ToxSci Advance Access originally published online on December 4, 2007
Toxicological Sciences 2008 102(1):138-149; doi:10.1093/toxsci/kfm292
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Apoptosis of Cultured Astrocytes Induced by the Copper and Neocuproine Complex through Oxidative Stress and JNK Activation
,1
* Institutes of Toxicology
Biochemistry
Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan
1 To whom correspondence should be addressed at Institute of Pharmacology, College of Medicine, National Taiwan University, Section 1, Jen-Ai Road, No. 1, Taipei 10043, Taiwan. Fax: +886-2-23915297. E-mail: syl{at}ha.mc.ntu.edu.tw.
Received September 8, 2007; accepted November 29, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Astrocytes play a critical neurotrophic and neuroprotective role in the brain, and improper function of these cells may contribute to the onset of neurodegenerative diseases. Because astrocytes are known to be enriched with Cu chaperone proteins, it is important to understand the factors that may lead to cytotoxic effects of Cu on astrocytes. In this report, we demonstrated a dramatic potentiating effect of neocuproine (NCP), a membrane permeable metal chelator, on Cu, but not Fe or Pb, in inducing apoptosis of cultured astrocytes. It was estimated that individually, CuCl2 and NCP only weakly exhibited cytotoxic effects on astrocytes, with EC50 of 180 and 600µM, respectively. However, NCP at a nontoxic concentration of 10µM markedly reduced EC50 of Cu to 0.35µM (physiological concentration) and Cu (10µM) reduced EC50 of NCP down to 0.06µM. The mechanisms underlying these dramatic potentiation effects are elucidated. NCP increased the intracellular concentration of Cu in astrocytes and a nonpermeable Cu chelator, bathocuproine disulfonate was able to abolish all of the apoptotic signaling. Cell death was determined to be via apoptosis due to increased reactive oxygen species production, mitochondrial dysfunction, depletion of glutathione and adenosine triphosphate, cytochrome c release, c-Jun N-terminal kinase, and caspase-3 activation, and poly-ADP-ribose polymerase degradation. This finding, coupled with our previous reports, suggests that metal chelators (NCP, dithiocarbamate and disulfiram) should be cautiously used as they may potentiate a cytotoxic effect of endogenous Cu on astrocytes. Their clinical implications in the etiology of neurodegenerative diseases deserve further investigation.
Key Words: neocuproine; Cu; apoptosis; astrocytes; oxidative stress; JNK; caspase-3.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Astrocytes are one of the major cell types in the brain and have a crucial neurosupportive role (Kuffler et al., 1984
; Zhu et al., 2006). They promote growth and survival of neurons by secreting neurotrophic factors (e.g., interleukin-6, glial derived neurotrophic factor), prevent neurotoxicity by uptake of excess glutamate and modulate ion homeostasis, thus maintaining proper neuronal function (Hansson and Ronnback, 1995). Astrocyte malfunction may contribute to the onset of neurodegenerative diseases (Forman et al., 2005). In this study, we have demonstrated that apoptosis of primary cultured astrocytes could be induced by a physiological concentration of Cu in the presence of a metal chelator—neocuproine (NCP). The possible molecular mechanisms of the toxic effect of the Cu/NCP complex on rat cortical astrocytes are elucidated.
Cu is an essential transition metal that modulates many biological processes (Camakaris et al., 1999) and is readily detectable in animal tissues (Kennedy et al., 1998). However, Cu could be dangerous due to its ability to increase oxidative stress. It has been suggested that Cu can produce hydroxyl radicals in the presence of hydrogen peroxide (H2O2) (Halliwell and Gutteridge, 1999), particularly in astrocytes due to the abundance of the Cu carrier protein-chaperone (Qian et al., 2005).
NCP is a metal chelator and frequently used as a protective agent against oxidative stress caused by Cu (Calderaro et al., 1993). By means of Cu chelating properties, NCP exerts various biological effects including inhibition of electrically stimulated mouse corpus cavernosum relaxation (Gocmen et al., 2000), facilitation of bladder contraction after purinergic nerve stimulation (Gocmen et al., 2005), inhibition of endogenous S-nitrosothiol decomposition by ultraviolet irradiation (Ogulener and Ergun, 2004), enhancement of NO-induced relaxation of mouse gastric fundus (De Man et al., 2001), suppression of the growth of Mycoplasma gallisepticum (Smit et al., 1981) and Escherichia coli B (Zhu and Chevion, 2000), and induction of anti-tumor effects when combined with Cu (Byrnes et al., 1992).
The mitogen-activated protein (MAP) kinase family that includes c-Jun N-terminal kinase (JNK), extracellular signal regulated kinase (ERK), and p38 kinase can be rapidly activated by various stress stimuli (Javelaud and Mauviel, 2005). Recent evidence suggests that the JNK/SAPK pathway may play an important role in triggering apoptosis in response to free radicals generated by ultraviolet (UV) radiation (Alder et al., 1995), inflammatory cytokines (Chen et al., 1996), or direct application of H2O2 (Yu et al., 1996). Caspase-3 is the downstream signaling pathway of activated JNK (Kim et al., 2005). A number of ICE/CED-3 protease targets have been identified, including the nuclear enzyme poly-ADP-ribose polymerase (PARP) (Zhu et al., 1997). We investigated whether these signal pathways were involved in the toxic effect of Cu/NCP complex in the primary culture of rat cortical astrocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Materials.
