A

mipio(t(oap©

K

f

0

Toxicology 243 (2008) 340–347

Available online at www.sciencedirect.com

Involvement of both mitochondrial- and death receptor-dependentapoptotic pathways regulated by Bcl-2 family in sodium

fluoride-induced apoptosis of thehuman gingival fibroblasts

Jin-Ha Lee, Ji-Yeon Jung, Yeon-Jin Jeong, Jae-Hong Park, Kyu-Ho Yang,Nam-Ki Choi, Sun-Hun Kim, Won-Jae Kim ∗

Dental Science Research Institute, 2nd Stage of Brain Korea 21 for School of Dentistry,Chonnam National University, Gwang Ju 500-757, South Korea

Received 28 August 2007; received in revised form 19 October 2007; accepted 20 October 2007Available online 4 November 2007

bstract

Sodium fluoride (NaF) has been shown to be cytotoxic and produces inflammatory responses in humans. However, the cellularechanisms underlying the NaF-induced cytotoxicity in periodontal tissues are unclear. This study examined whether or not NaF

nduces apoptosis in human gingival fibroblasts (HGF), and its underlying mechanisms by monitoring various apoptosis-associatedrocesses. NaF reduced the cell viability of HGF in a dose- and time-dependent manner. NaF increased TUNEL-positive cell andnduced apoptosis with concomitant chromatin condensation and DNA fragmentation in HGF. In addition, NaF increased the levelf cytochrome c released from the mitochondria into the cytosol, enhanced the caspase-9, -8 and -3 activities, the cleavage of polyADP-ribose) polymerase (PARP), and up-regulated the voltage-dependent anion channel (VDAC) 1. However, NaF did not affecthe production of reactive oxygen species (ROS) which is a strong apoptotic inducer. Furthermore, NaF up-regulated the Fas-ligandFas-L), a ligand of death receptor. Bcl-2, a member of the anti-apoptotic Bcl-2 family, was down-regulated, whereas the expression

f Bax, a member of the pro-apoptotic Bcl-2 family, was unaffected in the NaF-treated HGF. These results suggest that NaF inducespoptosis in HGF through both the mitochondria-mediated pathways regulated by the Bcl-2 family and death receptor-mediatedathway.

2007 Elsevier Ireland Ltd. All rights reserved.

eywords: NaF; HGF; Apoptosis; Bcl-2 family; Caspase; Fas-L

∗ Corresponding author. Tel.: +82 62 530 4880;ax: +82 62 530 4885.

E-mail address: wjkim@chonnam.ac.kr (W.-J. Kim).

1

cvraF

300-483X/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reservdoi:10.1016/j.tox.2007.10.026

. Introduction

Fluoride is widely used in dentistry to prevent dentalaries, even though the safety of fluoride is a contro-

ersial issue. Although there are rare adverse effectseported after long-term low dose fluoride ingestion,n overdose can cause serious acute toxicity (Li, 1993;DI Communication, 2000). Fluoride has been found

ed.

cology

2

2

pw(CDil(c

2

tauaG

2

rdBpNwTsaiai

2p

2pTaw5t

J.-H. Lee et al. / Toxi

to inhibit protein synthesis and cell cycle progres-sion (Holland, 1979; Aardema et al., 1989), as wellas induce apoptosis in epithelial lung cells and alve-olar macrophages (Hirano and Ando, 1996; Refsneset al., 1999). However, sodium fluoride (NaF)-inducedcytotoxicity in periodontal tissues and its underlyingmechanisms are unclear.

