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European Journal of Cell Biology 87 (2008) 389–397

0171-9335/$ - se

doi:10.1016/j.ej

�CorrespondE-mail addr

sungsu@cau.ac

www.elsevier.de/ejcb

Kynurenic acid attenuates MPP+-induced dopaminergic neuronal cell

death via a Bax-mediated mitochondrial pathway

Do Yeon Leea,b, Kyu-Sun Leec, Hyun Jung Leea, Yoo Hun Noha, Do Hee Kima,Jun Young Leed, Soo Hyun Choe, Ok Ja Yoonf, Won Bok Leea, Kyung Yong Kima,Yoon Hee Chunga,�, Sung Su Kima,�

aDepartment of Anatomy and Cell Biology, College of Medicine, Chung-Ang University, 221 Huksuk-dong, Dongjak-ku,

Seoul 156-756, Republic of KoreabDepartment of Herbal Resources Research, Korea Institute of Oriental Medicine, Daejeon, Republic of KoreacCenter for Regenerative Medicine, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon,

Republic of KoreadDepartment of Neuropsychiatry, College of Medicine, Seoul National University, Seoul, Republic of KoreaeDepartment of Family Medicine, College of Medicine, Chung-Ang University, Seoul, Republic of KoreafSchool of Advanced Materials Science and Engineering and Center for Advanced Plasma Surface Technology,

Sungkyunkwan University, Suwon, Republic of Korea

Received 25 October 2007; received in revised form 6 March 2008; accepted 10 March 2008

Abstract

Kynurenic acid (KYNA), a tryptophan metabolite in the kynurenine pathway, is protective against various insults.However, the molecular mechanism of this protective effect has not been identified. In this study, we examined theprotective effects of KYNA against 1-methyl-4-phenylpyridinium (MPP+), the best-characterized toxin inducingpathological changes resembling Parkinson’s disease (PD), using SH-SY5Y and SK-N-SH human neuroblastoma cells.Pre-treatment of KYNA attenuated MPP+-induced neuronal cell death in SH-SY5Y and SK-N-SH cells. MPP+-induced cell death was preceded by increases in Bax expression and mitochondrial dysfunction, such as collapse ofmitochondrial membrane potential (DCm), release of cytochrome c from mitochondria into the cytoplasm, andincreases in caspase-9/-3 activities. KYNA effectively inhibited all of these mitochondrial apoptotic processes. Ourresults indicate that KYNA plays a protective role by down-regulating Bax expression and maintaining mitochondrialfunction in MPP+-induced neuronal cell death, and suggest that KYNA may have therapeutic potential in PD.r 2008 Elsevier GmbH. All rights reserved.

Keywords: Kynurenic acid (KYNA); 1-methyl-4-phenylpyridinium (MPP+); Bax; Mitochondrial dysfunction; Neuronal apoptosis

e front matter r 2008 Elsevier GmbH. All rights reserved.

cb.2008.03.003

ing authors. Tel.: +822820 5690; fax: +82 2826 1265.

esses: yoonhee@cau.ac.kr (Y.H. Chung),

.kr (S.S. Kim).

Introduction

Parkinson’s disease (PD), the second most commonneurodegenerative disease, is characterized by posturalinstability, gait abnormality, bradykinesia, tremor at rest,and rigidity (Eberhardt and Schulz, 2003). Pathologically,

ARTICLE IN PRESSD.Y. Lee et al. / European Journal of Cell Biology 87 (2008) 389–397390

a progressive degeneration of dopaminergic neuronsin the substantia nigra pars compacta results in accelera-tion of dopaminergic neuronal cell loss and a deficiencyof dopamine in the striatum (Nicotra and Parvez,2002). The underlying cellular and molecular mechanismsin dopaminergic neuronal death are still unknown.Determining the cause of the disease, the signalingmechanisms of dopaminergic neuronal degenerationand developing new protective drugs are key scientificchallenges.