Dulbecco's modified Eagle's medium (DMEM), fetal calf serum, and other cell culture supplements were obtained from FALCON. The chemicals 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), CuCl2, CuCl, NCP, bathocuproine disulfonate (BCPS), vitamin C, catalase, superoxide dismutase, glutathione, and N-acetyl-cysteine (NAC) were purchased from Sigma (St Louis, MO). Benzyloxycarbonyl-Asp-Glu-Val-Asp(Ome)-fluoromethyl ketone (Z-DEVD-FMK) (Calbiochem, La Jolla, CA), Höechst 33258, 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)), and 2',7'-dichlorofluorescein diacetate (DCFHDA) (all from Molecular Probes, Eugene, OR) are water insoluble and were dissolved in dimethyl sulfoxide (DMSO). [
32P] ATP was obtained from Amersham Life Science, Ltd. Antibodies for JNK-1, ERK1, p38, Bcl-2, and PARP were purchased from Santa Cruz (Santa Cruz, CA). The final concentration of DMSO in the incubation medium was less than 0.5% to prevent a toxic effect of DMSO. Vehicle or the respective concentrations of DMSO were used as the control.
Cell culture.
Astrocytes were cultured from the brain tissue of 1-day-old Wistar rats (Tedeschi et al., 1986). Briefly, cortices were isolated, cleaned of white matter and meninges, minced, and digested with trypsin (0.3 mg/ml) for 25 min. The digestive process was terminated with a Type II-S trypsin inhibitor (0.3 mg/ml; Sigma). Subsequently, DNase (23.5 U/ml) was applied to digest extracellular oligonucleotides. The dissociated cells were diluted into DMEM supplemented with 10% fetal calf serum and seeded in culture dishes or on coverslips. Cells were grown to confluence (14 days) in an incubator at 37 ± 0.5°C, 5% CO2. The culture medium was changed every 3–4 days, at which time the culture dishes were gently shaken to remove the loosely adherent oligodendrocytes and microglia cells from the astrocyte monolayer. These cells were decanted with the medium, leaving a 95% pure culture of astrocytes as assessed by a previously described method, where positive immunolabeling for glial fibrillary acidic protein was used as an indicator of astrocyte presence (Amruthesh et al., 1993).
Cell viability assay.
Cultured astrocytes were exposed to a series of cytotoxic reagents for various time periods and the effects of these chemicals on the cells were determined with the MTT (Sigma) assay, previously described by Denizot and Lang (1986). In the mitochondria of living cells, the yellow MTT dye was reduced to purple formazan, which was then dissolved in glycine buffer containing DMSO, resulting in a colored solution. The absorbance of this solution was quantified by measuring optical density at 570 nm using an enzyme-linked immunosorbent assay reader (Dynatech MR-7000), which was proportional to astrocyte viability.
Determining the physiological concentration of Cu in astrocytes.
The physiological concentration of Cu in the cellular components of cultured astrocytes and in whole cells was analyzed. Cellular fractions (membrane, cytosol, and nuclei) were obtained by treating cells with different lysing buffers, following the protocol from Fernandes and Cotter (1994). Briefly, cultured astrocytes were treated with trypsin (0.3 mg/ml) and washed three times with 15mM 4-(2-hydroxyethyl) piperazine-1-ethesulfonic acid (HEPES) in 0.9% NaCl (wt/vol), pH 7.3. Contamination with external Cu was minimized as previously described (Zhang et al., 1993). The cell suspension was split into two parts, the first part (Suspension 1) being for preparation of the cytosolic and nuclear fractions and the second part (Suspension 2) for preparation of the membrane fraction. Both suspensions were centrifuged at 3000 x g for 5 min, yielding two pellets. The pellet from Suspension 1 was divided into two parts, Pellet A and Pellet B. Pellet A was resuspended in a hypotonic lysis buffer and the solution was held on ice for 15 min, followed by centrifugation at 13,000 x g for 1 min. The supernatant was considered to be the cytosolic fraction. Pellet B was resuspended in a high salt extraction buffer (20mM HEPES, pH 7.9, 420mM NaCl, 1.5mM MgCl2, 0.2mM ethylenediaminetetraacetic acid [EDTA], 25% vol/vol glycerol, 0.5mM phenylmethylsulfonyl fluoride [PMSF], 0.5mM dithiothreitol [DTT], 1 µg/ml aprotinin, 1 µg/ml leupeptin) and placed on ice for 15 min. The suspension was then centrifuged at 13,000 x g for 15 min and that supernatant was considered to be the nuclear fraction. The second pellet from Suspension 2 was lysed by radioimmunoprecipitation (RIPA) solution for 30 min on ice and then centrifuged at 9000 x g for 20 min. The supernatant was further centrifuged at 100,000 x g for 1 h, and the obtained pellet was resuspended in RIPA solution. This solution was considered to be the membrane fraction. An aliquot of cellular fractions was removed for a protein assay using a biocinchoninic acid (BCA) kit, and the remainder of cellular fractions was diluted in a solution of in 0.1N nitric acid. To determine the concentration of Cu in the lysed astrocytes, a model Z-8200 atomic absorption spectrophotometer (Hitachi, Tokyo), a graphite furnace (flameless mode) was used. The Cu standard was a commercially available solution of 1000 ppm (1000 mg/l). It was diluted to 10 ppm by adding nitric acid (0.1N), and the contents of Cu element were measured.
