TOXICOLOGICAL SCIENCES 102(1), 138–149 (2008)

doi:10.1093/toxsci/kfm292

Advance Access publication December 5, 2007

Apoptosis of Cultured Astrocytes Induced by the Copper andNeocuproine Complex through Oxidative Stress and JNK Activation

Sung-Ho Chen,* Jen-Kun Lin,† Shing-Hwa Liu,* Yu-Chih Liang,† and Shoei-Yn Lin-Shiau*,‡,1

*Institutes of Toxicology; †Biochemistry; and ‡Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan

Received September 8, 2007; accepted November 29, 2007

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 600mM, respectively. However, NCP at a nontoxic

concentration of 10mM markedly reduced EC50 of Cu to 0.35mM(physiological concentration) and Cu (10mM) reduced EC50 of

NCP down to 0.06mM. The mechanisms underlying these dra-

matic 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 adeno-

sine 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 di-

sulfiram) 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; oxidativestress; JNK; caspase-3.

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 neuro-

degenerative diseases (Forman et al., 2005). In this study, we

have demonstrated that apoptosis of primary cultured astro-

cytes 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 endog-

enous 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.,

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: þ886-2-23915297. E-mail:

syl@ha.mc.ntu.edu.tw.

� The Author 2007. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.For Permissions, please email: journals.permissions@oxfordjournals.org

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2005). A number of ICE/CED-3 protease targets have been

identified, including the nuclear enzyme poly-ADP-ribose poly-

merase (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.

MATERIALS AND METHODS

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), Hoechst 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). [c32P] 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 3 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 3

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 lg/ml aprotinin, 1 lg/ml leupeptin) and placed

on ice for 15 min. The suspension was then centrifuged at 13,000 3 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 3 g for 20 min. The

supernatant was further centrifuged at 100,000 3 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 Instru-

ment 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 lg/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 403 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% para-

formaldehyde 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 lg/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

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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 3 106) were incubated for

various lengths of time at 37 ± 0.5�C with the Cu/NCP, in the presence of

30lM of DCFHDA. Upon entering the cells, DCFHDA deesterased and turned

into a nonfluorescent polar derivative, 2#,7#-dichlorofluorescein. In the pres-

ence 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 mem-

brane potential was indicated by retention of the dye 3,3#-di-hexyloxacarbo-

cyanine (DiOC6(3)) (Pastorino et al., 1998). After treatment with cytotoxic

agents, cells were trypsinized and washed with PBS. Cells (1 3 106 cells in 500

ll 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 3 g

for 10 min and reaction buffer containing 0.15M imidazole (pH 7.4) was added

to 50 ll 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 de-

termined 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 3

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 lg/ml PMSF, 8 lg/ml aprotinin, and 2

lg/ml leupeptin, pH 7.4). Cells were passed through a 26G 3 1/2$ needle 10

times. Unlysed cells and nuclei were pelleted by centrifuging at 750 3 g for 10

min. The supernatant was further spun at 100,0003 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 3 g for 1 h and the

supernatant obtained from this step represented the cytosolic fraction. A 50-lg

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-cyto-

chrome 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 lg of protein) were incubated overnight at 4�Cwith 10 lg of polyclonal anti-JNK1, anti-ERK1, and anti-p38 antibodies. Cell

lysates were then incubated with 20 ll 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 b-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 lCi of [c-32 P] ATP, 20lM of ATP, 3.3lM of DTT, 3 lg 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 ll of 53 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 auto-

radiograms 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 lg/ml pepstatin A, and 10 lg/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 3 g for 5 min.

Cell lysates containing 50 lg of protein were incubated with either 50lM of

Ac-DEVD-AMC (the substrate of caspase-3) or 50lM 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 lg/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.