Cells undergoing apoptosis show distinct morpholog-ical and biochemical changes such as cell shrinkage,membrane blebbing, chromatin condensation and DNAfragmentation (Kerr et al., 1972; Wyllie et al., 1980).Apoptosis is triggered by a variety of stimuli that inducethrough the cell surface death receptors such as Fas,mitochondrial and endoplasmic reticulum stress. Cas-pases are cysteine proteases that cleave a critical set ofcellular proteins to initiate the apoptotic signal includ-ing several representatives involved in apoptosis (Roth etal., 2000; Tsujimoto and Shimizu, 2000). Among the 14known mammalian caspases, those involved in apoptosiscan be further subdivided into initiators (-2, -8, -9, and-10) and effector caspases (-3, -6, and -7) (Adams andCory, 1998; Crompton, 2000). The main mechanism forapoptosis through the pathways for the activating cas-pases can be divided into the death receptor-mediatedand mitochondria-mediated mechanisms. The mitochon-drial pathway is initiated by the release of cytochromec from the mitochondria into the cytosol, consequentlyresulting in the activation of caspase-9, which in turnactivates caspase-3 (Green and Reed, 1998). The deathreceptor pathway is stimulated by the cell surface deathreceptors such as the tumor necrosis factor (TNF) recep-tor and Fas. The receptors activated by ligands lead tocaspase-8 activation, with the subsequent activation ofcaspase-3 (Beer et al., 2000). The mitochondria- anddeath receptor-mediated pathways share the activationof caspase-3, which cleaves one of its substrates, poly(ADP-ribose) polymerase (PARP), resulting in apoptoticDNA fragmentation (Ogata et al., 1998). In addition,the Bcl-2 protein family plays a key role in apopto-sis (Tsujimoto and Shimizu, 2000). The Bcl-2 familycontrols the release of mitochondrial cytochrome cby regulating the mitochondrial permeability transition(PT) pore, which consists of the voltage-dependent anionchannel (VDAC) in the outer membrane, adenosinenucleotide translocated (ANT) in the inner membrane,and cyclophilin-D (Cyp-D) in the matrix assemblies(Ankarcrona et al., 1995; Krajewski et al., 1999).

This study examined the involvement of the

mitochondria- or death receptor-mediated apoptoticpathways, and investigated the roles of the Bcl-2 familyin the NaF-induced apoptosis of human gingival fibrob-lasts (HGF).

Ka3Pw

243 (2008) 340–347 341

. Materials and methods

.1. Cell culture and cell viability assay

The HGF were obtained from the healthy gingival tissue ofatients in Chonnam National University Hospital. The HGFere cultured in DMEM media supplemented with 10% FBS

Gibco BRL, USA), 1% streptomycin–penicillin under 5%O2 at 37 ◦C. NaF (Sigma, USA) was dissolved in distilledMEM and sterilized through a 0.2 �m filter. The cells were

ncubated in 96-well plates (5 × 103 cells/well) for 24 h, fol-owed by suction and replacement with media containing NaF5, 10, 20, 30, and 40 mM). After 6 h, the number of viableells was measured using a MTT assay.

.2. In situ apoptosis detection by TUNEL staining

The cells were treated with 20 mM NaF for 6 h andhen examined by the TUNEL method using an in situpoptosis detection kit (Chemicon, CA) according to the man-facturer’s instructions. The samples were visualized usingn LSM 510 confocal laser scanning microscope (Zeiss,ermany).

.3. Detection of ROS production and caspase activity

The production of ROS was monitored using a fluo-escence spectrometer (Hitachi F-4500, Japan) with 2′,7′-ichlorofluorescein diacetate (Sigma, USA) (Jakubowski andartosz, 2000). The cells were seeded in a 96-well micro-late (5 × 103 cells/well) for 24 h, and then incubated withaF (5, 10, 20, 30, and 40 mM) for 6 h. Then, the cellsere exposed to 10 �M DCF-DA for 10 min at 37 ◦C.he fluorescence excitation was at 480 nm and the emis-ion was collected at 530 nm. The caspases activity weressessed by an ELISA reader using a caspase-3, -9 activ-ty assay kit (Calbiochem, CA) and a caspase-8 activityssay kit (Santa Cruz, USA) according to the manufacturer’snstructions.

