* Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia 30602, and Research Center for Resistant Cells, College of Medicine, Chosun University, Gwangju 501-759, South Korea
Received September 21, 2004; accepted November 1, 2004
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ABSTRACT |
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Key Words: aluminum; maltolate; apoptosis; necrosis; caspase 3; neurodegeneration; neurotoxicity; Neuro-2a; p53; Bcl2; BAX.
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INTRODUCTION |
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Aluminum maltolate (Al-malt) is a lipophilic complex of aluminum that forms a metastable solution of pH 7. This Al complex is advantageous for use in in vitro mechanistic studies because it dose not form insoluble precipitates of Al-hydroxide at physiological pH, as do most other salt forms of Al (Martin, 1991). Maltolate is also a common component of the human diet. It is a by-product formed during sucrose pyrolysis or thermal degradation of starch (Johnson et al., 1969
) and can be found in coffee, soybean, baked cereals, and caramelized and browned foods (Gralla et al., 1969
). Given the extremely high affinity of maltolate for Al, there is a potential for Al-malt to form in the gastrointestinal tract. Therefore, investigating the enhanced toxicity of Al-malt is relevant to human health. Numerous studies have demonstrated that Al-malt is a potent neurotoxin. Intracisternal injection of Al-malt produced cytoskeletal alterations (Klatzo et al., 1965
; Savory et al., 1996
, 1998
) that were reminiscent of the tangles seen in AD. There is also evidence to suggest that maltolate may facilitate the entry of Al into the brain (van Ginkel et al., 1993
), thereby increasing the potential for neurotoxicity. Recent findings show that the neurofibrillary degenerations induced by Al also contain hyperphosphorylated tau proteins as in AD (Savory et al., 1998
). Increasing evidence suggests that oxidative stress and apoptosis are key events in the neuropathological changes following exposure to Al-malt (Savory et al., 1999
).
Al-malt-induced apoptosis in the rabbit hippocampus involves perturbation of the Bcl-2/Bax ratio (Savory et al., 1999). More recently, it was shown that the endoplasmic reticulum is involved in Al-malt-induced apoptosis (Ghribi et al., 2001a
). Translocation of Bax to the endoplasmic reticulum and a loss of Bcl-2 have been demonstrated in rabbits treated with Al-malt. These effects may be involved in the observed release of cytochrome c from the mitochondria in Al-malt-treated rabbits (Ghribi et al., 2002a
). Once released, cytochrome c can bind to Apaf-1 in the cytoplasm, forming a complex that can activate caspase 9 with subsequent activation of death-inducing caspase 3 (Li et al., 1997
). Ghribi et al. (2001a)
demonstrated that caspase 3 was activated following intracisternal injection of Al-malt, and the site of activation was the endoplasmic reticulum (Ghribi et al., 2001a
). These biochemical changes were shown to culminate in DNA fragmentation, as evidenced by terminal uridine nucleotide end labeling (TUNEL)-staining in the hippocampus of Al-malt-treated rabbit (Ghribi et al., 2002b
; Savory et al., 1999
). Collectively, these studies provide evidence for the involvement of apoptotic cell death in Al-induced neurodegeneration and strengthen the possible involvement of Al as an early factor in the development of AD. The pathway responsible for the observed shift in Bcl2/BAX ratio remains to be identified. One likely candidate is p53, which is known to differentially control expression of Bcl2 and BAX as well as other important genes involved in apoptosis.