RESULTS

Cytotoxic Effects of Various Metals either Alone or inCombination with Metal Chelators

The MTT reduction assay was used to evaluate the cytotoxic

effects of CuCl2, FeCl2, ZnCl2, Pb(NO3)2, or the metal che-

lators NCP and BCPS, on cultured cortical astrocytes harvested

from neonatal rats. Figure 1 shows that 1lM of Fe and 1lM of

Pb were nontoxic, Zn was moderately toxic (> 100lM) and Cu

was toxic at 100lM. Only high concentrations of NCP, CuCl2,

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or ZnCl2 induced cell death. IC50 of NCP, CuCl2 (Fig. 1), and

CuCl (Supplementary Fig. 1A) were 600 ± 27lM, 180 ±12.5lM, and 152 ± 10.3lM, respectively. Cytotoxicity of

CuCl2, ZnCl2, FeCl2, and Pb(NO3)2, in the presence of NCP

(0.1, 0.3, 1.0lM) was also studied. NCP had no effect on cyto-

toxicity 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 0.1lM of

NCP, or to 0.5 by 1lM of NCP, respectively (Fig. 2B). On the

other hand, 10lM of CuCl2 decreased the IC50 of NCP from

600 to 0.06lM (Supplementary Fig. 1). Cytotoxicity induced

by Cu (300lM), NCP (600lM), and the Cu/NCP (0.1lM/

1lM) 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).

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 (300lM), 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 (1–20lM),

partially inhibited toxicity of 0.03 and 0.1lM 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 cyto-

toxicity by 55 ± 4.3%, 53 ± 5.4%, and 49 ± 5.6%, respectively.

This was in comparison to the vehicle treatment, where CuCl2(10lM)/NCP (0.1lM) induced 28 ± 2.7% cytotoxicity.

The Cu/NCP Complex Increased the IntracellularConcentration of Cu

Changes within intracellular metal concentrations, caused by

various metal compounds either alone or in combination with

NCP, were determined. Individually, low (0.3lM) and high

(300lM) concentrations of NCP or CuCl2 (10lM) did not

significantly alter the intracellular Cu content, but a 3-h

incubation with a higher concentration of CuCl2 (300lM) 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 300lM

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 con-

tent 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 (0.3lM)/CuCl2 (10lM) induced DNA

breakage (hypodiploid cells) in a time- and concentration-

dependent manner. In contrast, neither 0.3lM of NCP nor

10lM 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.

The Cu/NCP Complex Induced Apoptosis of CulturedAstrocytes

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. Exten-

sive cell death, some by necrosis, took place from 6 to 24 h

after incubation (Figs. 5A, 5C, and Supplemental Fig. 2).

FIG. 1. Cytotoxic effects of various metal compounds, neocuproine, and

BCPS on cultured rat cortical astrocytes. The viability of astrocytes was

determined by MTT test after continuous incubation with CuCl2, FeCl2, ZnCl2,

Pb(NO3)2, or NCP, BCPS at 37 ± 0.5�C for 24 h. Data are presented as mean ±

SE (n ¼ 3–6). The vehicle control treatment was defined as 100%.

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Nuclear staining with the DNA binding fluorochrome Hoechst

33258 revealed that Cu/NCP, but not NCP (0.3lM) or Cu

(10lM) 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 (0.3lM)/Cu (10lM) and became

more obvious 6 h after treatment (Supplementary Fig. 2).

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 (300lM)

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 (300lM) 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 OxygenSpecies, Decreased the Concentration of GSH, andReduced 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

FIG. 2. Selective enhancement by NCP and its prevention by BCPS or by antioxidants of the cytotoxic effects of CuCl2 on cultured rat cortical astrocytes.

Cytotoxicity (% of control vehicle) on astrocytes was determined by MTT test after incubation at 37 ± 0.5�C for 24 h or for indicated times. Data are presented as

mean ± SE (n ¼ 3). (A) Concentration-dependent cytotoxicity of FeCl3, or ZnCl2, or Pb(NO3)2, in combination with NCP at 0.3lM. (B) Concentration-dependent

cytotoxicity of CuCl2 either alone, or in combination with NCP at 0.1, 0.3, and 1lM, respectively. *p < 0.05, **p < 0.01, ***p < 0.001 as compared with that

treated with CuCl2 alone. (C) Time course of cytotoxicity induced by 300lM CuCl2 alone, 600lM NCP alone, 0.1lM NCP þ 10lM CuCl2, or 1lM NCP þ 10lM

CuCl2. *p < 0.05, **p < 0.01, ***p < 0.001 as compared with 10lM CuCl2 alone. (D) Prevention by 300lM BCPS, 3mM vitamin C, or 3mM NAC of

cytotoxicity induced by NCP þ 10lM CuCl2. *p < 0.05, **p < 0.01, ***p < 0.001 as compared that treated with NCP plus 10lM CuCl2 without inhibitors.