The neurotoxin 1-methyl-4-phenylpyridinium (MPP+),a toxic metabolite converted from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine by monoamine oxidase-B inastrocytes (Markey et al., 1984), induces pathologicalchanges resembling PD (Jacquy et al., 1993). Themolecular mechanisms for MPP+-induced neuronal celldeath are still not clearly defined. It is thought thatneuronal apoptosis induced by MPP+ is mediated bydisruption of mitochondrial membrane potential (Kotakeand Ohta, 2003). Mitochondrial accumulation of MPP+

inhibits NADH dehydrogenase (complex I) of the electrontransport chain, thereby causing dopaminergic neurotoxi-city (Ramsay et al., 1991).

Kynurenic acid (KYNA) is one of the majorendogenous metabolites formed in the tryptophanmetabolic pathway, the kynurenine pathway (Wolfand Brown, 1971). It is a potential endogenousneuroprotective agent, found in the pathway fromtryptophan to NAD(H), and its phosphorylated form,NADP(H) (Stone, 2000). KYNA plays a protectiverole in neurodegenerative diseases such as PD,Huntington’s disease, cerebral ischemia, stroke, andepilepsy (Beal et al., 1992; Cozzi et al., 1999; Stone,2001). However, the neuroprotective effects of KYNAagainst MPP+-induced neurotoxicity are not welldefined.

Mitochondria are unique organelles in the consump-tion of oxygen, production of ATP, generation ofoxygen radicals, and mobilization of calcium (Melov,2000). Mitochondrial functions are controlled by theBcl-2 family proteins. Bcl-2 family proteins are sub-divided into two functional groups, an anti-apoptoticgroup, including Bcl-2 and Bcl–xL, and a pro-apoptoticgroup, including Bax and Bak (Gross et al., 1998).Mitochondrial involvement in apoptosis includes twocrucial events: the onset of multiple parameters ofmitochondrial malfunction (Bhagwat et al., 1995),and the release of cytochrome c from the intermem-brane space of the mitochondria into the cytoplasm(Green and Reed, 1998; Wang, 2001). Regulation ofchanges in mitochondrial membrane potential (DCm)and the release of cytochrome c from the mitochondriainto the cytoplasm are important processes duringapoptosis (Kroemer and Reed, 2000). The molecularmechanisms responsible for release of cytochrome c andthe collapse of DCm during apoptosis are unknown.

Moreover, a direct relationship between the release ofcytochrome c and stimulated changes in DCm has notbeen demonstrated.

In the present study, we examine whether KYNA canrescue MPP+-induced cell death in the SH-SY5Y andSK-N-SH human neuroblastoma cell line, and alsoinvestigate the underlying mechanisms.

Materials and methods

Cell culture and pharmacological treatments

Human neuroblastoma SH-SY5Y and SK-N-SH cellswere cultured at 37 1C in minimum essential medium(Gibco-BRL) supplemented with 10% heat-inactivatedfetal bovine serum (FBS) in a humidified 95% air, 5%CO2 incubator. The cells were transferred to low serummedia (1% FBS/MEM) 2 h before the treatment withMPP+. MPP+ and KYNA were obtained from SigmaChemical and dissolved in DMSO. KYNA was added tocells 2 h before MPP+ treatment.

Effect of the Bax antisense oligonucleotides on

neuronal viability

For down-regulating Bax expression, an antisense21-mer phosphothioate oligonucleotide was synthesizedand added to the medium (10 mM final concentration)6 h before addition of MPP+. The sequence of the Baxantisense oligonucleotide (50–TGCTCCCCGGACCC-GTCCATC–30) was complementary to the translationinitiation site of Bax mRNA.

Cell viability assay (alamarBlue test)

MPP+ and KYNA-treated cells were stained with10 ml alamarBlue (Serotec). Cells were incubated for3 h and absorbance was measured at 570 nm with anELISA reader (Molecular Devices). The backgroundabsorbance was measured at 600 nm and subtracted.The cell viability was defined as ((test sample count)�(blank count)/(untreated control count)�(blank count))�100.

Hoechst 33258 staining

Cells were fixed with 4% formaldehyde (freshlyprepared from paraformaldehyde) and then stainedwith 8mg/ml of Hoechst dye 33258 (Sigma) for 5min.Nuclear morphology was visualized using a fluorescencemicroscope (IX70 microscope, Olympus). Dead cellsand apoptotic bodies were determined by condensed orfragmented nuclei.