The concentration of Cu in whole astrocytes was determined with the inductive coupled plasma mass spectrometry (ICP-MS) instrument (Friel et al., 1990). An aliquot of collected cells was removed for a protein assay, and then the residue was resuspended in 0.1% Triton x-100/0.2% nitric acid for analysis. The commercial preparations for trace metals (National Research Council Canada) were used as standard. Samples and the standard were placed into a Teflon cup of a high-pressure microwave acid-digestion bomb (Parr Instrument Co., Moline, IL), microwaved twice, and the contents were analyzed for metal elements by the ICP-MS instrument (Elan 250, Scuex, Thornhill, Ontario, Canada). The data were processed by Lotus 123 software (Lotus, Cambridge, MA).
Flow cytometry.
Cultured astrocytes were treated with trypsin to loosen the adherent cells. Cells were then washed with ice-cold phosphate buffered saline (PBS) and fixed in 70% ethanol at –20°C for at least 1 h. The fixed cells were washed twice with PBS and incubated at 37 ± 0.5°C for 30 min with 1 mg/ml of RNase-A dissolved in 0.5 ml of 0.5% Triton X-100/PBS solution. Following the incubation, cells were stained with 0.5 ml of 50 µg/ml propidium iodide (PI) for 10 min, during which time the PI bound to the intracellular DNA. Upon excitation of the fluorescent dye by a FACScan flow cytometer (Becton Dickinson), the PI–DNA complex emitted a fluorescent signal that could be quantified.
Morphological features of astrocytes.
The morphology of cultured astrocytes was examined. After incubation with the applied reagents, astrocytes were fixed in 4% paraformaldehyde. Photomicrographs were obtained with a 40x objective lens on a cooled CCD camera (OlymPix 50 2500) adapted to a Zeiss Axiovert 135-TV microscope.
Hoechst staining of apoptotic bodies.
After exposure to the cytotoxic agents, cultured astrocytes were washed with PBS and fixed in 4% paraformaldehyde for 30 min at room temperature. The cells were then washed with PBS (twice), incubated in 0.5% Triton X-100 for 10 min, stained with Hoechst dye 33258 (3 µg/ml) for 40 min, and again washed with PBS. This process labeled apoptotic bodies within the cell's nucleus and an Olympus IMT-2 fluorescence microscope (UV excitation and 475 nm emission) was used to detect the green fluorescent signal.
Comet assay.
Apoptosis in astrocytes was quantified by single-cell microgel electrophoresis (comet assay). The comet assay is a method of measuring DNA strand breaks and has been used to quantify apoptotic cells in the past (Olive and Banath, 1995). In comparison with other methods of detecting apoptosis, the comet assay is considered to be more sensitive and able to detect DNA cleavage earlier. The comet assay was performed as described by Singh et al. (1994), but with some modifications. Briefly, cultured astrocytes were treated with the cytotoxic agent (e.g., Cu, NCP/Cu) and embedded in situ in 1% agarose (SeaKem Gold; FMC Bioproducts, Rockland, ME). The embedded cells were then placed in a refrigerated alkaline lysis buffer (2.5M NaCl, 1% Na-lauryl sarcosinate, 100mM EDTA, 10mM Tris base, 1% peroxide, and carbonyl-free Triton X-100) for 1 h, followed by a 15-min incubation in an electrophoresis buffer containing 300mM NaOH, 10mM EDTA, 0.1% hydroxyquinoline, and 0.02% DMSO, pH 10.0. The nuclei were electrophoresed for 18 min at 1 V/cm and stained with ethidium bromide (EtBr). A fluorescence microscope equipped with a rhodamine filter (Olympus Corp., Lake Success, NY) was used to examine the image.
Determination of free radical production.
The production of free radicals post exposure of the cultured astrocytes to the Cu/NCP complex was measured with flow cytometry (Sasada et al., 1996). Cells (1 x 106) were incubated for various lengths of time at 37 ± 0.5°C with the Cu/NCP, in the presence of 30µM of DCFHDA. Upon entering the cells, DCFHDA deesterased and turned into a nonfluorescent polar derivative, 2',7'-dichlorofluorescein. In the presence of H2O2 and other peroxides, this derivative became oxidized, forming a fluorescent compound, 2',7'-dichlorofluorescein, that emitted a fluorescent signal at 525 nm. A flow cytometer (Beckton Dickinson) was employed to measure cellular fluorescence intensity (excitation at 475 nm), which directly reflected the concentration of intracellular peroxides.
Measurement of mitocondrial membrane potential.
Mitochondrial membrane potential was indicated by retention of the dye 3,3'-di-hexyloxacarbocyanine (DiOC6(3)) (Pastorino et al., 1998). After treatment with cytotoxic agents, cells were trypsinized and washed with PBS. Cells (1 x 106 cells in 500 µl of PBS) were loaded with 50nM DiOC6(3) and incubated at 37 ± 0.5°C for 15 min. Fluorescence intensity of the DiOC6(3) dye was determined by FACScan flow cytometry (excitation at 475 nm and emission at 525 nm).
Glutathione determination.
Total concentration of glutathione (GSH) was monitored by a modification of the GSH reductase method (Griffith, 1980). Cells were trypsinized and washed with ice-cold PBS and lysed with RIPA solution. An aliquot was removed and 100% trichloroacetic acid was added to make a final concentration of 5%. The aliquot was centrifuged at 12,000 x g for 10 min and reaction buffer containing 0.15M imidazole (pH 7.4) was added to 50 µl of the supernatant. To the initiate the reaction, 5,5-dithiobis (2-nitrobenzoic acid) was added. Concentration of GSH was determined with a spectrophotometer at 412 nm, respective to the GSH standard.
ATP determination.