Neuronal cell lines are also sensitive to the toxic effects of Al-malt and may provide models to study the mechanisms involved in apoptosis. Al-malt-induced apoptosis has been demonstrated in differentiated PC-12 cells (Ohyashiki et al., 2002; Tsubouchi et al., 2001
) and human NT2 neuroblastoma cells (Griffioen et al., 2004
). Neuro-2a (N2a) murine neuroblastoma cells are also sensitive to Al treatment, which up-regulated the expression and accumulation of neurofibrillay tangle protein (Abreo et al., 1999
). This effect was associated with increased uptake of iron in N2a cells, possibly contributing to Al-induced oxidative stress and neurotoxicity. The objective of the present study was to develop an in vitro model that can be used to define the nature of Al-malt-induced cell death, apoptosis versus necrosis. The results clearly demonstrate the coinvolvement of apoptosis and necrosis in Al-malt neurotoxicity. The apoptotic cell death induced by Al-malt was dependent on de novo protein synthesis as evidenced by inhibition of apoptosis in the presence of cycloheximide (CHX). Gene expression studies showed altered expression of p53 and downstream signaling molecules, including Bax and Bcl-2, potentially implicating the p53 pathway in Al-malt-induced apoptosis. The present model will be useful in defining the pathways and mechanisms involved in Al-induced neurotoxicity.
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MATERIALS AND METHODS |
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Cell culture and treatment. Murine neuroblastoma Neuro-2a (N2a; CCL-131, American Type Culture Collection, Manassas, VA) cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS) and 1% penicillin-streptomycin (Pen-Strep®, GIBCO, Grand Island, NY). Cultures were maintained in plastic tissue culture vessels in a humidified atmosphere at 37°C with 5% CO2. Cells were seeded at 2 x 105/ml for each experiment and allowed to grow for 18 h prior to treatment with Al-malt. All treatments were performed in DMEM with 10% FCS for duration of 24 h unless stated otherwise.
The toxicity of both Al3+ and Al-malt was investigated in the present study. The speciation chemistry of Al is very complex, and aqueous solutions of Al3+ undergo extensive hydrolysis over time, yielding colloidal complexes that may influence the bioavailability of Al (Campbell et al., 1999). To control this, Al3+ stocks were made immediately prior to treatment by dissolving AlCl3 in deionized water at a concentration of 10 mM, and this stock was used to make the treatment solutions (11000 µM AlCl3). The pH of the treatment solutions was adjusted to 7 prior to addition to the cells. Several dietary ligands, including citrate, lactate, and maltolate, have a high affinity for Al and can influence both the solubility of the metal and its toxicity. Numerous studies have demonstrated that maltolate greatly enhances the toxicity of Al both in vitro (Levesque et al., 2000
; Ohyashiki et al., 2002
; Tsubouchi et al., 2001
) and in vivo (Savory et al., 1999
). Al-malt was prepared as previously described in detail (Bertholf et al., 1989
), and a 20 mM stock solution was prepared immediately prior to use. Al-malt forms a metastable solution with a pH of 7. Each molecule of Al binds three molecules of maltolate, and for this reason the maltolate control was added to cells at 3x molar concentrations. The role of de novo protein synthesis in the observed toxicity was examined using cell cultures pretreated with CHX (0.5 µM final concentration) for 30 min prior to treatment with Al-malt or maltolate. All solutions were filter sterilized using 0.22-µm syringe filters immediately after preparation.
Determination of Al cytotoxicity in N2a cells. Mitochondrial enzyme activity, an indirect measure of the number of viable respiring cells, was determined using the 3[4,5-dimethyl thiazolyl-2]2,5-diphenyl tetrazolium bromide (MTT) assay. N2a cells were seeded in 96-well culture plates at a density of 20,000 cells/well in 100 µl volume and allowed to grow for 18 h before treatments. Cells were treated by adding 100 µl of a 2x concentration of the respective reagent (AlCl3, Al-malt, or maltolate as detailed in figures), and 24 h later, MTT was added to each well at a final concentration of 0.5 mg/ml for 4 h. Media was removed and the purple formazan crystals were dissolved in 100% dimethyl sulfoxide. Absorbance at 570 nm was determined using a PowerWaveXTM absorbance microplate reader (Bio-Tek Instruments, Inc., Winooski, VT). Data are expressed as relative absorbance at 570 nm.
