Cadmium-induced apoptosis in rat hepatocytes does not necessarily involve caspase-dependent pathways

Toxicology in Vitro 20 (2006) 1331–1342www.elsevier.com/locate/toxinvit

Cadmium-induced apoptosis in rat hepatocytes does not necessarily involve caspase-dependent pathways

T.N.D. Pham a, M. Marion a, F. Denizeau z, C. Jumarie b,¤

a Département de Chimie, Centre TOXEN, Université du Québec à Montréal, C.P. 8888, Succ. Centre-Ville, Montréal, Qué., Canada H3C 3P8b Des Sciences Biologiques, Centre TOXEN, Université du Québec à Montréal, C.P. 8888, Succ. Centre-Ville, Montréal, Qué., Canada H3C 3P8

Received 30 January 2006; accepted 12 May 2006Available online 23 May 2006

Abstract

Cadmium (Cd) is a well-known hepatotoxic environmental pollutant. Depending on the exposure conditions, Cd may cause necrosisor apoptosis. Oxidative stress is believed to participate in Cd toxicity but the molecular signaling responsible for Cd-induced apoptosis innon-malignant liver cells still needs to be clariWed. Therefore we have studied apoptosis in primary cultures of rat hepatocytes incubatedwith low levels of Cd for short exposure times. Studies of nuclear morphology, chromatin condensation, and oligonucleosomal DNAfragmentation demonstrate that 1–5 �M Cd induces apoptosis as early as 6–12 h with minor eVects on MTT activity. A concomitant time-and concentration-dependent increase in caspase-9 and -3 activities was observed, whereas Cd did not aVect caspase-8 activity as much,suggesting a minor role of the death-receptor pathway. SigniWcant release of cytochrome c into the cytosol demonstrated the involvementof a mitochondrial-dependent apoptotic pathway. However, cell pre-treatment with caspase inhibitors (Z-VAD-fmk or Ac-DEVD-CHO)did not prevent apoptosis. Increases in the cytosolic levels of the mitochondrial apoptosis-inducing factor (AIF) were also observed:kinetics of cytochrome c and AIF release were similar. These results show that Cd-induced apoptosis in rat hepatocytes is time- and con-centration-dependent. The early apoptotic events involved mitochondrial-dependent pathways but not necessarily caspase-dependentsignaling.© 2006 Elsevier Ltd. All rights reserved.

Keywords: Cadmium; Rat hepatocytes; Apoptosis; Caspases; Cytochrome c; AIF

1. Introduction

Cadmium (Cd) is an environmental pollutant with manyindustrial uses throughout the world. Cadmium is toxic toseveral tissues, most notably causing hepatotoxicity and

Abbreviations: AIF, apoptosis-inducing factor; DEVD-AMC,AcAsp-glu-Val-Asp-(7-amino-4-methylcoumarin); Ac-LEHD-AFC, Ac-Leu-Glu-His-Asp-(7-amino-4-triXuoromethylcoumarin); Z-IETD-AFC, Z-Ile-Glu-Thr-Asp-(7-amino-4-triXuoromethylcoumarin); Ac-DEVD-CHO,Ac-Asp-Glu-Val-Asp-CHO; Z-VAD-fmk, Z-Val-Ala-Asp(Ome)-CH2F;MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PI,propidium iodine; DTT, dithiothretinol; GST, glutathion S-tranferase

* Corresponding author. Tel.: +1 514 987 3000x7680; fax: +1 514 9874647.

E-mail address: [email protected] (C. Jumarie).z Deceased March 24, 2004.

0887-2333/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.tiv.2006.05.005

nephrotoxicity following acute and chronic exposure,respectively. Many studies have described the acute toxiceVects of Cd, but very few have addressed the mechanism oftoxicity at the molecular level. During the last decade, Cdhas been shown to induce apoptosis in vivo (Habeebu et al.,1998; Harstad and Klaassen, 2002; Tzirogiannis et al.,2003) and in vitro (Hart et al., 1999; Achanzar et al., 2000;Kim et al., 2000; Li et al., 2000; Kondoh et al., 2002; Jimiet al., 2004) at various concentrations ranging from 1 to300 �M. Therefore, Cd-mediated toxicity is thought toinvolve, at least in part, the induction of apoptosis. How-ever, the apoptosis signaling induced by Cd is still unclear.