The intracellular concentration of ATP was determined by the luciferin–luciferase bioluminescent assay, as described by Tsai et al. (1997). In brief, ATP was extracted from astrocytes using 1 ml of 100mM Tris–EDTA buffer (pH 7.5) at 100°C for 10 min. After centrifugation at 30,000 x g for 20 min at 4°C, the ATP content of 0.3 ml of the supernatant was measured by an LKB 1251 luminometer (LKB-Wallac, Tarku, Finland). The sensitivity of the assay was approximately 1 pmol of ATP. ATP standards were used for calibration. Total protein levels were measured and the results expressed as nmol of ATP/mg of protein.
Assay of cytochrome c release.
Mitochondrial and cytosolic fractions were prepared by resuspending cells in ice-cold buffer A (250mM sucrose, 20mM HEPES, 10mM KCl, 1.5mM MgCl2, 1mM EDTA, 1mM ethylene glycol tetraacetic acid (EGTA), 1mM DTT, 17 µg/ml PMSF, 8 µg/ml aprotinin, and 2 µg/ml leupeptin, pH 7.4). Cells were passed through a 26G x 1/2'' needle 10 times. Unlysed cells and nuclei were pelleted by centrifuging at 750 x g for 10 min. The supernatant was further spun at 100,000 x g for 15 min. This pellet was resuspended in buffer A and the supernatant represented the mitochondrial fraction. This supernatant was then centrifuged at 100,000 x g for 1 h and the supernatant obtained from this step represented the cytosolic fraction. A 50-µg aliquot of protein from each sample was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After transferring to the nitrocellulose membrane, samples were probed with monoclonal anti-cytochrome c antibody coupled with horseradish peroxidase (HRP)–mouse secondary antibody. Immunoreactivity was detected by the enhanced chemiluminescence detection system (NEN; Life Science Products). Immunoblots were quantified by densitometry (Bio-Rad GS-700 Imaging Densitometer equipped with Molecular Analysis software, version 2.1; Bio-Rad Laboratories, Inc., Hercules, CA).
Measurement of MAP kinase activity.
Extracted cells were centrifuged to remove cellular debris, and protein content of the supernatant was measured by the BCA protein assay. JNK1, ERK1, and p38 were immunoprecipitated and kinase activity was measured using an immunokinase complex assay with the substrates GST-c-Jun, MBP, and GST-ATF2, respectively (Kyriakis et al., 1994). Briefly, cell lysates (200 µg of protein) were incubated overnight at 4°C with 10 µg of polyclonal anti-JNK1, anti-ERK1, and anti-p38 antibodies. Cell lysates were then incubated with 20 µl of Sepharose A–conjugated protein A for an additional 1 h. The beads were pelleted and washed three times with cold PBS containing 1% Nonidet P-40 and 2mM sodium orthovanadate, once with cold 100mM Tris–HCl (pH 7.5) buffer containing 0.5M LiCl, and once with cold kinase reaction buffer (12.5mM morpholinepropanesulfonic acid, pH7.5; 12.5mM β-glycerophosphate, 7.5mM MgCl2, 0.5mM EGTA, 0.5mM NaF, and 0.5mM sodium orthovanadate). The kinase reaction was performed in the presence of 1 µCi of [-32 P] ATP, 20µM of ATP, 3.3µM of DTT, 3 µg of the substrate GST-c-Jun-(1–135), MBP, and GST-ATF2 in kinase reaction buffer for 30 min at 30°C and stopped by addition of 10 µl of 5x Laemmli loading buffer. The samples were heated for 5 min at 95°C and analyzed by SDS-PAGE (12% polyacrylamide). Phosphorylated substrates (GST-c-Jun, GST-MBP, and GST-ATF2) were visualized by autoradiography. The optical density of autoradiograms was determined with the NIH Image program. The kinase activity was expressed as a fold of the control.
Caspase activity.
Cells were harvested and treated with the cytotoxic reagents. They were then washed with PBS and lysed in a solution containing 25mM HEPES (pH 7.5), 5mM MgCl2, 5mM EDTA, 5mM DTT, 2mM PMSF, 10 µg/ml pepstatin A, and 10 µg/ml leupeptin. The Promega CaspACE kit (Fluorometric Assay System; Madison, WI) was used to measure activity of caspases-1 and -3. Cells were lysed and centrifuged at 12,000 x g for 5 min. Cell lysates containing 50 µg of protein were incubated with either 50µM of Ac-DEVD-AMC (the substrate of caspase-3) or 50µM of Ac-YVAD-AMC (the substrate of caspase-1) at 30°C for 1 h. To measure caspase activity, levels of cleaved substrate were monitored using a spectrofluorometer (Hitachi F-4500) with excitation at 360 nm and emission at 460 nm. Caspase activity was expressed as a fold of the control.
Western blotting.
Equal amounts of lysate protein (50 µg/lane) were subjected to SDS-PAGE with 10% polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes. Nitrocellulose blots were first blocked with 3% bovine serum albumin (BSA) in PBST buffer (PBS with 0.01% Tween 20, pH7.4), and incubated overnight at 4°C with primary antibodies (JNK1, ERK1, p38, PARP, Bcl-2) in PBST containing 1% BSA. Immunoreactivity was detected by sequential incubation with HRP-conjugated secondary antibodies, and detected by the enhanced chemiluminescence technique.
Statistic analysis of the data.