Cytotoxicity was determined using the lactate dehydrogenase (LDH) release assay as previously described (He et al., 2002). N2a cells were seeded as described above for the MTT assay. Treatments were added after 18 h as 2x concentrations. Plates were then incubated for 24 h, following which 40 µl of the supernatant was added to a new 96-well plate to determine LDH release, and 40 µl of 6% Triton X-100 was added to the original plate for determination of total LDH. An aliquot of 100 µl of 4.6 mM pyruvic acid in 0.1 M potassium phosphate buffer (pH 7.5) was mixed with the supernatant using repeated pipetting. Then 100 µl of 0.4 mg/ml reduced ß-NADH in 0.1 M potassium phosphate buffer (pH 7.5) was added to the wells, and the kinetic change in absorbance at 340 nm was read for 1 min using a PowerWaveXTM absorbance microplate reader. This procedure was repeated with 40 µl of the total cell lysate to determine total LDH U/well. A change of 0.001 absorbance units/min is equivalent to 1 U/l of LDH activity (Jemmerson et al., 2002
). The percentage of LDH release was determined by dividing the LDH released into the media by the total LDH following cell lysis in the same well.
Determination of DNA synthesis as an index of cell proliferation. The effects of Al treatment on N2a cell proliferation and DNA synthesis was determined using a modification of the [methyl-3H]thymidine incorporation assay previously described (Johnson and Sharma, 2001). N2a cells were treated with Al-malt for 24 h, following which 25 µCi/ml of [methyl-3H]thymidine was added for an additional 6 h. Cells were then harvested onto glass fiber filter paper (Cambridge Technology, Watertown, MA) using a cell harvester (PHD, Cambridge Technology). The harvested cells were lysed with deionized water, and filters dried with 95% ethanol. The filter papers were placed in scintillation vials containing 2 ml of liquid scintillation cocktail (Ready-Solv, Beckman, Fullerton, CA) and counted in a liquid scintillation counter (Pharmacia, Turku, Finland). Proliferative responses (uptake of 3H-thymidine) were expressed as stimulation index [disintegrations per minute (DPM)treated/DPMcontrol].
Determination of apoptosis using the acridine orange/ethidium bromide assay. The ability of acridine orange and ethidium bromide dual-staining to differentiate live from apoptotic and necrotic cells has been useful in cell death research. The assay was performed as previously described (Kern and Kehrer, 2002). N2a cells were treated with Al-malt for 24 h, following which the cells were harvested by trypsinization. The floating and adherent cells were collected and combined. Cells (106) were centrifuged at 200 x g for 10 min at 4°C and resuspended in 1 ml PBS, and 2 µl of acridine orange and 2 µl of ethidium bromide (final concentration 200 ng/ml for each) were added to each tube, and the cells were stained for 5 min. Two-parameter fluorescence was acquired from 20,000 individual cells per sample using an EPICS XL/MCL flow cytometer (Coulter Cytometry, Hialeah, FL, USA) with a 488 nm argon-ion laser. Green emissions from acridine orange and red emissions from ethidium bromide were captured at 525 and 620 nm, respectively. Live, apoptotic, and necrotic populations were analyzed using WinMDITM flow analysis software. Discrimination between cell populations was based on previously published diagrams and descriptions (Kern and Kehrer, 2002
; Liegler et al., 1995
).
Determination of phosphatidyl serine externalization: annexin V binding assay. The externalization of phosphatidyl serine is an early event in apoptosis and serves as a signal for phagocytosis by macrophages (Anderson et al., 2002). Flow cytometry analysis was performed to determine this event using the annexin V binding assay (BD Biosciences Pharmingen, San Diego, CA). N2a cells were treated with Al-malt for 24 h, following which 100,000 cells were washed 2x in cold PBS and resuspended in 100 µl 1x binding buffer. Five µl of annexin V-FITC and 5 µl of PI (final concentrations 2.5 µg/ml) were added and gently mixed, followed by incubation protected from light for 15 min. Cells were acquired within 60 min using an EPICS XL/MCL flow cytometer. The fluorochrome was excited using the 488 nm line of argon-ion laser, and annexin V and PI emissions were monitored at 525 and 620 nm, respectively. A total of 20,000 events were acquired for each sample. The following controls were run at the time of assay (1) unstained cells, (2) annexin V only, and (3) PI only.
