A Mechanism for Zinc Toxicity in Neuroblastoma Cells

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Metabolic Brain Disease [mebr] pp1207-mebr-487146 May 4, 2004 20:35 Style file version Nov. 19th, 1999

Metabolic Brain Disease, Vol. 19, Nos. 1/2, June 2004 (C© 2004)

A Mechanism for Zinc Toxicity in Neuroblastoma Cells

Willie M. U. Daniels,1,4 Jacobus Hendricks,1 Ruduwaan Salie,2

and Susan J. van Rensburg3

Received September 16, 2003; accepted December 8, 2003

Zinc is an important component of proteins essential for normal functioning of the brain.However, it has been shown in vitro that this metal, at elevated levels, can be toxic tocells leading to their death. We investigated possible mechanisms of cell death caused byzinc: firstly, generation of reactive oxygen species, and secondly, the activation of the MAP-kinase pathway. Cell viability was assessed by means of the methyl-thiazolyl tetrazolium salt(MTT) assay and confirmed by tetramethylrhodamine methyl ester (TMRM) staining. Wemeasured the phosphorylation status of Erk and p38 as indicators of MAP-kinase activity,using Western Blot techniques. A time curve was established when neuroblastoma (N2α)cells were exposed to 100µM of zinc for 4, 12, and 24 h. Zinc caused a significant reductionin cell viability as early as 4 h, and indirectly stimulated the accumulation of reactive oxygenspecies as determined by 2.7 dichlorodihydrofluorescein diacetate (DCDHF) staining andconfocal microscopy. Investigation of the MAP-kinase pathway indicated that Erk wasdownregulated, while p38 was stimulated. Our results therefore led us to conclude that invitro, zinc toxicity involved the generation of reactive oxygen species and the activation ofthe MAP-kinase pathway.

Key words: Zinc toxicity; MAP-kinase pathway; Erk; p38; MTT; reactive oxygen species.

INTRODUCTION

Zinc is required as a catalyst for over 300 enzymes. It is important as a structuraland regulatory ion for enzymes, proteins, and transcription factors (Vallee and Falchuk,1993). Within the brain zinc is an essential nutrient for normal neurological functions. Forexample, high levels of zinc in the limbic system regulate both inhibitory and excitatorysynaptic transmission in the hippocampus and therefore may play an important role in theprocessing of memory formation (Takeda, 2000). Zinc uptake into neuronal cells occurs atthe level of the cell body as well as neuron terminal. It is incorporated into zinc-bindingproteins and transported into vesicles (Coleet al., 1999). The concentration of zinc in thesevesicles is approximately 300–350µM (Takeda, 2000). Zinc is therefore sequestered athigh concentrations in these presynaptic boutons (Perez-Clausell and Dansher, 1985), andwhen released with neuronal activity, is estimated to achieve peak synaptic concentrationsof several hundred micromolar (Howellet al., 1984). It has not yet been established why

1Department of Medical Physiology, University of Stellenbosch, Tygerberg, South Africa.2The MRC Diabetic Research Group, Parow, South Africa.3Department of Chemical Pathology, University of Stellenbosch, Tygerberg, South Africa.4To whom correspondence should be addressed at Department of Medical Physiology, University of Stellenbosch,P.O. Box 19063, Tygerberg 7505, Western Cape, South Africa. E-mail: [email protected]

790885-7490/04/0600-0079/0C© 2004Plenum Publishing Corporation

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80 Daniels, Hendricks, Salie, and van Rensburg

these high levels of zinc are not toxic to neurons in vivo, but it may be due to efficienthomeostatic mechanisms regulating the entry into, distribution in, and excretion from cellsand tissues (Vallee and Falchuk, 1993). There are no known disorders of excessive, toxic zincaccumulation in the body or brain in contrast to iron (haemochromatosis), copper (Wilson’sdisease), and other metals (Vallee and Falchuk, 1993). In neuronal cultures, however, zinchas been shown to be extremely toxic to cells especially at elevated levels (Armstronget al.,2001). Studies have reported that high levels of this metal damage cell membranes, alterenzyme specificity, disrupt cellular functions, and damage the structure of DNA (Bruinset al., 2000).