In recent years, the molecular mechanisms responsiblefor apoptosis have been elucidated. A family of intracellu-lar proteases, the caspases, which are directly or indirectlyresponsible for the morphological and biochemical changes

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1332 T.N.D. Pham et al. / Toxicology in Vitro 20 (2006) 1331–1342

characterizing apoptotic phenomena, has been identiWed.Morphologically, apoptosis is characterized by cell shrink-age, chromatin condensation and oligonucleosomal DNAcleavage followed by cell death. In mammalian cells, twomajor pathways for caspase activation are deWned as theextrinsic and the intrinsic pathways. The extrinsic pathwayinvolves the ligation of death receptors (e.g., TNF�-R1 andFas), resulting in caspase-8 activation. This initiatorcaspase activates other caspases (caspase 3, 7) referred to aseVector caspases. In the intrinsic pathway, various forms ofcellular stress result in mitochondrial alteration leading tomitochondrial membrane depolarization and the release ofcytochrome c. In the cytosol, cytochrome c binds to andactivates Apaf-1 which itself activates pro-caspase-9. Activecaspase-9 has been shown to directly cleave and activate theeVector protease, caspase-3. Once the caspase cascade isactivated, downstream molecules, such as caspase-activatedDNase (CAD) and Acinus, will conduct chromatin conden-sation and DNA laddering (Robertson and Orrenius,2000).

More recently, caspase-independent apoptosis has alsobeen observed in several cell types (Carmody and Cotter,2001; Marzo et al., 2001; LoeZer et al., 2002; Ahn et al.,2004). One of the proteins responsible for caspase-indepen-dent chromatin condensation is apoptosis-inducing factor(AIF) (Susin et al., 1999). AIF (57 kD) is a phylogeneticallyancient conserved Xavoprotein that is conWned to the mito-chondrial intermembrane space in healthy cells. Uponlethal signaling, AIF translocates from the mitochondria tothe nucleus, via the cytosol. Although this protein mayinduce caspase-independent peripheral chromatin conden-sation and large-scale DNA fragmentation, the molecularsignaling mechanism remains to be identiWed.

The apoptotic pathway induced by Cd remains contro-versial. In Cd-treated (10�M) rat Wbroblasts (Kim et al.,2000) and human leukemia cells (100 �M) (Li et al., 2000;Kondoh et al., 2002) caspase inhibitors were shown to pre-vent apoptosis, strongly suggesting that caspases play acentral role in Cd-induced cell death. On the other hand,numerous studies performed in vivo focusing on liver injury(Harstad and Klaassen, 2002), or in vitro on porcine kidneycells LLC-PK1 (10�M Cd) (Ishido et al., 1995), MRC-5human fetal lung Wbroblast cells (100 �M Cd) (Shih et al.,2003) and human hepatocarcinoma Hep3B (1–10 �M Cd)(Lemarié et al., 2004) suggested caspase-independent Cd-induced apoptosis. It is conceivable that Cd may inducediVerent apoptotic pathways in diVerent cell types anddepending on the exposure (cell treatment) conditions.

The aim of the present study was to investigate the mech-anism(s) involved in Cd-induced apoptosis in non-malig-nant liver cells using primary cultures of rat hepatocytes bytesting eVect of Cd on: (i) cell morphology; (ii) condensa-tion of nuclear chromatin; (iii) DNA ladder formation; (iv)activation of various caspases; and (v) release of cyto-chrome c or AIF in the cytosol. Our results show that Cdmay induce caspase activation as well as AIF release in thecytosol.