Determination of caspase 3 activation. Caspase 3-like activity was determined using the CaspACETM fluorometric activity assay (Promega Corporation, Madison, WI) with modifications as noted. Recently it was reported that approximately 15% and 58% of the active caspase 3 enzyme is released into the media within 4 and 24 h, respectively, following induction of apoptosis (Jemmerson et al., 2002). Traditional assays require a wash step prior to lysis of the cells that may result in the loss of the active enzyme and a false negative result. For this reason, we lysed the cells in the treatment media using 1% Triton X-100. N2a cells were treated in 24-well plates at a density of 200,000/ml for 24 h, following which Triton X-100 was added to a final concentration of 1%. The cells were lysed by trituration and centrifuged at 10,000 x g for 10 min to remove cell debris. The supernatant was assayed for active caspase 3-like activity using the CaspACETM system according to manufacturer's instructions. The fluorescence of 7-amino-4-methyl coumarin (AMC) following cleavage of the substrate was determined using a Spectramax Gemini fluorescent plate reader (Molecular Devices, Irvine, CA, USA). The fluorescence signal was digitized and analyzed using SoftMax ProTM (version 3.1.1, Molecular Devices), and the concentration of caspase 3-like activity determined from an AMC standard curve.
Cell cycle analysis. The cell cycle distribution of N2a cells treated with Al-malt was determined using flow cytometry. Cells were treated with Al-malt for 24 h and then floating and trypsinzed cells were resuspended in nuclear isolation media (NIM; 50 µg/ml PI, 1 mg/ml RNase A, 0.1% Triton X-100). Nuclear DNA content from 50,000 cells was determined using an EPICS XL/MCL flow cytometer with a 488 nm argon-ion laser and an emission wavelength of 620 nm. DNA histograms were analyzed using WinMDITM flow analysis software.
Analysis of cellular and nuclear morphology. Cellular morphology was examined using phase and scanning electron microscopy, and nuclear morphology was examined using an epifluorescence microscope following staining with Hoechst 33258 (H33258). Cells were seeded (2 x 105/ml) in 6-well plates for phase contrast and nuclear morphology imaging. Following 24-h treatment with Al-malt, cells were stained with H33258 (1 µg/ml) for 5 min, and phase and fluorescence microscopy was performed using an IX71 inverted microscope (Olympus America, Inc., Melville, NY). Digital images were captured using a MagnaFire SP® digital camera.
Scanning electron microscopy was performed on cells seeded on 13-mm ThermanoxTM cover slips (Nalge Nuc International, Rochester, NY) in 24-well plates and treated with Al-malt for 24 h. Following the treatment, cover slips were removed from the well and rinsed with ice cold PBS for 5 s and immediately plunge-frozen in liquid nitrogen. The frozen monolayers were freeze-dried for 18 h at 50°C and carbon coated using a Denton DV502A vacuum evaporator (Denton Vacuum, Moorsetown, NJ). This fixation method does not affect cellular morphology (Fernandez-Segura et al., 1999). Imaging was conducted using a LEO 982 field emission scanning electron microscope (Leo Electron Microscopy Inc., Thornwood, NY).
