ABSTRACT

    Concentrations of heavy metals, including mercury, have been shown to be altered in the brain and body fluids of Alzheimer's disease (AD) patients. To explore potential pathophysiological mechanisms we used an in vitro model system (SHSY5Y neuroblastoma cells) and investigated the effects of inorganic mercury (HgCl₂) on oxidative stress, cell cytotoxicity, b-amyloid production, and tau phosphorylation. We demonstrated that exposure of cells to 50 µg/L (180 nM) HgCl₂ for 30 min induces a 30% reduction in cellular glutathione (GSH) levels (n = 13, p < 0.001). Preincubation of cells for 30 min with 1 µM melatonin or premixing melatonin and HgCl₂ appeared to protect cells from the mercury-induced GSH loss. Similarly, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assays revealed that 50 µg/L HgCl₂ for 24 h produced a 50% inhibition of MTT reduction (n = 9, p < 0.001). Again, melatonin preincubation protected cells from the deleterious effects of mercury, resulting in MTT reduction equaling control levels. The release of b-amyloid peptide (Ab) 1-40 and 1-42 into cell culture supernatants after exposure to HgCl₂ was shown to be different: Ab 1-40 showed maximal (15.3 ng/ml) release after 4 h, whereas Ab 1-42 showed maximal (9.3 ng/ml) release after 6 h of exposure to mercury compared with untreated controls (n = 9, p < 0.001). Preincubation of cells with melatonin resulted in an attenuation of Ab 1-40 and Ab 1-42 release. Tau phosphorylation was significantly increased in the presence of mercury (n = 9, p < 0.001), whereas melatonin preincubation reduced the phosphorylation to control values. These results indicate that mercury may play a role in pathophysiological mechanisms of AD.

Key Words: Mercury – Oxidative stress – b-Amyloid – Tau – Melatonin – SHSY5Y neuroblastoma cells

INTRODUCTION

    The etiology of Alzheimer's disease (AD) appears to be multifactorial, with genetic factors playing an important role in the onset and progression of the disease. Mutations of the amyloid precursor protein (APP) and presenilin genes cause familial forms of early-onset AD (Sorbi et al., 1993 ; Tanzi et al., 1996). Allelic variants of the apolipoprotein E gene are associated with familial forms of late-onset AD. These findings have been extended to sporadic late-onset AD (Saunders et al., 1993). The impact of apolipoprotein E variants on early-onset AD is less impressive (van Duijn et al., 1995). In addition to genetic factors, other factors may also influence the risk of acquiring AD, such as head trauma, atherosclerosis, high maternal age, low education levels, and exposure to pesticides. Levels of various metals and trace elements such as iron, aluminum, zinc, copper, lead, and cadmium were shown to be altered in the brains and body fluids of AD patients. Also, mercury levels were shown to be elevated in postmortem AD brain tissue (Ehmann et al., 1986; Thompson et al., 1988) and in the blood of AD patients (Hock et al., 1998). (see AD & Hg abstracts)

    A hallmark of AD pathology is the deposition of amyloid plaques, consisting mainly of b-peptides, in the brain. b-Amyloid peptide (Ab) is derived from the proteolytic processing of the larger APP (Selkoe, 1994). Decreased activity in the -secretase pathway may lead to increased Ab levels. Ab has been implicated in oxidative stress and free radical production (Martin et al., 1996; Mattson et al., 1997). Furthermore, oxidative stress appears to mediate Ab toxicity by free radical production (Hensley et al., 1994; Behl, 1997; Pappolla et al., 1997).

    Another major pathological hallmark of AD is the presence of neurofibrillary tangles and their constituents, paired helical filaments, consisting mainly of hyperphosphorylated tau (Mandelkow and Mandelkow, 1998). Oxidative stress has also been shown to influence the phosphorylation state of tau protein: The exposure of primary human cortical cultures to Ab and the resultant induction of oxidative stress were found to increase tau phosphorylation (Busciglio et al., 1995; Takashima et al., 1996).

    Glutathione (GSH) is the most prevalent and important intracellular antioxidant (Wu et al., 1994). Its ability to scavenge both singlet oxygen and hydroxyl radicals provides a first line of antioxidant defense. Reduced GSH strongly modulates the redox state of cells, a role that is critical for cell survival (Bains and Shaw, 1997). The changes in cellular GSH levels thus serve as a sensitive measure of the oxidative state of cells (Toborek and Hennig, 1994).