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NCP (0.3lM)/Cu (10lM) 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

FIG. 3. Increases of cellular metal contents of cultured rat cortical

astrocytes after treatment with Cu/NCP complex. Astrocytes were treated with

NCP and CuCl2 either alone or in combination at 37 ± 0.5�C for 3 h, and then

the cellular metal contents were assayed by inductively coupled plasma mass

spectrometry (ICP-mass) (A). Cu contents of the subcellular fractions were

assayed by atomic absorption spectrophotometer (B and C). Data (mean ± SE)

are expressed as fold of control. The mean contents (ng/mg protein) of Cu, Fe,

Zn, and Pb of control cells are 14.36 ± 0.7, 251.32 ± 44.21, 126.05 ± 27.1, and

13.6 ± 1.9, respectively. The control Cu contents (ng/mg protein) of membrane

fraction, cytosol, nuclear fractions, and DNA are 2.17 ± 0.35, 9.8 ± 1.8, 1.25 ±

0.25, and 1.9 ± 0.4, respectively. Data are presented as mean ± SE (n ¼ 3). *p

< 0.05, **p < 0.01, as compared with the respective control.

FIG. 4. Time course of hypodiploidy cell formation induced by Cu/NCP

complex and its prevention by BCPS, NAC, and vitamin C in cultured rat

cortical astrocytes. Cells were treated with Cu/NCP for various times without

(A) or with 300lM BCPS, 3mM NAC, or 3mM vitamin C (B) for 24 h, and

then analyzed hypodiploidy cells by altered fluorescence intensity (sub G1

fraction as indicated by M1) after staining with PI coupled with flow cytometry.

Data are presented as mean ± SE (n ¼ 3). *p < 0.05, **p < 0.01, ***p < 0.001

as compared with those treated with respective Cu (10lM)/NCP. The vehicle

control treatment was defined as 100%.

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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.

There is increasing evidence that a reduction in mitochon-

drial transmembrane potential leads to altered mitochondrial

function and that mitochondrial malfunction may be linked to

apoptosis. We monitored the effects of Cu/NCP on mitochon-

drial transmembrane potential (Dwm) 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 Dwm. 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 (0.03–0.3lM, 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).

Differential Activation of the JNK1, ERK1, and P38 KinaseActivity Depends on Concentration of Cu/NCP andExposure 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

FIG. 5. Morphological changes and DNA fragmentation (comet tail) of cultured rat cortical astrocytes induced by Cu/NCP complex. Cells were treated with

vehicle (A, B), or 0.3lM NCP plus 10lM Cu2þ (C, D) for 24 h. Morphological changes were examined under phase contrast microscope (A, C). The apoptotic

bodies produced by respective treatments were examined by fluorescent microscope after fixation with 4% paraformaldehyde for 30 min at room temperature, and

then stained with Hoechst dye 33258 (B, D). Arrows indicate apoptotic nuclei. Cells were treated with either vehicle (E), or 0.1lM NCP plus 10lM Cu for 6 h (F).

The nuclear DNA was stained with EtBr and visualized under fluorescence microscopy. Scale bar ¼ 10 lm. (D#) The higher magnification of the box region of (B).

The results represent one of three independent experiments.

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involved in cell death induced by Cu/NCP, we examined

whether Cu/NCP or NCP alone (600lM) 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 pre-

vented activation of the JNK pathway (Fig. 8A).

In contrast, Cu/NCP or NCP (600lM) alone, just transiently

increased activation of the ERK pathway, as detected by

phosphorylation of its substrate, MBP. There was approxi-

mately 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 lM). Unlike JNK1 and ERK1, degradation of the p38

protein occurred at 24 h (Fig. 8C).

Cu/NCP Stimulated Activation of Caspase-3 andDegradation 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

(600lM), and CuCl2 (300lM) 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).