Real-time PCR analysis of gene expression. N2a cells were treated with Al-malt for 24 h, followed by preservation of RNA with RNAlater (Qiagen, Valencia, CA) according to manufacturer's directions. Total cellular RNA was isolated using RNeasy kits (Qiagen) according to the instructions provided. One microgram of RNA was reverse-transcribed using oligo dT and 60 U of Superscript II (Life Technologies, Grand Island, NY). Real-time Sybr Green primers (see Table 1) for Bcl-2, Bax, Mdm2, p53 and GAPDH (reference gene) were designed using Beacon Designer (PREMIER, Biosoft International, Palo Alto, CA). Template was combined with primers and 2X Taqman® Universal Sybr Green PCR Master Mix (contains PCR buffer, passive reference dye ROX, deoxynucleotides, uridine, uracil-N-glycosylase, and AmpliTaq Gold® DNA polymerase; Perkin-Elmer, Applied Biosystems, Foster City, CA) in a 25-µl reaction volume, and amplification was performed using an ABI Prism® 7900HT (PE, Applied Biosystems) real-time thermal cycler. Cycling conditions were 2 min at 50°C; 10 min at 95°C; 50 cycles of 92°C for 30 s; 60°C for 1 min. Real-time fluorescence detection was performed during the 60°C annealing/extension step of each cycle. Melt-curve analysis was performed on each primer set to ensure that no primer dimmer or nonspecific amplification was present under the optimized cycling conditions. The fold difference in mRNA expression between treatment groups was determined using the relative quantification method utilizing real-time PCR efficiencies (Pfaffl et al., 2002) and normalized to the housekeeping gene, GAPDH, thus comparing relative CT changes between control and experimental samples. Prior to conducting statistical analyses, the fold change from the mean of the control group was calculated for each individual sample (including individual control samples to assess variability in this group).
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RESULTS |
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DISCUSSION |
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The manner in which a cell dies can have a great impact on the resulting response in the surrounding tissue. Death by necrosis has been shown to act as a natural adjuvant, inducing oxidative stress and the production of numerous pro-inflammatory cytokines (Anderson et al., 2002). This is due to the indiscriminant release of the cytoplasmic contents from the dying cells. The result is an area of inflammation leading to immune-mediated damage of innocent cells surrounding the initial insult. In contrast, death by apoptosis is a controlled event, usually with minimal loss of membrane integrity until the later stages termed secondary necrosis. Instead, the cytoplasmic contents are systematically degraded from within. This type of cell death often involves phagocytosis by resident tissue macrophages and the release of anti-inflammatory cytokines (Fadok et al., 1998
). However, a recent study suggested that this contention may not always apply. Jemmerson et al. (2002)
showed that cytochrome c, active caspase 3, and LDH were all released from apoptotic cells and that release began within 2 h of the initiating stimulus. Previously, it was thought that all of these proteins were degraded in the cytoplasm of apoptotic cells. Therefore, apoptotic cell death may also substantially contribute to death-induced secondary inflammation and damage to surrounding tissues. The findings of this study confirm that LDH and active caspase 3 are released from apoptotic cells, since inhibition of apoptosis with CHX reduced LDH release and caspase 3 activity in the media. Results presented here support the hypothesis that the apoptosis seen in vivo following treatment with Al-malt could contribute to secondary immune-mediated damage and further neurodegeneration.
Caspases are important mediators of apoptosis, and caspase activation has been demonstrated in Al-malt-induced neurodegeneration (Ghribi et al., 2001a, 2002a
,b
). Our in vitro N2a cell model also showed strong caspase 3 activation in response to Al-malt treatment. Inhibition of protein synthesis using CHX inhibited apoptotic cell death and the activation of caspase 3 in Al-malt-treated cells, implicating de novo protein synthesis in the activation of this death effector. Protein synthesis could affect the regulation of caspase 3 in two ways: (1) new pro-caspase 3 synthesis and (2) synthesis of other proteins required for caspase 3 activation. Our results show that the expression of BAX mRNA is increased in N2a cells following treatment with Al-malt. BAX is an important mediator of the mitochondrial intrinsic pathway leading to caspase 3 activation and apoptosis and likely represents one of the pro-apoptotic factors down-regulated by CHX in this system.