    Melatonin has been shown to be a metal-chelating agent, binding aluminum, cadmium, copper, iron, lead, and zinc (Limson et al., 1998). Levels of melatonin and its precursors decrease with age within the body, thus potentially contributing to increased levels of heavy metals, including mercury, in the aged and AD subjects (Limson et al., 1998).

    To elucidate possible pathophysiological links with AD, we investigated the effects of mercury on the redox state of cells (GSH), cell cytotoxicity [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)], Ab production (Ab 1-40 and Ab 1-42 levels in culture supernatants), and tau phosphorylation (levels of phosphorylated tau in cell lysates) in an in vitro SHSY5Y neuroblastoma cell culture system. In addition, we studied the potential protective effects of the metal chelator and antioxidant melatonin in the above system.

MATERIALS AND METHODS

Cell culture: SHSY5Y neuroblastoma cells were grown in minimum essential medium (MEM) containing 10% fetal calf serum (FCS), 100 µg/ml streptomycin sulfate, 100 U/ml penicillin G, and L-glutamine (designated complete medium) in a humidified air/5% CO₂ chamber at 37°C. At 16 h before treatment cells were washed free of FCS-containing medium and further incubated in FCS-free MEM containing the neuroblastoma growth supplement N₂. All cell culture materials were purchased from Gibco, Life Technologies unless otherwise stated.

Capillary electrophoretic GSH measurements Cells were grown in complete medium to 70-80% confluency in 75-cm2 Nunc culture flasks, after which they were subjected to metal treatment in FCS-free MEM containing N2 supplement. Cells were exposed to various concentrations of inorganic mercury in the form of mercuric chloride (HgCl₂) for 30 min at 37°C in the presence or absence of a 30-min preincubation with 1 µM melatonin (Sigma). After treatment, culture medium was removed, and the cells were washed two times with ice-cold phosphate-buffered saline containing no calcium and magnesium. Lysis buffer, composed of 10 mM sodium phosphate containing 25 mM N-ethylmaleimide (Sigma), was pipetted onto the cells, after which they were scraped off the culture flask using a cell scraper. The cell suspension was sonicated on a Vibra-Cell sonicator (Sonics & Materials, Danbury, CT, U.S.A.) for 20 s to lyse all cells. Homogenates were centrifuged at 13,000 rpm at 4°C for 10 min, and the supernatants were filtered through Ultrafree (Biomax 10 K) spin filters (Millipore). Protein concentration was assessed by protein analysis by the method of Bradford (1976), and all samples were diluted to equal protein concentrations. Filtrates were analyzed on a Celect-P150 (50-µm x 50-cm) capillary column (Supelco) at 18 kV and 30°C for 60 min in an Applied Biosystems model 270A-HT capillary electrophoretic apparatus. Detection was at 200 nm, and the changes in reduced glutathione (GSH) levels, as the area under the peak, were calculated by the Applied Biosystems software. The results are presented as a ratio of GSH/internal standard, with the internal standard having a statistically unaffected value throughout the assay and culture conditions. All GSH capillary electrophoretic assays were performed in quadruplicate.

MTT reduction assay: SHSY5Y cells were seeded into 96-well culture plates and allowed to attach. Serum-free medium containing varying nanomolar concentrations of inorganic mercury in the presence or absence of a 1 µM melatonin preincubation was added to the cells for 24 h. MTT was added to all wells and allowed to incubate in the dark at 37°C for 6 h, followed by cell lysis and spectrophotometric measurement at 590 nm. All MTT assays were performed in triplicate.

Preparation of samples for Ab and tau-ELISAs SHSY5Y neuroblastoma cells were cultured as mentioned above. Cells were exposed for between 1 and 9 h to 50 µg/L (180 nM) mercuric chloride in the presence or absence of a 12-h preincubation with 1 µM melatonin. After the desired incubation times cell culture supernatants were removed and processed for Aß analysis, whereas cells were scraped off the culture plates, lysed, and assayed for phosphorylated tau. For Ab-ELISA, culture supernatants were removed and centrifuged to precipitate any cellular debris. Proteinaceous material was precipitated with 10% trichloroacetic acid (Fluka, Buchs, Switzerland) for 30 min at 4°C followed by centrifugation at 40,000 g. The resultant pellets were resuspended in a 150 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 2 mM EDTA, and Complete Mini protease inhibitors (Roche Biochemicals) and normalized to equal protein concentrations with the protein assay of Bradford (1976). For tau-ELISA, cells were washed free of residual medium with phosphate-buffered saline, scraped off the culture plate, centrifuged into a pellet, and lysed with lysis buffer [150 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 2 mM EDTA, Complete Mini protease inhibitors, 1 mM sodium orthovanadate (Sigma), and 2 µM okadaic acid (Roche Biochemicals)]. Cell lysate protein concentrations were normalized using the protein assay of Bradford (1976).