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Detection of Bax/Bcl-2 expression and cytochrome crelease from mitochondria

For measuring the expression level of Bax/Bcl-2protein and the release of cytochrome c, immunoblot-ting was performed with rabbit anti-Bax or anti-Bcl-2antibodies (1:1000 dilution) or goat anti-cytochrome c

antibody (1:1000 dilution) (Santa Cruz Biotechology).Anti-b-actin antibody (SantaCruz Biotechology; 1:1000dilution) was used as loading control. Protein prepara-tion and immunoblotting were performed as described(Lee do et al., 2006).

Analysis of mitochondrial membrane

potential (DWm)

The changes in DCm were estimated using tetramethylr-hodamine ethyl ester (TMRE) (Molecular Probes, Eugene,OR, USA). Carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP) was used as a positive controlfor complete mitochondrial depolarization.

Caspase substrate cleavage assay

Caspase-3 and caspase-9 activities were measured asdescribed (Lee do et al., 2006). Cell extracts (20mg protein)were incubated with 0.5mM Ac-LEHD-AMC (caspase-9assay) or 0.5mM Ac-DEVD-AMC (caspase-3 assay) in afinal volume of 100ml at 37 1C for 30min. Assays were readby fluorometry (excitation at 380nm, emission at 460nm).Enzymatic activity is expressed as arbitrary units.

Statistical analysis

All data were expressed as mean7SD. Statisticalsignificance was determined via ANOVA followed byScheffe’s post-hoc tests. A p value of o0.05 wasconsidered to be significant.

Results

KYNA potently attenuates MPP+-induced neuronal

apoptosis

In this study, the neuroprotective effect of KYNA onMPP+-induced dopaminergic neuronal cell death wasevaluated using the alamarBlue assay. As shown inFig. 1A, MPP+-induced neuronal cell death in a time-and dose-dependent manner. This MPP+-induced neuro-nal cell death was significantly attenuated by pre-treatmentwith KYNA (Fig. 1B). To examine the cell death patternin detail, we also examined morphological changes.MPP+ caused apoptotic features, including retraction ofneurites, cell shrinkage, and membrane blebbing, which

were markedly blocked by pre-treatment with KYNA(Fig. 1C). These findings indicate that KYNA attenuatesMPP+-induced neuronal apoptotic cell death.

KYNA down-regulates the level of Bax protein

induced by MPP+

We examined whether Bcl-2 family proteins, pro-apoptotic Bax and anti-apoptotic Bcl-2, were involved inMPP+-induced neuronal apoptosis, and whether KYNAcould block multiple events associated with this signaltransduction pathway. Bax protein levels significantlyincreased at 3h after treatment with MPP+, and enhance-ment was sustained even 12h after treatment (Fig. 2A,upper panel), whereas anti-apoptotic Bcl-2 protein levelsdid not markedly change until 12h after MPP+ treatment(Fig. 2A, lower panel). KYNA significantly inhibitedMPP+-induced up-regulation of Bax protein (Fig. 3A,upper panel), but had little effect on the expression of Bcl-2protein (Fig. 3A, middle panel). To determine whetherMPP+-induced apoptosis was mediated by Bax, weblocked the expression of Bax protein by pre-incubatingSH-SY5Y cells with a Bax-specific antisense oligonucleo-tide. After pre-treatment with Bax antisense oligonucleo-tide, SH-SY5Y cells were exposed to MPP+ and cell deathwas evaluated with the alamarBlue assay. As shown inFig. 2C, Bax antisense oligonucleotide treatment rescuedSH-SY5Y cells from MPP+-induced cell death. Inhibitionof Bax expression significantly increased cell viability andprovided neuroprotection against MPP+.

KYNA attenuates MPP+-induced mitochondrial

damage in a Bax-dependent manner

For analyzing the effect of MPP+-induced neuro-toxicity in mitochondria, we examined DCm and thelocalization of cytochrome c. MPP+ exposure triggeredthe collapse of DCm whereas pre-treatment of KYNAsignificantly blocked depolarization of DCm (Fig. 4A, B).KYNA alone did not influence DCm. MPP+-induced a4.5-fold larger cytochrome c release than untreatedcontrols, and KYNA attenuated this release to someextent (Fig. 5). These data indicate that KYNA effectivelyinhibited MPP+-induced mitochondrial dysfunction, asmeasured by loss of DCm and release of cytochrome c.Pre-treatment with Bax antisense oligonucleotiedes atte-nuated MPP+-induced DCm reduction (Fig. 4A, B) andcytochrome c release (Fig. 5C), indicating that Bax plays arole in MPP+-induced mitochondrial dysfunction.