The involvement of de novo protein synthesis in toxicant-induced apoptosis has been demonstrated previously (Chow et al., 1995). Our finding of a marked decrease in the neurotoxicity of Al-malt in the presence of the protein synthesis inhibitor CHX suggests that Al-malt-induced apoptosis is at least partially dependent on de novo protein synthesis. Several candidate proteins have been identified from in vivo studies in the rabbit brain. The pro-apoptotic protein BAX has been implicated in Al-malt-induced neurodegeneration in the rabbit (Ghribi et al., 2001a
, 2002a
; Savory et al., 1999
). Present data demonstrates that Al-malt increased the expression of BAX in N2a cells. Studies with other chemicals including nitric oxide (NO) showed that apoptosis can be accompanied by an increase in BAX protein synthesis (Chen et al., 2002
). Inhibition of protein synthesis partially inhibited the apoptosis suggesting that de novo synthesis of BAX may be responsible for the apoptosis induced by NO. The demonstration of increased BAX immunoreactivity in the CA1 region of the hippocampus (Ghribi et al., 2002b
; Savory et al., 1999
) of rabbits treated with Al-malt and our data showing increase BAX mRNA expression strongly support a role for new BAX synthesis in the induction of apoptosis in these models. We also observed a dramatic down-regulation of Bcl2 expression following treatment of N2a cells with Al-malt. A similar down-regulation of Bcl2 immunoreactivity is observed in the brain of aged rabbits following treatment with Al-malt (Savory et al., 1999
). It is clearly evident that the intrinsic pathway of apoptosis involving altered Bcl2/BAX ratio is critical to Al-malt apoptosis.
The tumor suppressor gene product p53 is an important activator of the intrinsic apoptotic pathway. Here, we demonstrate for the first time that Al-malt induces the expression of p53 in neuronal cells. Up-regulation of p53 is commonly observed following exposure to DNA-damaging agents such as UV light. Increased production of growth arrest and DNA damage inducible factor 153 suggests that DNA damage may be induced in the brain of Al-malt-treated rabbits (Ghribi et al., 2001b) and could lead to activation of p53. Tumor suppressor p53 has been shown to differentially regulate Bcl2 and BAX levels both in vitro and in vivo (Miyashita et al., 1994b
). Increased production of BAX following p53 activation is due to p53 being a direct transactivator of BAX gene expression (Miyashita and Reed, 1995
). Importantly, a p53-dependent negative response element is present in the 5' untranslated region of the Bcl2 gene and is responsible for potent transcriptional down-regulation following p53 activation (Miyashita et al., 1994a
). Our observations of decreased Bcl2 and increased BAX expression concomitant with increased p53 expression suggest that apoptosis induced by Al-malt in N2a cells may be controlled by the p53 pathway, and further studies are warranted.
In conclusion, the results of the present study strongly suggest that apoptosis is a prominent form of cell death in N2a cells treated with Al-malt, thus supporting previous in vivo findings. However, necrotic cell death was also apparent in N2a cells and may play a role in the neurotoxicity of Al-malt observed in vivo. The substantial release of intracellular enzymes observed in these cells even when dying by apoptosis has implications for the involvement of secondary damage induced by immune activation and inflammation in Al-malt neurotoxicity. Furthermore, the inhibitory effect of CHX on Al-malt-induced cell death indicates that de novo protein synthesis is intimately involved in the resulting neurotoxicity. The observed shift in Bcl2/BAX ratio confirms in vivo findings, and our data suggest that p53 is a potential mediator of this effect. Detailed investigation of the p53 pathway in response to treatment with Al-malt will greatly improve our understanding of the mechanisms involved in Al neurotoxicity and may provide insight into the etiology of neurodegenerative diseases including AD. Such research may also lead to effective strategies for therapeutic intervention in human neurodegenerative diseases. The present in vitro model shows many of the molecular characteristics present in the brains of rabbits treated with Al-malt and thus is an important in vitro alternative for further mechanistic studies.
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ACKNOWLEDGMENTS |
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NOTES |
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2 To whom correspondence should be addressed at Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, GA 306027389. Fax: (706) 542-3015. E-mail: rpsharma{at}vet.uga.edu.
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