2. Materials and methods

2.1. Materials

Cadmium (CdCl2, Sigma Chemical Co., St. Louis, MO,USA) was dissolved in water, sterilized with 0.22�m Wlters,and added to cultures at the indicated time and concentra-tions. Cell culture reagents Williams’ medium E (WME),minimal essential medium (MEM), modiWed Leibovitz-15 (L-15) (Crabb and Li, 1985) and gentamicin were obtained fromGibco/Life Technology (Burlington, Ont, Canada). Fetalbovine serum (FBS) was purchased from Immunocorp(Montréal, Que., Canada). N,N,N�,N�-Tetramethyl-ethylen-ediamine (Temed), acrylamide, bisacrylamide and proteinmolecular weight standards were obtained from Bio-Rad(Mississauga, Ont., Canada). Proteinase K was from RocheDiagnostic (Mannheim, Germany). The DNA standard 100bp ladder was purchased from Amersham PharmaciaBiotech (Piscataway, NJ, USA). All Xuorescent substratessuch as Ac-Asp-glu-Val-Asp-(7-amino-4-methylcoumarin)(Ac-DEVD-AMC) for caspase-3, Ac-Leu-Glu-His-Asp-(7-amino-4-triXuoromethylcoumarin) (Ac-LEHD-AFC) forcaspase-9, Z-Ile-Glu-Thr-Asp-(7-amino-4-triXuoromethy-coumarin) (Z-IETD-AFC) for caspase-8, as well as thecaspase-3-speciWc inhibitor Ac-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO), the pan-caspase inhibitor Z-Val-Ala-Asp-(Ome)-CH2F (Z-VAD-fmk), and anti-glutathion S-tranferase(GST) antibody (Ab-2) were purchased from Calbiochem(La Jolla, CA, USA). Insulin, Hoechst 33258, 3-(4,5-dimeth-ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), pro-tease inhibitor cocktail, Ribonuclease A and all otherchemicals used in this study were obtained from Sigma–Ald-rich (St. Louis, MO, USA) and were of cell culture grade.The anti-caspase-3 rabbit polyclonal (H-277), the anti-apop-tosis-inducing-factor (AIF) mouse monoclonal (E-1), and theanti-�-tubulin mouse monoclonal (D-10) antibodies werepurchased from Santa Cruz Biotechnology (Santa Cruz, CA,USA). The mouse monoclonal anti-cytochrome c (clone7H8-2C12) antibody was from Pharmingen (San Diego, CA,USA).

2.2. Hepatocyte isolation, cell culture and exposure conditions

Hepatocytes were isolated from male Sprague–Dawleyrats (Charles River Laboratories, Wilmington, MA) weigh-ing 140–180 g by a two-step collagenase perfusion tech-nique (Seglen, 1976). The hepatocytes were puriWed byisodensity Percoll centrifugation, and cell viability wasdetermined by propidium iodine exclusion assay (2 �g PI/mlphosphate buVer) using Xow cytometry (Becton Dicking-son) (Reader et al., 1993). Only cell preparations for whichthe viability was higher than 85% were used. Isolated hepa-tocytes were plated on collagen-coated Petri dishes (60 mmdiameter) at a density of 2£ 106 viable cells/cm2 in WMEcontaining 0.2 �M bovine insulin, 50 �g/ml gentamicin, andsupplemented with 10% FBS. Cultures were maintained at

T.N.D. Pham et al. / Toxicology in Vitro 20 (2006) 1331–1342 1333

37 °C in a humidiWed 5% CO2–air atmosphere. Following a2-h incubation, the cultures were washed with serum-freeMEM and were then kept in L-15 medium for 20 h beforebeing used for subsequent experiments.

Cell cultures were then washed with MEM and suppliedwith L-15 medium containing 1, 5 or 10 �M Cd for speciWctimes. In some cases, hepatocytes were preincubated withthe pan-caspase inhibitor (Z-VAD-fmk, 100 �M) or thecaspase-3 (-like) protease inhibitor (Ac-DEVD-CHO,20 �M) (Brauns et al., 2005; Cao et al., 2005; Zhang et al.,2006) for 1 h prior to incubation with Cd.