.4. Isolation of total RNA and reverse transcriptionolymerase chain reaction (RT-PCR)

The total RNA in the cells after being treated with 5, 10 and0 mM NaF for 6 h was extracted by homogenizing using aolytron homogenizer in Trizol reagent (Gibco BRL, USA).he cDNA was synthesized by mixing 2 �g of total RNAnd 2 �l of oligo-dT (10 pmoles) with 50 �l of RNase-freeater, and incubating them at 42 ◦C for 1 h and 94 ◦C formin. The PCR products were generated in a PCR buffer con-

aining 10 pmoles primer using a PCR-premix kit (Bioneer,

orea). PCR was initiated in a thermal cycle programmedt 95 ◦C for 5 min, 95 ◦C for 40 s, 55 ◦C for 40 s, 72 ◦C for0 s, followed by amplification for 30 cycles on a GeneAmpCR system (PerkinElmer 2400). The following primer pairsere used: for Fas-L, 5′-CAGCCCCTGAATTACCCATATC-

3 cology

3(5Case

2

bCCPtftmce52wat

cdaoSAobmU

2

n

3

ai

FTHi

42 J.-H. Lee et al. / Toxi

′ (sense primer), 5′-CACTCCAGAGATCAAAGCAGTTC-3′

antisense primer); for GAPDH, as the internal control,′-TGCATCCTGCACCACCAACT-3′ (sense primer) and 5′-GCCTGCTTCACCAC TTC-3′ (antisense primer). Themplified products were visualized on a 1.5% agarose gel andtained with ethidium bromide. The intensity of the bands wasxamined using NIH Scion Image Software.

.5. Western blot analysis

The cells were lysed using the standard procedures aftereing exposed to the indicated NaF concentration for 6 h.ell lysates were made in a NP-40 lysis buffer (30 mM Tris-l, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1% NP-40, 1 mMMSF, and protease inhibitor mixture containing 1 �g/ml apro-

inin and leupeptin). The amount of cytochrome c releasedrom the mitochondria into the cytosol was determined usinghe cytosolic fractions, according to the previously described

ethod (Boulares et al., 2002). To determine the level ofytosolic cytochrome c, the pellet was resuspended in anxtraction buffer containing 220 mM mannitol, 68 mM sucrose,

0 mM PIPES–NaOH (pH 7.4), 50 mM KCl, 5 mM EGTA,mM MgCl2, and 1 mM DTT. The lysates (100–300 �g)ere determined using a BCA protein assay (Pierce, USA)

nd separated by 12% polyacrylamide gel electrophoresis andransferred to a nitrocellulose membrane (Amersham Pharma-

ac1f

ig. 1. NaF-induced the apoptotic cell death of HGF. The cell viability was dehe HGF were incubated with different NaF concentrations (A) and 20 mMGF treated with 20 mM NaF for 6 h (C) and TUNEL-positive cells were d

ndependent experiments. *p < 0.05, compared with the untreated control.

243 (2008) 340–347

ia Biotech, UK). The membrane was blocked in 5% nonfatry milk and incubated with the primary antibodies for 1 ht room temperature. The primary antibodies were rat mon-clonal anti-cytochrome c (Pharmingen, USA), PARP (Cellignaling, USA), Bax, Bcl-2, and VDAC (Santa Cruz, USA).fter incubation with the specific peroxidase-coupled sec-ndary antibodies (Sigma, USA) for 1 h, the antibody-stainedands were detected using an ECL enhanced chemilu-inescence detection kit (Amersham Pharmacia Biotech,K).

.6. Statistical analysis

The Student’s t-test was used to determine statistical sig-ificance.

. Results

The cytotoxic effect of NaF on HGF was determinedt various NaF concentrations and times. As shownn Fig. 1A and B, NaF inhibited the cell viability in

dose- and time-dependent manner, respectively. Theell viability after treatment with 5–40 mM NaF for2 h decreased from 100% to 11%, and the IC50 valueor NaF-induced growth inhibition was estimated to be

termined using a MTT assay as described in the methodology section.NaF for the indicated times (B). TUNEL staining was done in the

etermined (D). The data is represented as the mean ± S.E.M of five

J.-H. Lee et al. / Toxicology

Fig. 2. ROS levels in the NaF-treated HGF. Cells loaded with DCFwere incubated with NaF for 6 h and the intracellular levels of ROS

cmnmp

catpttaBiitNSc1icin HGF.

were determined by measuring the level of DCF-DA fluorescence.The data is a represented as the mean ± S.E.M of five independentexperiments.