Ab-ELISA: Ab 1-40 and Ab 1-42 levels were determined by a sandwich ELISA. In brief, microtiter plates (Maxisorb; Nunc) were sensitized with streptavidin (Roche Biochemicals) overnight. Primary capture antibody, biotinylated 6E10 (1 µg/ml; Senetek, Maryland Heights, MO, U.S.A.), specific for Ab 1-17 (Kim et al., 1990) was added for 8 h. Concentrated (50-fold) cell culture supernatants were diluted to 1 mg/ml with assay buffer [50 mM Tris-HCl (pH 7.5) containing 140 mM NaCl, 5 mM EDTA, 0.05% Nonidet P-40, 0.25% gelatin, and 1% bovine serum albumin] and incubated for 24 h at 4°C. Discrimination between Ab 1-40 and Ab 1-42 was made possible by the use of peroxidase-labeled BAP-17 and BAP-15 antibodies specific for Ab 1-40 and Ab 1-42, respectively (Brockhaus et al., 1998). Each assay plate included a standard curve with highly purified Ab 1-40 and Ab 1-42 (Dobeli et al., 1995). After color development with tetramethylbenzidine (Roche Biochemicals), the plates were analyzed on a Labsystems Multiskan RC plate reader using Genesis software (Labsystems). There was no cross-reactivity between Ab 1-40 and Ab 1-42, and the assay was not sensitive to Ab 1-43. All Ab-ELISAs were performed in triplicate.

Tau-ELISA: Phosphorylated tau levels were determined by a solid-phase, noncompetitive sandwich ELISA as described previously (Herrmann et al., 1999). All tau-ELISAs were performed in triplicate.

Statistical analysis: Statistical analysis of data was performed using the Mann-Whitney U test plus Bonferroni's multiple comparisons. Statistical significance was assumed at p < 0.05.

RESULTS

    Mercury induced cytotoxicity in a concentration-dependent manner from as little as 10 µg/L (36 nM) to as much as the maximal concentration tested, 5,000 µg/L (18 µM), over a 24-h period (Fig. 1A). The lowest, near-physiological concentration to induce consistently between 35 and 50% inhibition of MTT reduction (cell cytotoxicity) was 50 µg/L (180 nM), which was therefore used for all subsequent experiments (Fig. 1). Exposure of cells to 1 µM melatonin before administration of mercury resulted in a protective effect with MTT metabolism not significantly different from mercury-free controls (Fig. 1B). However, the premixing of melatonin and mercury did not protect against mercury cytotoxicity (Fig. 1B).

    Click on image to enlarge Figure

FIG. 1. MTT reduction by SHSY5Y cells. A: MTT reduction over 24 h in the presence of increasing concentrations of mercury. B: Effect of mercury (50 µg/L) over 24 h in the presence or absence of a 12-h melatonin (1 µM) preincubation. Included is a group in which melatonin and mercury (Mel + Mer) were premixed for 30 min before administration. Data are mean ± SEM (bars) values of nine observations (from triplicate independent assays). All concentrations with the exception of 1 µg/L showed a significant difference from the control group: p < 0.005 for A. * p < 0.001, significantly different from the control and melatonin groups; [UNK] p < 0.004, significantly different from the melatonin plus mercury group for B. Statistics were calculated by Mann-Whitney U test with Bonferroni's multiple comparison.

    GSH measurements showed that 50 µg/L mercury, for as little as 30 min, was able to induce a 30% reduction in cellular GSH levels (Fig. 2A). The effect of mercury on GSH levels was also found to be concentration-dependent, with increasing GSH loss correlating to increasing mercury concentrations (Fig. 2B). Preincubation of cells for 30 min with 1 µM melatonin protected cells from the deleterious effect of mercury (Fig. 2C). Furthermore, the premixing of 1 µM melatonin and 50 µg/L mercury for 30 min before administration also resulted in a protective effect (Fig. 2C).

FIG. 2. Effect of mercury on SHSY5Y cellular GSH (reduced) levels. A: Changes in GSH levels over time after exposure to mercury (50 µg/L). B: Changes in GSH levels after a 30-min incubation with increasing concentrations of mercury. C: Influence of mercury (50 µg/L) after a 30-min preincubation with 1 µM melatonin. Included is a group in which melatonin and mercury (Mel + Mer) were premixed for 30 min before administration. Data are mean ± SEM (bars) values of 13 observations (from
quadruplicate independent assays). * p < 0.001, significantly different from the zero-time point for A. * p < 0.001, significantly different from the zero concentration for B. * p < 0.001, significantly different from the control and melatonin groups for C. Statistics were calculated by Mann-Whitney U test with Bonferroni's multiple comparison.