Data were expressed as mean values ± SEM. Statistical analysis was carried out with a one-way ANOVA followed by Dunnett's test to assess statistical significance (*p < 0.05) between treated and untreated groups in all the experiments. The IC50 value of cytotoxicity was obtained from nonlinear regression analyses. Errors associated with IC50 values were estimated from means of the errors of experimental data.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Cytotoxic Effects of Various Metals either Alone or in Combination with Metal Chelators
The MTT reduction assay was used to evaluate the cytotoxic effects of CuCl2, FeCl2, ZnCl2, Pb(NO3)2, or the metal chelators NCP and BCPS, on cultured cortical astrocytes harvested from neonatal rats. Figure 1 shows that 1µM of Fe and 1µM of Pb were nontoxic, Zn was moderately toxic (> 100µM) and Cu was toxic at 100µM. Only high concentrations of NCP, CuCl2, or ZnCl2 induced cell death. IC50 of NCP, CuCl2 (Fig. 1), and CuCl (Supplementary Fig. 1A) were 600 ± 27µM, 180 ± 12.5µM, and 152 ± 10.3µM, respectively. Cytotoxicity of CuCl2, ZnCl2, FeCl2, and Pb(NO3)2, in the presence of NCP (0.1, 0.3, 1.0µM) was also studied. NCP had no effect on cytotoxicity of FeCl2, only slightly increased the cytotoxic effect of Pb(NO3)2, and moderately increased the cytotoxic effect of Zn. However, this chelator dramatically increased Cu cytotoxicity on the cultured astrocytes (Fig. 2A and Supplementary Fig. 1). The IC50 of CuCl2 was decreased from 180 to 2.5 by 0.1µM of NCP, or to 0.5 by 1µM of NCP, respectively (Fig. 2B). On the other hand, 10µM of CuCl2 decreased the IC50 of NCP from 600 to 0.06µM (Supplementary Fig. 1). Cytotoxicity induced by Cu (300µM), NCP (600µM), and the Cu/NCP (0.1µM/1µM) complex was time dependent, with the maximum toxic affect attained after a 24-h incubation (Fig. 2C). There was a positive correlation between cytotoxicity and an increase in Cu concentration (Fig. 3A).
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A number of substances were tested for their capacity to inhibit cytotoxicity of the Cu/NCP complex. It was found that the nonpermeable Cu chelator, BCPS (300µM), completely protected the astrocytes from cytotoxic affects of Cu/NCP (Fig. 2D). Among the antioxidants tested, NAC (3mM) and Vitamin C (3mM), but not GSH (3mM) and vitamin E (1–20µM), partially inhibited toxicity of 0.03 and 0.1µM of the Cu/NCP complex (Fig. 2D and data not shown). Fetal calf serum (20–25%), BSA (0.3–3%), and catalase (600 units/ml, data not shown) were partially effective in attenuating Cu/NCP cytotoxicity by 55 ± 4.3%, 53 ± 5.4%, and 49 ± 5.6%, respectively. This was in comparison to the vehicle treatment, where CuCl2 (10µM)/NCP (0.1µM) induced 28 ± 2.7% cytotoxicity.
The Cu/NCP Complex Increased the Intracellular Concentration of Cu
Changes within intracellular metal concentrations, caused by various metal compounds either alone or in combination with NCP, were determined. Individually, low (0.3µM) and high (300µM) concentrations of NCP or CuCl2 (10µM) did not significantly alter the intracellular Cu content, but a 3-h incubation with a higher concentration of CuCl2 (300µM) and especially, the Cu/NCP complex, significantly increased the intracellular Cu concentration (Fig. 3A). The extent of increase in Cu concentration was similar in the membrane, cytosolic, and nuclear fractions (Fig. 3B). Furthermore, the concentration of Cu in the DNA fraction increased by sevenfold within 30 min of incubation with the Cu/NCP complex and was maintained at a high level over a 0.5- to 24-h incubation, reaching a maximum 21-fold rise after 1 h (Fig. 3C). In contrast, addition of 300µM of Zn, Pb, or Fe did not increase the intracellular concentration of these metals. The elevation profiles of Cu, Pb, and Fe were closely correlated with cytotoxicity, as detected by the MTT reduction test. BCPS abolished both the elevation of Cu content and cytotoxicity (Figs. 2D and 3). These findings suggest that the Cu/NCP complex entered the cells and dramatically increased the intracellular Cu concentration, distributing the metal to various subcellular fractions, thus inducing cytotoxicity.
The Cu/NCP Complex Induced Hypodiploidy in Astrocytes
Subdiploid quantities of DNA were measured by staining with PI and analyzed by flow cytometry. Figures 4A and 4B show that NCP (0.3µM)/CuCl2 (10µM) induced DNA breakage (hypodiploid cells) in a time- and concentration-dependent manner. In contrast, neither 0.3µM of NCP nor 10µM of Cu induced hypodiploidy, even after a 48-h incubation (Fig. 4B). This subdiploid effect was successfully blocked by BCPS and was well correlated with cytotoxicity, as detected by the MTT assay illustrated in Figure 1.
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The Cu/NCP Complex Induced Apoptosis of Cultured Astrocytes
Mature astrocytes (14DIV) were tightly attached to the culture plate and formed a confluent layer of flat cells (Fig. 5A). However, after treatment with the Cu/NCP complex, astrocyte morphology dramatically changed. Cells began to dissociate from the culture plate within 1 h after treatment, and continued to progressively detach as cell shrinkage, nuclear condensation, and apoptotic body formation occurred. Extensive cell death, some by necrosis, took place from 6 to 24 h after incubation (Figs. 5A, 5C, and Supplemental Fig. 2). Nuclear staining with the DNA binding fluorochrome Hoechst 33258 revealed that Cu/NCP, but not NCP (0.3µM) or Cu (10µM) alone, induced apoptotic morphology, which included condensation and fragmentation of the nuclei (Figs. 5B, 5D, and Supplementary Fig. 2). The nuclear changes began as early as 3 h after treatment with NCP (0.3µM)/Cu (10µM) and became more obvious 6 h after treatment (Supplementary Fig. 2).