20 mM NaF for 24 h (Fig. 1A). Approximately 60% ofcells survived exposure to 20 mM NaF for 6 h (Fig. 1B).Based on these observations, treatment of 20 mM NaFfor 6 h was selected in future mechanistic studies.TUNEL-positive cells were enhanced in HGF treatedwith 20 mM NaF for 6 h. In TUNEL staining, the HGFshowed apoptotic morphological changes including

chromatin condensation, DNA fragmentation, whichdemonstrates that fluoride induces apoptosis in HGF(Fig. 1C).

it

Fig. 3. Caspases activation and PARP cleavage in the NaF-treated HGF. For tNaF for the indicated times, and the cell lysates were measured using a coloand caspase-3 (C) activity was measured after the 20 mM NaF treatment. C12% polyacrylamide gel electrophoresis and the lower band (85 kDa) indicatmean ± S.E.M of five independent experiments. *p < 0.05, **p < 0.01, compa

243 (2008) 340–347 343

To determine the involvement of ROS in NaF-inducedell death of HGF, the level of ROS production waseasured using DCF-DA. Fig. 2 shows that NaF did

ot affect the ROS level in HGF. Overall, NaF directlyay induce apoptotic cell death in HGF not through

roducing ROS.In order to determine if the initiator and effector

aspases are involved in the apoptotic progression, thectivities of capases-9, -8 and -3 were measured usingheir synthetic peptide substrates, LEHD-pNA, IETD-NA, and DEVD-pNA, respectively. Fig. 3A shows thathe caspase-9 activity was enhanced after the 20 mM NaFreatment and peaked at 6 h, suggesting that NaF-inducedpoptosis in HGF may occur through the mitochondria.esides, NaF-induced an increment in caspase-8 activ-

ty in a time-dependent manner (Fig. 3B) even if itsncrement was lower than that of caspase-9. In addi-ion, NaF-induced caspase-3 activation after the 20 mMaF treatment and its activity peaked at 6 h (Fig. 3C).ubsequently, NaF-induced the cleavage of PARP into alear 85 kDa fragment when the HGF was treated with0 and 20 mM NaF for 6 h (Fig. 3D). These resultsndicate that NaF activates caspase-3 leading to theleavage of PARP, subsequently resulting in apoptosis

The release of cytochrome c from the mitochondrianter-membranous space into the cytosol is a key event inhe activation of caspase-9, which subsequently initiates

he caspase-9, -8, and -3 activities, the cells were treated with 20 mMrimetric assay kit (A–C). The level of caspase-9 (A), caspase-8 (B)leavage of PARP was measured using Western blot analysis using

es the cleaved fragment of PARP (D). The data is represented as thered with the untreated control.

344 J.-H. Lee et al. / Toxicology 243 (2008) 340–347

Fig. 4. Increase in the levels of cytochrome c released from themitochondria into the cytosol and the expression of VDAC-1 in theNaF-treated HGF. The cytosolic cytochrome c (A) and VDAC-1 (B) inHGF treated with different concentrations of NaF for 6 h were analyzedby Western blot. These data were quantified through densitometry anditt

tSttVcaioa

asrNN

FsNp

TNrN

The expression ratio of Bax to Bcl-2 was determinedbecause its ratio is a critical key in regulating cytochromec release from the mitochondria to the cytosol. Bcl-2was shown to be down-regulated in the NaF-treated HGF

Fig. 6. Altered Bax/Bcl-2 expression in NaF-treated HGF. The expres-

ntensity of the bands is expressed as the mean ± S.E.M relative to con-rol of triplicate experiments (relative intensity = 1.0) after normalizinghe bands to actin. p < 0.05, compared with the untreated control.

he caspase cascade involving caspase-3 (Reed, 1997;lee et al., 1999). As shown in Fig. 4A, NaF enhanced

he release of cytochrome c from the mitochondria intohe cytosol in a dose-dependent manner. Furthermore,DAC-1, a cytochrome c releasing channel in the mito-

hondria, was up-regulated in the NaF-treated HGF indose-dependent manner (Fig. 4B). Taken together, it

s concluded that NaF induces an increase in the releasef cytochrome c from the mitochondria to the cytosolccompanied by the up-regulation of VDAC.