Incubation of SHSY5Y cells with 50 µg/L mercury resulted in an increase in Ab 1-40 release, over time, with a peak (15.3 ng/ml, p < 0.001 as compared with controls) release after 4 h. This represents a threefold increase in Ab 1-40 release as compared with untreated control cells (Fig. 3A). Ab 1-42 release into the culture supernatant also increased as compared with untreated control cells with maximal release after 6 h (9.3 ng/ml, p < 0.001 as compared with controls) of mercury exposure (Fig. 3B). Noteworthy is the concentration and time difference between Ab 1-40 and Ab 1-42 release induced by mercury.

FIG. 3. Time course of the effect of mercury (50 µg/L) on secreted Ab 1-40 and Ab 1-42 levels from SHSY5Y cells with or without a 12-h melatonin (1 µM) preincubation. A: Changes in Ab 1-40 levels. Mercury-treated cells showed significantly higher Ab 1-40 levels from 3 h of exposure through to the end of the experiment as compared with control cells (p < 0.001) and melatonin-pretreated cells (p < 0.004). Melatonin-pretreated cells exposed to mercury (Mel + Mer) also had higher levels than untreated control cells (p < 0.004). B: Changes in Ab 1-42 levels. Mercury-treated cells showed significantly higher Ab 1-42 levels between 4 and 6 h of exposure as compared with control cells and melatonin-pretreated cells (p < 0.004). Data are mean ± SEM (bars) values of nine observations (from triplicate independent assays). Statistics were calculated by Mann-Whitney U test with Bonferroni's multiple comparison.

    Preincubation of cells with 1 µM melatonin for 12 h resulted in a significant attenuation of the mercury-induced Ab 1-40 release. This was true for all the time points examined. The attenuated levels were, however, always above control values (Fig. 3A). Similarly, melatonin preincubation resulted in an attenuation of mercury-induced Ab 1-42 release compared with untreated control levels (Fig. 3B). In all instances melatonin alone did not affect normal Ab 1-40 and Ab 1-42 release as levels were within control values (data not shown). These results suggest that melatonin may have different effects on the metabolic pathways regulating Ab 1-40 and Ab 1-42 release.

    Mercury's effect on tau was to induce an approximately twofold increase in phosphorylation as compared with untreated controls (n = 9, p < 0.001) over a 9-h period (Fig. 4). Pretreatment of cells with 1 µM melatonin for 12 h before addition of mercury resulted in a significant (n = 9, p < 0.001) decrease of phosphorylation to control levels. Melatonin alone did not affect tau phosphorylation as levels were within control values (Fig. 4).

 FIG. 4. Time course of the effect of mercury (50 µg/L) on phosphorylated tau levels in the presence or absence of a 12-h melatonin preincubation of
SHSY5Y cells. Data are mean ± SEM (bars) values of nine observations (from triplicate independent assays). The mercury-treated cells showed significantly higher phosphorylated tau levels compared with controls and melatonin plus mercury (Mel + Mel)- and melatonin-treated cells (p < 0.001 by Mann-Whitney U test with Bonferroni's multiple comparison).

DISCUSSION

    Mercury toxicity and intoxication are well documented and reported pharmacological phenomena. Chronic neurotoxicity is reported at concentrations >35 µg/L, whereas acute toxicity occurs at concentrations >200 µg/L (Hock et al., 1998). However, the effects of low levels of mercury such as those found in teeth amalgams or in the environment remain unclear. In this study, we have examined the effect of mercury as an inducer of oxidative stress and the resultant effect on Ab production and phosphorylated tau levels in neuroblastoma cells. Furthermore, we demonstrated that these effects are reduced and/or reversed by the pineal indoleamine melatonin.

    A 24-h exposure to 50 µg/L mercury induced significant cell cytotoxicity in neuroblastoma cells. Treatment of cells with melatonin before administration of mercury greatly reduced the mercury-induced cytotoxicity. Mercury treatment of cells produced another as yet undocumented phenomenon, that of inducing oxidative stress, as measured by the loss of reduced GSH from cells. This was a rapid process, requiring only 30 min of exposure to mercury. Similarly, pretreating the cells with melatonin or premixing mercury and melatonin before administration protected cells from the mercury-induced oxidative stress. Melatonin's mechanism of action is at present unclear; however, melatonin is known to bind heavy metals (Limson et al., 1998) and to increase intracellular GSH levels through an up-regulation of GSH-synthesizing enzymes (Todoroki et al., 1998 ). It is thus possible to speculate on two mechanisms for melatonin's antioxidant action, namely, (a) melatonin as a chelating agent binding mercury, thus eliminating its cytotoxic properties, or (b) melatonin causing production of increased levels of intracellular antioxidants such as GSH (Todoroki et al., 1998). It is further not excluded that both these mechanisms could be operating simultaneously.