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Apoptosis of the astrocytes could be quantified by the comet assay. The attached and still viable cells were embedded, lysed, and subjected to electrophoresis. Fragments of DNA from apoptotic cells migrated in the agarose away from the nucleus, forming an image that resembled a comet tail. Cells treated for 6 h with Cu/NCP or a high concentration of CuCl2 (300µM) produced a distinct comet-like pattern, characterized by a bulging tail of fragmented DNA disconnected from the remnant nuclei of apoptotic cells (Fig. 5F, and Supplementary Fig. 3). A 15-min preincubation with BCPS (300µM) abolished the morphological changes and presence of the comet-like tail (Supplementary Figs. 2 and 3). These results suggest that Cu/NCP and a high concentration of CuCl2 triggered an apoptotic pathway, and this was the mechanism of cell death.
Cu/NCP Increased the Production of Reactive Oxygen Species, Decreased the Concentration of GSH, and Reduced the Mitochondrial Transmembrane Potential
Production of reactive oxygen species (ROS) and depletion of GSH are well known to be closely related to cell death in various biological systems. As shown in Figure 6A, Cu/NCP increased ROS production in a concentration- and time-dependent manner with a rapid onset at 1 h and reaching a peak at 6 h after application of Cu/NCP. It was also found that NCP (0.3µM)/Cu (10µM) rapidly decreased the concentration of GSH to 65 ± 6.1% within 1 h and to 25 ± 3.2% at 24 h, in comparison with the control (Fig. 6C). Furthermore, Cu/NCP also decreased the ATP content in a time-dependent manner with a slight decrease at 1 h and a decline to 32 ± 3.1% after 3 h, in comparison with the control (Fig. 6C). Because production of free radicals, as well as depletion of GSH and ATP, occurred prior to the morphological changes and signs of cytotoxicity, it is conceivable that the mechanism of Cu/NCP toxicity is mediated by oxidative stress.
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There is increasing evidence that a reduction in mitochondrial transmembrane potential leads to altered mitochondrial function and that mitochondrial malfunction may be linked to apoptosis. We monitored the effects of Cu/NCP on mitochondrial transmembrane potential (
m) using the fluorescent probe DiOC6 coupled with flow cytometric analysis. DiOC6 is a positively charged fluorescent dye that localizes to the mitochondria. The greater the concentration of dye sequestered in the mitochondrial matrix, the higher the m. A decrease in accumulation of DiOC6 within the mitochondria is reflective of a decrease in mitochondrial permeability transition potential, which refers to regulated opening of a large, nonspecific pore in the inner mitochondrial membrane (Pastorino et al., 1998). The MPT refers to the regulated opening of a large, nonspecific pore in the inner mitochondrial membrane. Treatment of astrocytes with Cu/NCP induced a gradual but significant decline in mitochondrial transmembrane potential (Fig. 6B). The onset of this decline was dependent on concentration of NCP (0.03–0.3µM, Fig. 6B). BCPS abolished this effect of the Cu/NCP complex as well.
Cu/NCP Stimulated Cytochrome c Release in a Time-Dependent Manner
The process of cell death may involve release of cytochrome c from the mitochondria, subsequently leading to apoptosis by activation of various caspases. Cu/NCP caused cytochrome c to be released into the cytosol of the astrocytes in a time-dependent fashion (Fig. 7). As early as 1 h after the decrease of mitochondrial membrane potential, cytochrome c began to gradually accumulate in the cytosol, and this continued for about 6 h. Again, this process could be blocked by BCPS (Fig. 7).
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Differential Activation of the JNK1, ERK1, and P38 Kinase Activity Depends on Concentration of Cu/NCP and Exposure Time
The JNK pathway has been implicated as the signaling pathway of cell death in response to several types of stress stimuli (Chen et al., 1996
; Kyriakis et al., 1994; Verheij et al., 1996). To test the hypothesis that MAP kinase activity may be involved in cell death induced by Cu/NCP, we examined whether Cu/NCP or NCP alone (600µM) initiated the JNK pathway. This was done by detecting phosphorylation of the substrate c-Jun and examining expression of the JNK1 protein. Kinase activity was markedly increased (by about 10-fold) after 1 h incubation with Cu/NCP and reached a plateau at 6 h (an increase of about 30-fold), which was sustained for 24 h (Fig. 8A, data of 24 h is not shown). However, expression of the JNK1 protein remained unaltered. BCPS successfully prevented activation of the JNK pathway (Fig. 8A).
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In contrast, Cu/NCP or NCP (600µM) alone, just transiently increased activation of the ERK pathway, as detected by phosphorylation of its substrate, MBP. There was approximately a twofold increase in kinase activity after 3 h of incubation, and after 6 h, this activity gradually decreased and eventually returned to basal levels at 24 h (Fig. 8B). BCPS blocked activation of this pathway as well. As with JNK1, expression of the ERK1 protein did not significantly change.
On the other hand, p38 kinase was not activated during the early incubation stages (1–3 h) but a late activation of p38 appeared at 6–12 h after addition of either Cu/NCP or NCP (600 µM). Unlike JNK1 and ERK1, degradation of the p38 protein occurred at 24 h (Fig. 8C).