In order to determine if the death receptor-mediatedpoptosis pathway is involved in NaF-induced apopto-

is, the levels of Fas-L mRNA, a ligand of the deatheceptor, were determined by RT-PCR after a 20 mMaF treatment for 6 h. Fas-L was up-regulated in theaF-treated HGF in a dose-dependent manner (Fig. 5).

siwtw

ig. 5. Enhanced Fas-L level in NaF-treated HGF. The mRNA expres-ion of Fas-L was analyzed by RT-PCR in HGF treated with 20 mMaF for 6 h. The data is represented as the mean ± S.E.M of five inde-endent experiments. p < 0.05, compared with the untreated control.

he results showed that the Fas-L are up-regulated inaF-induced apoptosis of HGF, suggesting that the death

eceptor-mediated apoptotic pathway is involved in theaF-induced apoptosis of HGF.

ion of Bax and Bcl-2 were analyzed by Western blot analysis afterncubating the HGF with NaF for 6 h (A). The ratio of Bax and Bcl-2as determined using a densitometer (B). The data is represented as

he mean ± S.E.M of five independent experiments. p < 0.05, comparedith the untreated control.

cology

tP

m(idtlTmttdtscm

lsttt2tistws8Hdisbt

ottBscaBl

J.-H. Lee et al. / Toxi

while Bax was unaffected (Fig. 6A), and the Bax/Bcl-2ratio increased in a dose-dependent manner (Fig. 6B).

4. Discussion

Fluorides have been known to exert a variety of effectsin different cell types. In bone cells, fluorides elicit poten-tially beneficial effects by stimulating growth of bonecells (Caverzasio et al., 1998). However, in other cells,NaF exhibited the cytotoxic effects including inhibitionof protein synthesis (Aardema et al., 1989), alterationsin cellular metabolism (Curnutte et al., 1979), inductionof inflammatory cytokines (Refsnes et al., 1999), andapoptosis (Hirano and Ando, 1996). Though there havebeen many epidemiological, pathogenetic, clinical andcytogenetic studies associated with fluoride toxicity (Li,1993; Kleinsasser et al., 2001; Refsnes et al., 2003), thecytotoxic mechanism by fluoride in periodontal tissue isstill not completely understood.

In the present study, NaF was found to decrease thecell viability of HGF in a dose- and time-dependent man-ner and gave rise to apoptotic morphological changesincluding chromatin condensation, and DNA fragmen-tation in HGF, demonstrating that fluoride inducesapoptosis in HGF. Previous report was demonstrated thatoxidative stress was induced in fluoride group in rat oralmucosal cells (He and Chen, 2006). However, our resultsshowed that intracellular concentration of ROS was notchanged by NaF, indicating that NaF induces apoptosisof HGF not through producing ROS.

Caspases, which are cysteine proteases, play animportant role in apoptosis. In general, caspase-3 is akey and common protease in both mitochondria- anddeath receptor-dependent pathways (Earnshaw et al.,1999; Bal-Price and Brown, 2000). Previous studieshave reported that fluoride induces apoptosis in humanleukemia HL-60 cells by activating caspase-3 (Anuradhaet al., 2000; Anuradha et al., 2001). The present studyshowed that caspase-3 activity was enhanced in NaF-treated HGF, in concordance with previous reports inother cells. This result implies that caspase-3 plays apivotal role in NaF-induced apoptosis of HGF. PARPis a target of caspase protease activity associated withapoptosis: During apoptosis, PARP is cleaved by acti-vated caspase-3 from its 116 kDa intact form into 85and 25 kDa fragments. It has been also reported that itis cleaved before or concomitant with degradation ofnuclear DNA into nucleosomal fragments (Kaufmann et

al., 1993), and PARP inhibitors delay or prevent apop-tosis (Kaufmann et al., 1993). The present study clearlyshowed that NaF-induced the cleavage of PARP into aclear 85 kDa fragment, thus indicating that NaF induces

earc

243 (2008) 340–347 345

he activation of caspase-3 and then the cleavage ofARP, thereby causing apoptosis in HGF.