2.3. MTT assay

Cell viability was determined by a colorimetric assay basedon the ability of viable cells to metabolize 3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). MTT is ayellow tetrazolium salt which forms a blue formazan dye pre-cipitate that can be extracted using organic solvent when it isreduced by the mitochondria of metabolically active cells(WaterWeld et al., 1998). Twenty-hour-old hepatocytes,seeded at a density of 2£104 cells/well on collagen coated 96-well plates, were exposed to various levels of Cd for 4h, 12 hor 24 h or to 2ng/ml TGF-� known to induce apoptosis inhepatocytes (Diez-Fernandez et al., 2002). Three hours beforethe end of the cell incubation periods, the culture mediumwas removed and 0.2 ml of MTT (from a stock of 5mg/ml)was added to each culture well. After an additional 3-h incu-bation at 37°C, the medium was removed and formazan crys-tals were dissolved in 0.2 ml DMSO for 30min at 37 °C. Theoptical density (DO) of each well was measured at 560nmusing a microplate reader (Molecular Devices, USA). The lin-ear relationship between DO measurements and cell densityhave been veriWed under our experimental conditions. Thedata are expressed as a percentage of control viability mea-sured in untreated cells.

2.4. Hoechst 33258 Xuorescence

Apoptotic morphological changes in the nuclear chro-matin were detected by staining with the DNA bindingXuorochrome Hoechst 33258 (bisbenzimide). Hoechst33258 exhibits Xuorescence enhancement upon binding toA-T rich regions of double stranded DNA. Cd- or TGF-�-treated hepatocytes were washed three times with phos-phate-buVer saline (PBS) prior to incubation with Hoechst33258 (50 �M in PBS) for 10 min in the dark at room tem-perature. Afterward, Hoechst was removed and cells werewashed with PBS prior to visualization (green Xuorescence,Ex: 395. Em: 470 nm) using an inverted Xuorescence micro-scope (model IM, Carl Zeiss Canada) with the followingcriteria. Normal nuclei were those in which the chromatinwas unaltered and spread uniformly throughout the entirenucleus. Condensed chromatin was located at the peripheryof the nuclear membrane and appeared as a half-moonform. Fragmented chromatin was characterized by ascattered, drop-like structure, located on the area of the

original nucleus. The nuclei of apoptotic cells appearedsmaller and shrunken when compared to intact cells. Insome cases, cells were also stained with PI (60�g/ml) tovisualize necrotic cells (red Xuorescence). No necrotic cellscould be observed for any of the incubation with 1�M Cd,whereas few necrotic cells (less than 10%) could be visual-ized for the 24 h-exposure to 5 �M Cd exclusively for some,but not all, samples (data not shown).

2.5. DNA fragmentation assay

Oligonucleosomal DNA fragmentation was detected byagarose gel electrophoresis. Following incubation with Cdor TGF-�, cells were washed twice with PBS and lysed with500 �l of modiWed buVer (pH 8.0) containing 10 mM Tris,5 mM EDTA and 0.5% SDS for 30 min on ice. RNase A(100 �g/ml) was added to the cell lysate for an additional30 min at 37 °C. Proteins were removed by incubation withproteinase K (150�g/ml) at 54 °C for 4 h. DNA sampleswere then heated for 1 h at 65 °C. After these digestionsteps, DNA was precipitated in 3 M sodium acetate pH 5.3(1:10 vol.) and cold ethanol (1:2 vol.) overnight at ¡20 °C.Afterwards, samples were centrifuged for 15 min at 15,000g.The pellets, which contain the precipitated DNA, were dis-solved in 30 �l of Tris–EDTA buVer (10 mM Tris–HCl,1 mM EDTA, pH 7.4). DNA quantiWcation was assessedspectrophotometrically (Beckman DU® 650) at 260 nm.DNA samples (10 �g) were Wnally separated by 1.5% aga-rose gel electrophoresis with Tris–Borate/EDTA buVer at100 V for 1 h 30, then stained with ethidium bromide andtransilluminated with UV light for photography.