Cu/NCP Stimulated Activation of Caspase-3 and Degradation of PARP in a Time-Dependent Manner
Caspases are believed to play a central role in mediating various apoptotic responses. In order to detect enzymatic activity of caspase-3 during induction of cellular death by Cu/NCP, we used the fluorogenic peptide Ac-DEVD-AMC as a specific substrate for caspase-3. Caspase activity was monitored following treatment of astrocytes with Cu/NCP for various time intervals. As shown in Figure 9A, Cu/NCP, NCP (600µM), and CuCl2 (300µM) caused an increase in enzymatic activity of caspase-3, but not caspase-1. This activity initiated 6 h after the start of incubation, reached a maximum at 12 h, and persisted to 36 h after the initial exposure. BCPS abolished activation of caspase-3 (Fig. 9A). The caspase-3 inhibitor (Z-DEVD-FMK) could partially reduce the cytotoxic effect of Cu/NCP complex (data not showed).
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The nuclear enzyme PARP is critical in many cellular systems undergoing apoptosis, and is one of the targets for ICE/CED-3 protease, cleaving the endogenous 116-kDa PARP protein into an 85-kDa fragment. Exposure of the astrocytes to Cu/NCP caused degradation of the 116-kDa PARP protein into the 85-kDa fragment in a time-dependent manner and BCPS effectively inhibited this cleavage (Fig. 9B). As a negative control, Bcl-2 protein was detected using the Bcl-2 antibody and no significant changes were found after treatment with the Cu/NCP complex (data not shown).
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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In this study, we demonstrated a dramatic potentiating effect of NCP on Cu cytotoxicity in primary cultures of astrocytes and attempted to elucidate the mechanisms by which this occurs. NCP is a metal chelator and a potential inhibitor of oxidative stress in biological systems. Results indicated that the Cu/NCP complex initially caused elevation in intracellular Cu concentration, followed by generation of free radicals, depletion of GSH, and reduction of mitochondrial membrane potential. Subsequently, cytochrome c was released, the JNK pathway and caspase-3 were activated, and finally, degradation of PARP occurred. These time-dependent sequential events eventually lead to apoptosis of the cultured astrocytes.
The Cu/NCP complex was shown to be a potent cytotoxin for the L1210 lymphoma cell line (Mohindru et al., 1983) and also as an inhibitor of transcription in both prokaryotic and eukaryotic cells (Perrin et al., 1994). In M. gallisepticum, Cu/NCP inhibited growth rate and prevented oxidation of nicotinamide adenosine dinucleotide. It was also reported to decrease accessible sulfhydryl groups as an ultimate consequence of Cu, rather than NCP, toxicity (Smit et al., 1981). Byrnes et al. (1992) reported that inhibition of growth and induction of single strand DNA breaks in Ehrlich tumor cells was caused by the Cu/NCP complex through generation of hydroxyl radicals due to removal of Cu (I) from the Cu/NCP complex and its reaction with H2O2. Furthermore, this study demonstrated that the Cu/NCP complex increased membrane permeability, as assayed by the tryptan blue exclusion test, and increased oxidative stress, as detected by Electron Spin Resonance spectroscopy in Ehrlich tumor cells. Almeida et al. (1999) showed that NCP and H2O2 caused lethal synergistic effects in Escherichia coli. However, the signaling pathway by which the Cu/NCP complex induces cell death has not been previously elucidated.
Upon comparison of the cytotoxic potential of various metal compounds in combination with NCP, it was found that Cu was unique in its ability to induce cytotoxicity when complexed with NCP. NCP did not have the same potentiating effect on any of the other metals tested (Fe, Zn, Pb). These results are closely correlated with a marked increase of Cu uptake by the astrocytes exposed to Cu/NCP, whereas combining the other metals with NCP did not have the same effect (Fig. 3A). Interestingly, Byrnes et al. (1992) reported that Cu increased the uptake of NCP in tumor cells. Thus, NCP and Cu may work synergistically with each other, potentially explaining the cytotoxic effects of the Cu/NCP complex observed in this study. Low concentration of NCP (0.3µM) showed a prominent increase in Cu transport, which was accompanied with cytotoxicity. Therefore, it was considered that NCP behaved as a more selective and efficient Cu chelator in our study (Figs. 2A and 2B). This contention is in agreement with findings from other studies, where chelating agents such as pyrrolidine dithiocarbamate (Chen et al., 2000) and disulfiram (Chen et al., 2001) were examined on cortical astrocytes.
Cu is an essential metal in animals. Mice serum normally contains 0.3–0.6 ppm (Massie et al., 1993) and human serum contains 1 ppm (16µM) (Graham et al., 1991). We examined the potentiating effect of NCP on Cu at the physiological concentration (1–10µM) (Fig. 2 and Supplemental Fig. 1) and found that NCP (0.03–10µM), combined with a fixed concentration of Cu (1–10µM = 0.06–0.6 ppm), exerted severe cytotoxic effects on the cultured astrocytes. These were dependent on the concentration of the toxic agent and on incubation time. Application of cyclohexamide or actinomycin D could not block cytotoxicity induced by the Cu/NCP complex or by NCP alone. It appeared that RNA and protein synthesis was not required for this toxic mechanism (data not shown). The appearance of hypodiploidy in cells, nuclear condensation, development of apoptotic bodies, and the comet-like tail of fragmented DNA suggested that cell death induced by Cu/NCP was at least in part mediated by apoptotic processes.