Several studies have revealed the involvement ofitochondria in apoptosis of various cellular systems

Bossy-Wetzel et al., 1998). Many apoptotic stimulinduce the release of cytochrome c from the mitochon-ria into the cytosol, subsequently activating caspase-9hrough binding to the CED-4 homolog Apaf-1, fol-owed by activation of caspase-3 (Cecconi et al., 1998).he present study showed that NaF resulted in an incre-ent of cytochrome c release from the mitochondria into

he cytosol in a dose-dependent manner and enhancedhe activity of caspase-9. Taken together, mitochondria-ependent apoptotic pathway has been definitely proveno be involved in NaF-induced apoptosis of the HGF,ince the cytochrome c released into the cytosol andaspase-9 are the major molecules associated withitochondria-dependent pathway.Once the death receptors have been activated by their

igands, the death receptors recruit the adaptor molecule,uch as Fas-associated death domain (FADD) throughhe activation of caspase-8. Recent studies have shownhat NaF regulates the expression of Fas and Fas-L inhe process of osteoclast-like cell apoptosis (Sun et al.,002). These previous studies suggested a possibilityhat death receptor-mediated apoptosis pathway may benvolved in caspase-3 activated in NaF-induced apopto-is of the HGF. However, there has so far been no studyo examine the death receptor-mediated apoptosis path-ay in NaF-induced apoptosis of HGF. The present study

howed that Fas-L levels were up-regulated and caspase-activity was elevated in NaF-induced apoptosis ofGF. Therefore, the present study demonstrates first evi-ence that death receptor-mediated pathway is involvedn NaF-induced apoptosis of HGF. These findings furtheruggest that NaF-induced apoptosis may be mediated byoth mitochondria- and death receptor-dependent apop-osis pathways with involvement of caspases cascade.

The Bcl-2 family is a well-characterized regulatorf cytochrome c release from the mitochondria intohe cytosol by regulating the mitochondrial PT porehat is composed of VDAC, ANT and Cyp-D. Thecl-2 family is classified into anti-apoptotic proteins

uch as Bcl-2 and Bcl-XL, which reduce the level ofytochrome c release (Gottlieb et al., 2000; Howard etl., 2002) and pro-apoptotic proteins such as Bax andak, which induce the release of cytochrome c and a

oss of the mitochondrial membrane potential (Starkov

t al., 2002). Therefore, ratio of pro-apoptotic and anti-poptotic Bcl-2 family may be a pivotal cue to theelease of cytochrome c from the mitochondria into theytosol. In the present study, Bcl-2 was shown to be

3 cology

dtaIiwifasNiaihiaa

add

R

A

A

A

A

A

B

B

B

B

C

C

C

C

E

F

G

G

H

H

H

H

J

K

K

K

K

LO

R

46 J.-H. Lee et al. / Toxi

own-regulated, whereas Bax was not affected in NaF-reated HGF, suggesting that Bcl-2 family may playn important role in NaF-induced apoptosis of HGF.nterestingly, the level of the VDAC-1 protein, whichs a major component of the mitochondrial PT pore,as increased, indicating that VDAC-1 may be involved

n the increase in the level of cytochrome c releasedrom the mitochondria into the cytosol in NaF-inducedpoptosis. This is first evidence showing that the expres-ion of VDAC, particularly VDAC-1, was altered in theaF-induced apoptosis of HGF. From these results, it

s suggested that the Bcl-2 family and VDAC-1 playkey role in the increase in the cytochrome c level

n the cytosol during NaF-induced apoptosis. However,ow Bcl-2 regulates VDAC-1 expression was not studiedn the present experiment which its underlying mech-nism is further needed to research in NaF-inducedpoptosis.