We were able to investigate the interaction betweenDCm and cytochrome c release by pre-incubatingSH-SY5Y cells with bongkrekic acid or cyclosporin A,which are widely used mitochondrial membrane transi-tion pore inhibitors (Maciel et al., 2004). After pre-treatment with bongkrekic acid (5 mM) or cyclosporin A

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(5 mM), SH-SY5Y cells were exposed to MPP+ and thelevel of cytochrome c release was evaluated. As shown inFig. 5C, neither of these inhibitors significantly affectedMPP+-mediated cytochrome c release.

KYNA attenuates MPP+-induced caspase

activation through down-regulation of Bax proteins

To determine whether MPP+-induced cell deathrequired activation of apoptotic proteases, we measured

the activities of caspase-9 and -3. MPP+ treatmentincreased caspase-9 activity (about 3.7-fold) and cas-pase-3 activity (about 4.3-fold), (Fig. 6A, B). KYNAsignificantly inhibited the activities of both caspases by50% compared with MPP+ alone. Bax-antisense treat-ment showed an effect similar to KYNA on MPP+-induced caspase activation. These results indicate thatKYNA inhibits both MPP+-induced caspase-9 and -3activities through blocking a Bax-dependent mitochon-drial pathway.

KYNA also potently attenuates MPP+-induced

neuronal apoptosis in SK-N-SH cells

In order to examine the neuroprotective role ofKYNA against MPP+ in another cultured neuronalcell line, we evaluated the effect of KYNA on MPP+-induced neuronal cell death in SK-N-SH cells. Expect-edly, in SK-N-SH cells, MPP+-induced neuronal celldeath in a time-dependent manner and this MPP+-induced neuronal cell death was significantly attenuatedby pre-treatment with KYNA (Fig. 7A). Moreover,KYNA significantly inhibited MPP+-induced caspases-3 activity (B) and blocked MPP+-induced depolariza-tion of DCm (Fig. 7C, D). KYNA alone did notinfluence DCm. These data indicate that KYNAeffectively inhibited MPP+-induced neuronal cell deathvia regulation of mitochondrial dysfunction and caspaseactivation in SK-N-SH as well as SH-SY5Y cells.

Discussion

The selective neurotoxin MPP+ has been widely usedto create in vitro and in vivo animal models of PD.MPP+ causes selective destruction of the nigrostriataldopaminergic pathway, which is similar to that observedin PD, and inhibits mitochondrial NADH-linked

Fig. 1. KYNA protects against the neuronal apoptosis

induced by MPP+ in SH-SY5Y cells. (A) SH-SY5Y cells were

exposed to MPP+ for 48 h and cell viability was assessed by

the alamarBlue assay. Data are given as the means7SD

obtained from three independent experiments. (B) SH-SY5Y

cells were pre-treated with KYNA (5 or 10mM) for 2 h,

followed by 750mM MPP+ treatment for 24 h. The cell

viability was assessed using the alamarBlue assay. *po0.05

versus vehicle alone; **po0.05 versus 750mM MPP+ alone.

(C) Morphological changes associated with MPP+ toxicity

and the effect of pre-incubation with KYNA. SH-SY5Y cells

were exposed to MPP+ for 24 h in the absence or presence of

10 mM KYNA and viewed under a phase-contrast microscope

(a–c) or stained with Hoechst 33258 and visualized by

fluorescence microscopy (d–f). (a,d) Control; (b,e) MPP+;

(c,f) KYNA+MPP+. Bar: 20 mm (a–c), 10 mm (d–f).