Studies of radiolabeled 67Cu, [14C]/NCP, and [3H]/NCP (Smit et al., 1981) showed that NCP is a cell permeable Cu-specific chelator, which can form complexes with Cu2+ and serve as a vehicle for transporting Cu from a nontoxic Cu-medium to complex to Cu/NCP and might dissociate within the L1210 cells. The mechanism by which the Cu/NCP complex induced cytotoxicity of cultured astrocytes may be as follows. NCP formed a complex with Cu via the thiol groups on the metal chelator, and upon entering the cells, the Cu/NCP complex either functioned in its bound form or dissociated to NCP and free Cu. Then ROS, produced by either the Cu/NCP complex or the free Cu ions, attacked the mitochondrial membrane, decreasing the m and oxidizing GSH, thus leading to a decline in GSH and ATP concentration (Fig. 6).
Mitochondrial dysfunction has been demonstrated to play a critical role in inducing apoptosis (Rego and Oliveira, 2003) and depolarization of the mitochondrial membrane as a result of opening of the permeability transition pores is an early, irreversible event during apoptosis (Parrado et al., 2004). The decrease of GSH in this study was similar to the finding in M. gallisepticum, where this phenomenon was claimed to be an ultimate consequence of Cu rather than NCP toxicity (Smit et al., 1981). However, because glia cells synthesize GSH, we could not exclude the possibility that the decrease in GSH concentration was reflective of reduced synthesis, for example as cells were in the process of dying. The decrease of m and an increase of ROS generation caused by Cu/NCP in this study were consistent with similar findings from other studies, where H2O2/Cu, menadione/Cu, and NCP/menadione combinations had similar effects (Gyulkhandanyan et al., 2003). Because BCPS abolished both the decrease of m and cytotoxic effects of cultured astrocytes induced by Cu/NCP, it is proposed that Cu/NCP induced the apoptotic process by increasing the intracellular concentration of Cu, which decreased m, and generated ROS. Furthermore, it is not surprising that serum or BSA partially reduced Cu or Cu/NCP toxicity as albumin can bind to Cu and then decrease cellular uptake of the metal.
The MAP kinase family, which includes JNK, ERK, and p38 kinase, can be rapidly activated by various stress stimuli (Javelaud and Mauviel, 2005). Recent reports suggest that the JNK/SAPK pathway may play an important role in triggering apoptosis in response to inflammatory cytokines (Chen et al., 1996), free radicals generated by UV-C, gamma radiation (Alder et al., 1995), or direct application of H2O2 (Yu et al., 1996), alkylating agents such as N-nitrosoguanidine (Derijard et al., 1994), and DNA-damaging agents like cisplatin (Potapova et al., 1997). Additionally, lipopolysaccharides can activate the ERK and p38 kinases in astrocytes (Schumann et al., 1998). Caspase-3 may be the downstream signaling pathway of activated JNK (Kim et al., 2005) and some ICE/CED-3 protease targets have been identified, including the nuclear enzyme PARP, which could be activated by staurosporine, leading to apoptosis in astrocytes (Keane et al., 1997). The results from this paper showed that Cu/NCP differentially activated JNK, ERK, and p38 kinase with a more profound and sustained activation of JNK (Fig. 8), followed by caspase-3 activation, then degradation of PARP (Fig. 9) and eventually apoptosis. The downstream of JNK activation signaling could be activation of Bid which translocated to the mitochondria and induced cytochrome c release and induced apoptosis (Luo et al., 1998; Tournier et al., 2000), or directly activated caspase-3, or was mediated by AP1 and caspase-8 activation (Lauricella et al., 2006). Activation of the JNK, ERK and p38 pathways by Cu/NCP was Cu dependent, because chelation of extracellular Cu was prevented by BCPS. MAP kinase activation and apoptosis of cultured astrocytes confirmed the essential role in cell death signaling of the earlier rise in intracellular Cu concentration, induced by the Cu/NCP complex. Because the GSH precursor NAC could partially block the cytotoxic activity of Cu/NCP and inhibit JNK kinase activation (data not shown), it is reasonable to infer that signaling by free radicals is essential to initiation of the JNK signaling cascade as suggested by Xia et al. (1995).
There is increasing evidence that deterioration of astrocytes plays an important role in aging and neurodegenerative disease. Human studies and mouse models of neurodegenerative diseases such as Alzheimer's disease (Forman et al., 2005), Parkinson's disease (Saura et al., 2003), amyotrophic lateral sclerosis (Guo et al., 2003), and Huntington's disease, in which astrocyte-specific proteins and pathways have been manipulated, have revealed astrocyte-specific pathologies that contribute to neurodegeneration. Our finding implies that the NCP/Cu complex may induce oxidative neurotoxicity of the brain through damaging the neuroprotective astrocytes.
In conclusion, our results from this study indicate that a very low concentration of (0.03–10µM) NCP facilitates the uptake of Cu at the physiological concentration (1–10µM) to exert profound pro-oxidant toxic effects on rat cortical astrocytes. Based on the time course of sequential events and on the fact that BCPS and antioxidants can block apoptotic signaling, it is proposed that death of cultured astrocytes, as induced by the Cu/NCP complex, is due to an initial elevation of intracellular Cu concentration, which triggers an increase in free radical production, a decrease in m mitochondrial membrane potential, and depletion of GSH and ATP. These events are followed by downstream activation of JNK and caspase-3, and finally PARP degradation. Clinical implications of this study are that the potentially clinically useful drug NCP can behave like a toxic pro-oxidant which may indirectly produce neurotoxicity by damaging protective astrocytes, especially in patients with Wilson's disease.
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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National Science Council (NSC 95-2320-B-002-102).
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