In summary, these results suggest that NaF inducespoptosis in HGF through the both mitochondria-ependent pathways mediated by the Bcl-2 family andeath receptor-dependent pathways.

eferences

ardema, M.J., Gibson, D.P., LeBoeuf, R.A., 1989. Sodium fluoride-induced chromosome aberrations in different stages of the cellcycle: a proposed mechanism. Mutat. Res. 223, 191–203.

dams, J.M., Cory, S., 1998. The Bcl-2 protein family: arbiters of cellsurvival. Science 281, 1322–1326.

nkarcrona, M., Dypbukt, J.M., Bonfoco, E., Zhivotovsky, B., Orre-nius, S., Lipton, S.A., Nicotera, P., 1995. Glutamate-inducedneuronal death: a succession of necrosis or apoptosis dependingon mitochondrial function. Neuron 15, 961–973.

nuradha, C.D., Kanno, S., Hirano, S., 2000. Fluoride induces apopto-sis by caspase-3 activation in human leukemia HL-60 cells. Arch.Toxicol. 74, 226–230.

nuradha, C.D., Kanno, S., Hirano, S., 2001. Oxidative damageto mitochondria is a preliminary step to caspase-3 activation influoride-induced apoptosis in HL-60 cells. Free Radic. Biol. Med.31, 367–373.

al-Price, A., Brown, G.C., 2000. Nitric-oxide-induced necrosis andapoptosis in PC12 cells mediated by mitochondria. J. Neurochem.75, 1455–1464.

eer, R., Franz, G., Schopf, M., Reindl, M., Zelger, B., Schmutzhard,E., Poewe, W., Kampfl, A., 2000. Expression of Fas and Fas ligandafter experimental traumatic brain injury in the rat. J. Cereb. Blood.Flow. Metab. 20, 669–677.

ossy-Wetzel, E., Newmeyer, D.D., Green, D.R., 1998. Mitochondrialcytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrialtransmembrane depolarization. EMBO J. 17, 37–49.

oulares, A.H., Zoltoski, A.J., Sherif, Z.A., Yakovler, A., Smul-son, M.E., 2002. Roles of DNA fragmentation factor and poly(ADP-ribose) polymerase-1 in sensitization of fibroblasts to tumornecrosis factor-induced apoptosis. Biochem. Biophys. Res. Com-mun. 290, 796–801.

R

243 (2008) 340–347

averzasio, J., Palmer, G., Bonjour, J.P., 1998. Fluoride: mode ofaction. Bone 22, 585–589.

ecconi, F., Alvarez-Bolado, G., Meyer, B.I., Roth, K.A., Gruss, P.,1998. Apaf1 (CED-4 homolog) regulates programmed cell deathin mammalian development. Cell 94, 727–737.

rompton, M., 2000. Mitochondrial intermembrane junctional com-plexes and their role in cell death. J. Physiol. 529, 11–21.

urnutte, J.T., Babior, B.M., Karnovsky, M.L., 1979. Fluoride-mediated activation of the respiratory burst in human neutrophils:a reversible process. J. Clin. Invest. 63, 637–647.

arnshaw, W.C., Martins, L.M., Kaufmann, S.H., 1999. Mammaliancaspases: structure, activation, substrates, and functions duringapoptosis. Annu. Rev. Biochem. 68, 383–424.

DI Communication, 2000. Mouth rinses and dental caries. Int. Dent.J. 52, 337–345.

ottlieb, E., Vander Heiden, M.G., Thompson, C.B., 2000. Bcl-xlprevents the initial decrease in mitochondrial membrane poten-tial and subsequent reactive oxygen species production duringtumor necrosis factor alpha-induced apoptosis. Mol. Cell. Biol.20, 5680–5689.

reen, D.R., Reed, J.C., 1998. Mitochondria and apoptosis. Science281, 1309–1312.

e, L.F., Chen, J.G., 2006. DNA damage, apoptosis and cell cyclechanges induced by fluoride in rat oral mucosal cells and hepato-cytes. World J. Gastroenterol. 21, 1144–1148.

irano, S., Ando, M., 1996. Apoptotic cell death following exposure tofluoride in rat alveolar macrophages. Arch. Toxicol. 70, 249–251.

olland, R., 1979. Fluoride inhibition of protein synthesis. Cell Biol.Int. Rep. 3, 701–705.