In the brain, zinc is mainly contained in glutaminergic neurons (Crawford and Conner,1973) and therefore experiments investigating the mechanisms of zinc toxicity have mainlyfocused on this neurotransmitter system. One such study proposed that zinc modulatedglutamate transport, influencing its removal from the synaptic cleft. This increase in synap-tic glutamate levels could therefore serve as an indirect mechanism for zinc to causeneuronal death (Spiridonet al., 1998). However, despite its intrinsic neurotoxicity, ad-dition of 500 µM zinc to neuronal cultures blocked acute neuronal swelling and celldeath caused by the glutamate agonistN-Methyl-D-aspartate (NMDA; Peterset al.,1987).

It was also suggested that as zinc is released with glutamate, the metal might translo-cate to postsynaptic neurons via voltage-sensitive calcium channels or calcium permeableglutamate receptors. Increase in intracellular zinc can cause mitochondrial dysfunctionwith subsequent generation of reactive oxygen species (Sensiet al., 1999). Similarly Weisset al. (2000) demonstrated zinc activation of protein kinase C resulting in the productionof oxygen-derived free radicals. Interestingly, low concentrations of zinc protected primarycortical neurons against Aβ toxicity, while higher levels enhanced the toxic effects of Aβpeptide (Lovellet al., 1999).

This study was undertaken to confirm the involvement of O2-derived free radicals inzinc toxicity in cultured neurons. Since free radicals have the potential to activate manysignal transduction pathways (Duganet al., 1997; Liet al., 1999; May and Ghosh, 1998), wedecided to investigate whether members of the family of mitogen-activated protein kinases(MAP-kinases) were implicated in zinc-induced cell death. We focused on the activationof extracellular-signal regulated kinase (Erk) usually associated with cell proliferation andp38 usually linked to cell stress (Mielke and Herdegen, 2000).

METHODS AND MATERIALS

Cell Culture

All experiments were done with N2α-Neuroblastoma cells, grown in RPMI – 1640medium (W/glutamine, W/O NaHCO3 and W/O Antibiotics, GibcoBRL/13108–015) at37◦C, 96% humidity, 5% CO2, supplemented with 10% Fetal Bovine Serum. Cells wereplated at a density of 0.85× 106/mL in a total volume of 3 and 2 mL of RPMI medium in60 and 35 mm culture dishes, respectively. Confluence (80%) of cells for 60 and 35 mmcell culture dishes were reached within 3 and 2 days, respectively.

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Zinc Toxicity in Neuroblastoma Cells 81

Treatment of Cells

To establish the optimal conditions for our investigations, concentration curves andtime curves were performed. We investigated the effects of zinc on N2α-Neuroblastomacells viability at different time intervals i.e. 4, 12, and 24 h. The cells were also subjectedto different concentrations of zinc (10, 50, 100, and 300µM) for a time period of 4 h.

Cell Viability

Cell viability was assessed by two methods. First the methyl–thiazolyl tetrazolium salt(MTT; Sigma/M-2128) assay was performed. To ensure the reliability of our observationswe also assessed cell viability by fluorescence staining with tetramethylrhodamine methylester (TMRM), whose uptake into the cell and mitochondria is dependent on transmembranepotentials (Mattsonet al., 1995). Modified MTT assays were done in 35-mm Petri dishescontaining 1 mL of medium (Denizot and Lang, 1986; Mosmann, 1983). Test reagents wereadded after maximal cell growth: 500µL of a 5 mg/mL MTT stock in phosphate–bufferedsaline was added per petri dish for an incubation period of 60 min. Following the incubationperiod, a solution of 1.5 mL containing 1% HCl in isopropanol and 0.1% Triton X-100was added to dissolve the converted formazan complex. This solution was collected and itsabsorption read at 540 nm.