    The release of both Ab 1-40 and Ab 1-42 into the culture medium was increased by exposure of SHSY5Y cells to mercury. Melatonin preincubation resulted in a significant decrease in Ab release. The mercury-induced increase in Ab release may be caused through mercury's deleterious action on essential kinase enzymes involved in the -secretase pathway of APP metabolism. The result could be that cells are pushed toward the b-secretase pathway of APP metabolism, resulting in an increase in Ab release. Mercury has previously been shown to be a potent inhibitor of enzymes, especially those containing sulfhydryl groups (Edstrom and Mattsson, 1976). Protein kinase C activity in vitro and in brain tissue is markedly reduced in a concentration-dependent manner by mercury (Rajanna et al., 1995). Phorbol ester binding to protein kinase C is also inhibited by micromolar concentrations of mercury (Rajanna et al., 1995). It is thus possible that increased levels of mercury reduce protein kinase C-mediated -secretase activity with the consequence of increased Ab formation because protein kinase C mediates activation of the -secretase pathway. Mercury induces both Ab production and oxidative stress; thus, the chelation of mercury by melatonin could shift the APP metabolism back toward the -secretase pathway, reducing Ab production and the concomitant oxidative stress-inducing effects of mercury and Ab. Ab-Fibrillogenesis is also inhibited by melatonin, thereby potentially reducing the toxic buildup of Ab 1-40 and Ab 1-42 fibrils (Pappolla et al., 1998). Furthermore, melatonin has been shown to reduce the release of soluble APP from cells in culture and to reduce the levels of APP mRNA and other housekeeping protein mRNAs (Song and Lahiri, 1997). These data suggest that melatonin may be involved in metabolic mechanisms regulating APP and other essential cellular protein production, over and above its antioxidant capacity.

    In a similar fashion mercury induced an increase in tau phosphorylation as compared with untreated cells. Melatonin treatment was able to protect cells from the mercury-induced tau hyperphosphorylation. Mercury's influence on tau phosphorylation remains unclear; however, it may be an indirect effect via oxidative stress and Ab production. Both Ab and oxidative stress have been shown to influence tau phosphorylation (Busciglio et al., 1995 ; Takashima et al., 1996). It is speculated that Ab's action is through the activation of glycogen synthase kinase-3 concomitant with an inhibition of phosphatidylinositol 3-hydroxykinase (Busciglio et al., 1995; Takashima et al., 1996). Phosphatidylinositol 3-hydroxykinase has further been shown to regulate glycogen synthase kinase-3 negatively via activation of protein kinase B and its pathway (Burgering and Coffer, 1995; Cross et al., 1995). Mercury could thus exert an effect within this pathway not unlike that in the -secretase pathway of APP metabolism.

    In conclusion, our data suggest that inorganic mercury could be involved in some of the pathophysiological aspects of AD. The pineal indoleamine melatonin is able to counteract, at least in part, these effects and needs further investigation as a potential therapeutic agent.

Acknowledgments

    The technical support of Fides Meier (Psychiatric University Hospital, Basel, Switzerland) and Choung Ly is gratefully acknowledged. We thank Dr. P. Frey, Novartis Pharma, Basel, for the gift of tau protein and Christoph Sturzinger (Biozentrum, Basel) for help with capillary electrophoresis. C.H. and F.M.-S. are supported by grant 3200-52908.97 of the NF Metals Project, Schweizerischer National-fonds, and Kanton Basel Stadt, C.B. by the AETAS Foundation for Gerontological Research and the EU Biomed I Action Programme MOLGERN, and P.R. by the FNRS, Belgium.

Footnotes

    Abbreviations used: Ab, b-amyloid peptide; AD, Alzheimer's disease; APP, amyloid precursor protein; FCS, fetal calf serum; GSH, glutathione; MEM, minimum essential medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. 

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Received July 13, 1999; revised manuscript received September 1, 1999; accepted September 1, 1999.

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For more information on the toxic effects of mercury and the possible relaionship between mercury exposure and Alzheimer's Disease see the Mercury Toxicity slide show.