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Fig. 2. Effects of MPP+ on Bax and Bcl-2 expression and

effect of Bax antisense oligonucleotide treatment on viability

of MPP+-treated SH-SY5Y cells. (A) SH-SY5Y cells were

treated with 750 mM MPP+ for the indicated times, and the

presence of Bax or Bcl-2 in cell lysates was determined by

immunoblotting. The result shown is representative of at least

three independent experiments. (B) Quantification of (A) by

densitometry. Relative Bax or Bcl-2 levels in control cells were

set as 1. *po0.05 versus 0 h. (C) Treatment of SH-SY5Y cells

with 750mM MPP+ for 24 h reduced cell viability by �50%,

and pre-treatment with Bax antisense oligonucleotide (Bax-

As+MPP+) reduced this toxicity. BaxAs alone resulted in no

significant change in cell viability compared with vehicle-

treated controls. Data are the mean7SD from three indepen-

dent experiments. *po0.05 versus vehicle alone; **po0.05

versus 750 mM MPP+ alone.

Fig. 3. The effects of KYNA on Bax and Bcl-2 expression in

MPP+-treated SH-SY5Y cells. (A) SH-SY5Y cells were pre-

treated with 10 mM KYNA for 2 h before incubation with

750 mMMPP+ for the indicated times, and the presence of Bax

or Bcl-2 in cell lysates was determined by immunoblotting.

Blots shown are representative of at least three independent

experiments. The result shown is representative of at least three

independent experiments. (B) Quantification of (A) by

densitometry. Relative Bax or Bcl-2 levels in control cells

were set as 1. *po0.05 versus 0 h. **po0.05 versus 750 mMMPP+.

D.Y. Lee et al. / European Journal of Cell Biology 87 (2008) 389–397 393

electron transport at complex I, resulting in the loss ofATP production and subsequent cell death (Kotake andOhta, 2003). Although the neurotoxin MPP+ induces

apoptosis in several neuronal cell types, its exactmechanism of toxicity is still not resolved.

In this study, we elucidate the mechanism of MPP+-induced cell death and the protective effects of KYNAagainst MPP+ toxicity in human neuroblastoma SH-SY5Y and SK-N-SH cells. The human neuroblastomacell line, SK-N-SH, which was originally establishedfrom a bone marrow biopsy of a neuroblastoma patient,and its subclone SH-SY5Y cell line were reported toexpress substantial levels of dopamine-b-hydroxylase(Biedler et al., 1978). Thus, these cells are a reliable invitro model for the study of MPP+-induced neurotoxi-city (Chetsawang et al., 2007; Jung et al., 2007; Lu et al.,2006). The damage is time- and dose dependent in bothcultured dopaminergic neurons, and apoptotic featuresincluding caspase activation, DNA fragmentation andnuclear condensation markedly increase when cells areexposed to MPP+ (Fan et al., 2006; Lu et al., 2006).

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Fig. 4. Effects of pre-treatment with 10mM KYNA or 10 mMBax antisense oligonucleotide (BaxAS) on mitochondrial

membrane potential (MMP) in SH-SY5Y cells treated with

MPP+ (750mM). (A) Cells plated on glass coverslips were

maintained for 6 h at 37 1C in either culture medium alone

(CTL) or medium supplemented with MPP+, KY-

NA+MPP+, KYNA, BaxAs+MPP+, or 5mM FCCP as

indicated. Cells were then loaded with 100 nM TMRE and

evaluated by fluorescence microscopy. Bar: 10 mm. (B)

Quantification of MMP by fluorometry (TECAN GENios).

Fluorescence levels are expressed as arbitrary units. The data

are the means7SEM of four separate experiments. *po0.05

versus vehicle alone; **po0.05 versus MPP+ alone.

Fig. 5. Release of cytochrome c from mitochondria in KYNA-

and/or MPP+-treated cells. (A) SH-SY5Y cells were pre-

treated with 10mM KYNA for 2 h before incubation with

750 mM MPP+ for the indicated times. Cell lysates (20 mgprotein) were separated by SDS-PAGE and immunoblotted

for detection of cytochrome c. The result shown is representa-

tive of at least three independent experiments. (C) Cytochrome

c release after treatment of cells with either 750mM MPP+,

10 mM Bax antisense oligonucleotide (BaxAs), 5mM bongk-

rekic acid (BA), or 5 mM cyclosporin A (CysA) followed by

MPP+ for 2 h. (B, D) Quantification of (A) and (C),

respectively, by densitometry. Relative cytochrome c release

levels in control cells were set as 1. *po0.05 versus control.