Confocal Microscopy

The confocal microscope was used for a dual purpose. First cell viability was establishedwith TMRM staining and secondly, the accumulation of hydrogen peroxide was investigatedby 2.7 dichlorodihydrofluorescein diacetate (DCDHF) fluorescence. DCDHF is reducedby ROS, but preferably hydrogen peroxide (Mattsonet al., 1995). For the microscopicinvestigations, the cells were loaded with 1 mL of loading buffer (5-mL loading buffer:12.5µL of 1µmol/L TMRM, 5µL of 1µmol/L DCDHF in RPMI) 10 min prior to analysisby scanning laser confocal microscopy. In all the experiments the cells were monitored withordinary brightfield microscopy for 10–15 min after which they were then exposed to two,8-s laser scans, in order to visualize membrane potential (TMRM) and ROS accumulation(DCDHF fluorescence).

Western Blotting for MAP-kinase: p38 and Erk

After being exposed to 4 h of zinc (100µM), cells were harvested for Western Blotanalysis of MAP-kinase activity (Erk and p38). Cells were lysed in lysis buffer containing:20 mM Tris (MERCK); 1 mM EGTA; 20 mM pNPP (Sigma); 50 mM NaF; 100µMNaVO4; 1 mM Dithiothreitol (DTT; Boehringer Mannheim Gmbit); 10µg/mL Aprotinin(Sigma); 10µg/mL Leupeptin (Sigma); 1 mM Phenylmethyl-sulfonyl fluoride (PMSF,Sigma). The lysates were diluted in Laemmli sample buffer (62.5 mM Tris, 4% SDS, 10%

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glycerol, 5% mercaptoethanol, 0.1% bromophenol blue, pH 6.8) and boiled for 5 min. Thelysate protein content was determined using the Bradford technique (Bradford, 1976) and10-µg protein was subsequently separated by electrophoresis on a 12% polyacrylamidegel, using the standard Bio-RAD Mini-PROTEAN II system. The separated proteins weretransferred to a PVDF membrane (ImmobilonTM-P from Millipore). These membraneswere stained with Ponceau S red (reversible stain) to visualize the proteins and to ensurethat comparable amount of proteins were loaded. To assess the quality and quantity of thetransfer, the membranes were laser-scanned and densitometrically analyzed (UN-SCAN-IT,Silkscience). Nonspecific sites on the membranes were blocked with 5% fat-free milk in Tris-buffered saline containing 0.1% Tween-20 (TBST). The activated p38 and Erk MAP-kinaseswas identified with an appropriate primary phospho-antibody. Membranes were washedwith large volumes of TBST (5× for 5 min) and the immobilized primary antibody wasconjugated with a diluted horseradish peroxidase-labeled secondary antibody (AmershamLIFE SCIENCE). After thorough washing with TBST, membranes were covered with ECLTMdetection reagents and quickly exposed to an autoradiography film (Hyperfilm ECL,RPN 2103) to detect light emission through a nonradiocative method (ECLTMWesternblotting from Amershan Pharmacia Biotech FIIMS). Protein bands were densitometricallyanalyzed and activated enzyme values were corrected for minor differences in proteinloading. Each band was evaluated densitometrically using a UN-SCAN-ITTMgel Versions5.1 for Windows. The mean densities of at least three experiments were obtained andcompared to controls.

Statistical Analyses

Data are expressed as means± SEM values. The significance of differences betweenthe groups were assessed by one-way ANOVA followed by Bonferroni’s test for multiplecomparisons.

RESULTS

From Fig. 1, we observed that exposure of N2α cells for 4 h tozinc at concentrationsof 100µM and greater, caused a significant decrease in MTT reduction when comparedto serum controls (p < 0.001, Bonferroni’s Multiple Comparison Test). The significantreduction in cell viability by 100µM of zinc was sustained for 24 h (Fig. 2).