oward, S., Bottino, C., Brooke, S., Cheng, E., Giffard, R.G., Sapol-sky, R., 2002. Neuroprotective effects of Bcl-2 overexpressionin hippocampal cultures: interactions with pathways of oxidativedamage. J. Neurochem. 83, 914–923.

akubowski, W., Bartosz, G., 2000. 2,7-Dichlorofluorescin oxidationand reactive oxygen species: what does it measure? Cell Biol. Int.24, 757–760.

aufmann, S., Desnoyers, S., Ottaviano, Y., Davidsonm, N., Poirier,G., 1993. Specific proteolytic cleavage of poly (ADP-ribose) poly-merase: an early marker of chemotherapy induced apoptosis.Cancer Res. 53, 3976–3985.

err, J.F., Wyllie, A.H., Currie, A.R., 1972. Apoptosis: a basic biologi-cal phenomenon with wide-ranging implications in tissue kinetics.Br. J. Cancer. 26, 239–257.

leinsasser, N.H., Weissacher, H., Wallner, B.C., Kastenbauer, E.R.,Harreus, U.A., 2001. Cytotoxicity and genotoxicity of fluoridesin human mucosa and lymphocytes. Laryngorhinootologie 80,187–190.

rajewski, S., Krajewska, M., Ellerby, L.M., Welsh, K., Xie, Z.,Deveraux, Q.L., Salvesen, G.S., Bredesen, D.E., Rosenthal, R.E.,Elskum, G., Reed, J.C., 1999. Release of caspase-9 from mitochon-dria during neuronal apoptosis and cerebral ischemia. Proc. Natl.Acad. Sci. U.S.A. 96, 5752–5757.

i, Y., 1993. Fluoride: safety issues. J. Indiana Dent. Assoc. 72, 22–26.gata, S., Takeuchi, M., Okumura, K., Taguchi, H., 1998. Apopto-

sis induced by niacin-related compounds in HL-60 cells. Biosci.Biotechnol. Biochem. 62, 2351–2356.

eed, J.C., 1997. Cytochrome c: can’t live with it-can’t live without it.

Cell 91, 559–562.

efsnes, M., Becher, R., Lag, M., Skuland, T., Schwarze, P.E.,1999. Fluoride-induced interleukin-6 and interleukin-8 synthe-sis in human epithelial lung cells. Hum. Exp. Toxicol. 18,645–652.

cology

S

S

J.-H. Lee et al. / Toxi

Refsnes, M., Schwarze, P.E., Holme, J.A., Lag, M., 2003. Fluoride-induced apoptosis in human epithelial lung cells (A549 cells): roleof different G protein-linked signal systems. Hum. Exp. Toxicol.22, 111–123.

Roth, J.A., Feng, L., Walowitz, J., Browne, R.W., 2000. Manganese-induced rat pheochromocytoma (PC12) cell death is independent

of caspase activation. J. Neurosci. Res. 61, 162–171.

Slee, E.A., Harte, M.T., Kluck, R.M., Wolf, B.B., Casiano, C.A.,Newmeyer, D.D., 1999. Ordering the cytochrome c-initiated cas-pase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8 and-10 in a caspase-9-dependent manner. J. Cell Biol. 144, 281–292.

T

W

243 (2008) 340–347 347

tarkov, A., Polster, B., Fiskum, G., 2002. Regulation of hydrogenperoxide production by brain mitochondria by calcium and Bax. J.Neurochem. 83, 220–228.

un, Y.M., Yang, F.J., Li, Y.M., Lu, B., Zhu, M., Qiu, M.C., 2002.Expression of Fas, FasL, and NF-kappa B in the process ofosteoclast-like cell apoptosis effected by sodium fluoride. Zhong-

guo Yi Xue Ke Xue Tuan Xue Bao 24, 491–494.

sujimoto, Y., Shimizu, S., 2000. Bcl-2 family: Life-or-death switch.FEBS Lett. 466, 6–10.

yllie, A.H., Kerr, J.F., Currie, A.R., 1980. Cell death in the signifi-cance of apoptosis. Int. Rev. Cytol. 68, 251–306.