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

FIG. 6. Cu/NCP induces oxidative stress in the cultured rat cortical

astrocytes. Cu/NCP increases reactive species (A), decreases the mitochondrial

membrane potential (B), and reduces the contents of glutathione and ATP (C)

that was assessed in the cultured astrocytes as described in ‘‘Materials and

Methods.’’ Cells were treated with NCP (0.03, 0.1, 0.3lM) plus CuCl2 (10lM)

without or with BCPS 300lM for various times. Then an aliquot of the cells

was labeled with the fluorescent dye either DCFHDA (A) or DiOC6 (B) for

analyzing the fluorescent intensity by flow cytometry. In (B), the curves a, b,

and c show the treatments with control, BCPS 300lM plus NCP 0.3lM and

CuCl2 10lM, and NCP 0.3lM plus CuCl2 10lM, respectively. The reduced

glutathione and ATP content (C) of cell extracts was determined by

spectrophotometer at 412 nm and luciferin–luciferase bioluminescent assay,

respectively. The vehicle control treatment was defined as 100%. The contents

of reduced glutathione and ATP in the control astrocytes treated with vehicle

are 6.2 ± 0.8 and 5.7 ± 0.6 nmol/mg protein, respectively. Data are presented as

mean ± SE (n ¼ 3). *p < 0.05, **p < 0.01, ***p < 0.001 as compared with the

respective control.

FIG. 7. Effects of Cu/NCP complex on cytochrome c release inhibited by

BCPS in cultured rat cortical astrocytes. Cells were treated with Cu/NCP

complex for various times. Cell extracts were prepared at the indicated time

points. Cu/NCP triggered cytochrome c release from mitochondria into the

cytosol in a time-dependent manner. The results represent one of three

independent experiments.

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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).

DISCUSSION

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, de-

pletion 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 conse-

quence 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

FIG. 8. Differential activation of the JNK1, ERK1, and P38 kinase activity

depends on concentration of Cu/NCP and exposure time. The cell lysates were

prepared and immunoprecipitated with 10 lg of polyclonal anti-JNK1 antibody

(A), anti-ERK 1 antibody (B), or p38 antibody (C), followed by 20 ll of

Sepharose A–conjugated protein A. The kinase reaction was performed by the

procedures as described in ‘‘Materials and Methods.’’ The top panel represents

the autoradiogram of [cc32P] ATP incorporation into exogenous GST-c-Jun (1–

135) (A), GST-MBP (B), or GST-ATF-2 (C), respectively. The amount of cell

lysates used was 200 lg of protein in each lane. The bottom panel is

immunoblot performed with antibody specific to anti-JNK1 antibody (A), anti-

ERK 1 antibody (B), or anti-p38 antibody (C). The fold induction in this figure

is presented as the ratio of JNK activity to JNK1 protein, ERK activity to ERK1

protein, or p38 activity to p38 protein against time, respectively. Data are

presented as mean ± SE (n ¼ 3).

FIG. 9. Effects of Cu/NCP complex on both the caspase activation and

cleavage of PARP blocked by BCPS in cultured rat cortical astrocytes. Cells

were treated with NCP 0.3lM and CuCl2 10lM either alone or in combination

for various times as indicated, and then caspase activities were analyzed as

described in ‘‘Materials and Methods.’’ The vehicle control treatment was

defined as 100% (A). The immunoblots were performed with antibody specific

to PARP (B). The amount of cell lysates used was 50 lg of protein in each lane.

The ratios of PARP fragment 85 KDa to intact PARP 116-KDa protein are also

shown. Data are presented as mean ± SE (n ¼ 3). *p < 0.05 compared with the

respective control.

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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 inEscherichiacoli. 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 (0.3lM)

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 (16lM) (Graham et al., 1991). We examined

the potentiating effect of NCP on Cu at the physiological

concentration (1–10lM) (Fig. 2 and Supplemental Fig. 1) and

found that NCP (0.03–10lM), combined with a fixed concen-

tration of Cu (1–10lM ¼ 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 con-

densation, 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 Cu2þ 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 mem-

brane, decreasing the Dwm 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 Dwm 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 Dwm 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 Dwm, 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

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(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 activa-

tion 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 manipu-

lated, have revealed astrocyte-specific pathologies that con-

tribute 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 (0.03–10lM) NCP facilitates the uptake

of Cu at the physiological concentration (1–10lM) 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 Dwm mitochondrial membrane poten-

tial, 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.

SUPPLEMENTARY DATA

Supplementary data are available online at http://toxsci.

oxfordjournals.org/.

FUNDING

National Science Council (NSC 95-2320-B-002-102).

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DEATH SIGNALING PATHWAY INDUCED BY CU/NCP 149

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