In Fig. 3 confocal micrographs show the effects of 100µM zinc on TMRM andDCDHF staining of N2α cells. In the control cells the TMRM staining Fig. 3(A) (left)was high showing that the cells were healthy and their membranes intact. The same cellsexhibited low DCDHF staining Fig. 3(B) (right) reflecting that the presence of reactiveoxygen species was minimal. The damaging effects of 100µM zinc can clearly be seenin Fig. 3(C) and (D). The most dramatic observations were the loss of neurites, decreasedTMRM staining (Fig. 3(C)) and strong DCDHF staining (Fig. 3(D)).

To gain more insight into the mechanism of toxicity, we subjected N2α cells to zinc(100µM), and investigated the phosphorylation of candidates of the MAP-kinase pathway(Erk and p38). In Fig. 4 densitrometric analysis of the western blots clearly showed thatzinc reduced Erk/44 when compared to the serum control band. Phosphorylation of Erk/42

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Figure 1. A concentration curve illustrating the effect of increasing concentrations of zinc on cellviability at 4 h. At concentrations of 100 and 300µM zinc significantly decreased MTT reduction.Experiments were done in triplicate. Results are the mean± SEM of three experiments. Significantlydifferent from serum controls, (∗ p < 0.001, Bonferroni’s Multiple Comparison Test).

Figure 2. A graph showing the effects of Zn on cell viability. N2α cells were exposed to 100µM zincfor 4, 12, and 24 h, respectively. Experiments were done in triplicate. Results are the mean± SEM ofthree experiments. Significantly different from serum controls, (∗ p < 0.001, Bonferroni’s MultipleComparison Test).

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Figure 3. Confocal micrographs showing TMRM (A) and DCDHF (B) fluorescence of serum controlN2α cells and TMRM (C) and DCDHF (D) fluorescence of N2α cells exposed to 100µM Zn for 4 h.

was also markedly decreased by zinc treatment (Fig. 4). In contrast to Erk, zinc stimulatedthe phosphorylation of p38 dramatically (Fig. 5).

DISCUSSION

Since zinc was shown to be neurotoxic at concentrations that may be present in thesynaptic cleft, the purpose of our study was to elucidate whether zinc toxicity involvedthe generation of reactive oxygen species and the activation of the MAP-kinase signal

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Figure 4. A representative graph generated by densitometric analysis showing the effects of zinctreatment on the phosphorylated status of Erk. N2α cells were exposed to Zn (100µM) for 4 h. Thecells were then harvested for the determination of Erk phosphorylation. The experiments were donein triplicate and repeated on four separate occassions. Above is a representative autoradiograph ofthe Western Blots obtained.

transduction pathway. From the concentration curve, we observed that zinc concentrationsless than 100µM were unable to affect cell viability. However, at concentrations of 100µMand higher, this metal was extremely toxic to the cells as reflected by a significant decreasein MTT reduction. This deleterious effect of zinc on the mitochondrial function of thecells was noted as early as 4 h of exposure. Subsequent confocal microscopic investigationrevealed that the zinc treatment also reduced the TMRM staining significantly, confirmingthe MTT result.

Zinc simultaneously caused the accumulation of reactive oxygen species within treatedcells as reflected by increased DCDHF fluorescence, suggesting that this may be one ofthe mechanisms by which zinc exerted its toxic effects. Our results compared well with thefindings of Sensiet al. (1999) who demonstrated that high levels of zinc (300µM) wereable to generate reactive oxygen species in cultured cortical neurons. A number of proposalshave been forwarded to explain how zinc could generate reactive oxygen species (Brownet al., 2000; Sensiet al., 1999; Weisset al., 1993, 2000). It has been proposed that zinc canenter glutamatergic neurones via calcium-permeable AMPA/kainate channels. Increases inintracellular zinc levels can then cause cell death by activation of protein kinase C andthe subsequent generation of reactive oxygen species (Takeda, 2000; Weisset al., 2000).Experimental evidence has shown that modest fluctuations of intracellular zinc (3.2µMzinc) may impair the regulation of mitochondrial energy metabolism by causing inhibition