**po0.05 versus MPP+ alone.

D.Y. Lee et al. / European Journal of Cell Biology 87 (2008) 389–397394

Our present study clearly shows that exposure toMPP+ resulted in a decrease in cell viability, and thatcell death involved Bax-dependent mitochondrial signal-ing, a finding consistent with previous works of othergroups (Dennis and Bennett, 2003; Hartmann et al.,2001).

KYNA acts as an endogenous anticonvulsant andneuroprotective agent in the central nervous system(Scharfman and Ofer, 1997; Yamamoto et al., 1995).

Furthermore, KYNA has an aromatic ring, whichenhances its lipophilicity (Fukui et al., 1991), andsubstitutions at the 6-position of the ring alter theinhibition of several distinct glutamate receptors(Leeson et al., 1991). In addition, KYNA is anendogenous compound which is synthesized predomi-nantly in astrocytes (Szerb and Butterworth, 1992), andaccumulates in the brain if given systemically (Stone,2001). These properties indicate that KYNA exhibitssufficient blood-brain barrier permeation for clinicalapplication. Previous reports have suggested that MPP+

inhibits the activity of kynurenine aminotransferase(KAT), leading to depletion of KYNA (Luchowskaet al., 2003; Luchowski et al., 2002). Nicotinylalanine, aspecific inhibitor of kynureninase or kynurenine hydro-xylase, leads to an increase in KYNA levels in the brain,resulting in reduced production of neurotoxic metabo-lites such as 3-hydroxykynurenine and quinolinic acid inthe kynurenine pathway (Stone, 2001).

Therefore, we aimed to evaluate the effect ofKYNA on MPP+-induced neurotoxicity. We observeda reduction in apoptotic cell death, as evidenced byamelioration of morphological indicators and directmeasurement of Bax expression and mitochondrial dys-function, such as collapse of mitochondrial membranepotential (DCm), release of cytochrome c, and increasesin caspase-9/-3 activities.

Ogawa et al. (1992) showed that KYNA levels weresignificantly decreased in the pars compacta of the

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Fig. 6. The effects of KYNA, Bax antisense oligonucleotide

(BaxAs), and caspase inhibitors (all 10mM) on MPP+-induced

activation of the caspase cascade. Caspase-9 (A) and caspase-3

(B) activity induced by MPP+ (750mM) were measured as

described in Materials and methods. Data are shown as

means7SEM of three separate experiments. *po0.05 versus

vehicle alone; **po0.05 versus MPP+ alone.

Fig. 7. KYNA protects against neuronal apoptosis induced by

MPP+ via regulation of mitochondrial membrane potential

(DCm) and caspase activation in SK-N-SH cells. (A) Viability

assay. (B) Caspase-3 activity assay. (C, D) Mitochondrial

membrane potential (MMP). For experimental details, see

description for SH-SY5Y cells (Figs. 1,4,6).

D.Y. Lee et al. / European Journal of Cell Biology 87 (2008) 389–397 395

substantia nigra of PD brain tissue. Furthermore,KYNA level is associated with a decrease in neuronalactivity of dopaminergic neurons (Schwieler et al.,2006). Thus, there are several evidences for a neuropro-tective effect of KYNA treatment. However, this is thefirst time that the protective effect of KYNA has beendescribed in an in vitro PD model.

Also, our results showed that down-regulation of Baxexpression rescued MPP+-induced mitochondrial dys-

function. In addition, there were no statisticallysignificant effects on MPP+-induced cytochrome c

release when DCm reduction was inhibited by bongk-rekic acid or cyclosporin A (Fig. 5C). These resultssuggest that MPP+-induced mitochondrial dysfunctionis initiated by Bax-mediated membrane poring ratherthan the collapse of DCm. This is in line with previousreports (Gonzalez-Polo et al., 2003; Antonsson et al.,1997).

Acknowledgment

This work was supported by a Grant (20050-401034812) from BioGreen 21 Program, Rural Devel-opment Administration, Republic of Korea.

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