentration following 15 sec exposure to 90 mM K+. n =3-9; * = significant difference from K+ stimulated cells - p < 0.05.

Data from Sensi et al., 1997

Table IV.

Increase in TBARS concentrations after 30 min exposure to 300 µM Zn2+ or Fe2+. n = 3.

Data from Kim et al. (1999).