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Figure 5. A representative graph generated by densitrometric analysis showing the effects of varioustreatments on the phosphorylated status of p38. N2α cells were exposed to Zn (100µM) for 4 h. Thecells were then harvested for the determination of p38 phosphorylation. The experiments were donein triplicate and repeated on four separate occassions. Above is a representative autoradiograph of theWestern Blots obtained.

of theα-ketoglutarate dehydrogenase complex (Brownet al., 2000). The electrons of theα-ketoglutarate dehydrogenase complex enter the electron transport chain as NADH atcomplex I. Thus zinc can interfere with the ATP production process rendering redox activemetals in the mitochondrion more prone to generate oxygen-derived free radicals. Sincezinc is redox-inactive as a result of its chemical properties, previous studies involving zinc infree radical production led to the hypothesis that zinc possibly displaces redox-active metalssuch as iron or copper from their binding sites, allowing them to participate in reactionswhich cause free radical production (Halliwell and Gutteridge, 1989). This in turn may leadto the activation of the apoptotic cascade within the affected cells (Cassarino and Bennett,1999).

Our findings showed that control cells exhibited high levels of Erk (44 and 42) phospho-rylation and low concentrations of phosphorylated p38. These results were in accordancewith reported data demonstrating that Erk was usually activated in circumstances that fa-vored cell growth and cell proliferation, whereas p38 activation was considered part of theneuronal stress response (Mielke and Herdegen, 2000). Zinc reduced Erk phosphorylationand enhanced phosphorylation of p38. Our confocal micrograph data showed that zinc wasinvolved in the generation of reactive oxygen species. It may therefore be possible that

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reactive oxygen molecules served as the activators of the MAP-kinase pathway. Such amechanism was plausible as other researchers have shown hydrogen peroxide to enhanceMAP-kinase activity (Samantaet al., 1998). The activation was however differential in thatErk was downregulated, while p38 was stimulated. This observation clearly demonstratedthe intricate interactions that exist between the members of the MAP-kinase pathway. Otherstudies have derived similar conclusions (Abe and Saito, 2000; Clerket al., 1997; New andHan, 1998; Xueet al., 2000) showing how Erk is more involved in neural survival whereasp38 is stimulated by a variety of stress stimuli causing neuronal death. It therefore seemsthat the particular pathway that is engaged to bring about the observed effects may bedetermined by the specific ligand used and the particular conditions of the experiment.

Zinc could also have stimulated p38 by mechanisms other than by reactive oxygenspecies. For instance it has been shown that activation of protein kinase C can also in-crease MAP-kinase activity by promoting Raf. In turn, Raf can phosphorylate and activateMEK1/2 which can subsequently stimulate p38 (Abe and Saito, 2000). Thus zinc couldmediate its neurotoxic effects by p38 also via other initiators in addition to reactive oxygenspecies.

While we are aware that no pathological condition has yet been ascribed to high levelsof zinc, our findings indicate that inappropriately high levels of zinc are toxic to culturedneurons. Kisilevsky (2000) pointed out that in vitro models do not necessarily duplicateprecisely what occurs in vivo, and that care must be taken to ensure that results of such modelsystems can be correlated with events occurring in vivo. In this regard it has been foundpreviously that zinc supplementation, 30 mg/day over 6 months (Potocniket al., 1997) and20 mg/day over 15 months (Van Rensburg and Potocnik, unpublished results) improvedcognition, i.e. attenuated neuronal degeneration, in patients with Alzheimer’s disease.

In conclusion, our results demonstrated that elevated concentrations of zinc causedextensive cell injury in cultured neurons. The damaging effects involved the generation ofreactive oxygen species, activating p38, but down regulating Erk. Further studies are neces-sary to elucidate the mechanisms whereby neurons under normal conditions, are protectedfrom zinc injury in the brain.

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A Mechanism for Zinc Toxicity in Neuroblastoma Cells

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