2.6. Measurement of caspase-like protease activity

Caspase-3, -8 or -9-like protease activities were assessedby measuring the proteolytic cleavage of the Xuorometricand speciWc substrates Ac-DEVD-AMC, Z-IETD-AFC orAc-LEHD-AFC, respectively. BrieXy, after incubation withCd for the indicated times, the hepatocytes were scraped oVthe Petri dishes with a rubber policeman and resuspended in200�l homogenization buVer (250 mM sucrose, 1 mMEDTA, 10 mM sodium pyrophosphate, 10 mM Tricine,2 mM MgCl2, pH 8.0). The cell suspensions were gently son-icated for 10 min on ice, and homogenates were centrifuged(L8-70M Ultracentrifuge, Beckman Instruments Inc., CA,USA) at 100,000g for 1 h at 4 °C. All caspase assays werecarried out with 200�g protein from each sample on micro-plates with the appropriate protease assay buVer and sub-strate. Assays for caspase-3 were performed in proteasebuVer [20 mM HEPES, 10% glycerol, 2 mM dithiothretinol(DTT), pH 7.5] using 50�M Ac-DEVD-AMC as the sub-strate. For caspase-8 and caspase-9, 100�M Z-IETD-AFCand Ac-LEHD-AFC were used as substrate, respectively,whereas the protease buVer (pH 7.5) contained: 100 mMHEPES and 10 mM DTT. The liberation of AMC(�exD380, �emD460) or AFC (�exD400, �emD505) as aresult of substrate cleavage was monitored for 60 min. A


SpectramaxGemini spectroXuorometer (Molecular Devices,Sunnyvale, CA) was used and the corresponding activity(Vmax/min) values were obtained by the SOFTmax Pro Soft-Ware (Molecular Devices). Data are expressed relative tocontrol activity measured in untreated cells.

2.7. Immunodetection of cytochrome c, caspase-3, and AIF

Following incubation with Cd, cells were washed twicewith ice-cold PBS and harvested in 200�l ice-cold homoge-nization buVer (250 mM sucrose, 1 mM EDTA, 10 mMsodium pyrophosphate, 10 mM tricine, 2 mM MgCl2) sup-plemented with protease inhibitors ([4-(2-aminoethyl)-ben-zene-sulfonylXuoride] hydrochloride, apotinin, bestatin,leupeptin, pepstatin A). Cells were homogenized using ahand potter (Kontes glass Co, Duall® 22, Fisher, Que., Can-ada) and were centrifuged at 1500g for 5 min. The resultingsupernatants were subjected to a two-step centrifugation:10,000g for 10 min, and then at 100,000g for 1 h both at 4 °C.The resulting supernatants representing the cytosolic frac-tion (which purity was veriWed by the enrichment of GSTestimated by immunodetection) were used to detect cyto-chrome c, caspase-3, and AIF by Western blotting. To doso, 20–30�g protein from each sample were mixed with Lae-mmli’s sample buVer [0.5 M Tris–HCl, 10% glycerol, 10%SDS, 0.01% bromophenol blue, 12.5% �-mercaptoethanol,pH 6.8] (Laemmli, 1970) which was subsequently boiled for5 min and loaded onto a 12% SDS–polyacrylamide gel. Pro-teins were transferred to a polyvinylidene diXuoride mem-brane using a semidry blotting apparatus (Milipore, Seattle,USA) and the non-speciWc sites were blocked overnight at4 °C with 5% non-fat milk in Tris-buVer saline with Tween(TBS + T) (2 mM Tris, 13.7 mM NaCl and 0.1% Tween 20,pH 7.4) under mild agitation. The membranes were thenwashed with TBS + T for 1 h, and primary antibody wasadded for 1 h at room temperature (anti-caspase-3 1:500;anti-cytochrome C 1:1000 and anti-AIF 1:500, diluted inTBS + T 1% BSA). Thereafter, the membranes were washedfor 1 h prior to incubation for a subsequent 1 h with appro-priate secondary antibodies conjugated to horseradish per-oxidase (diluted 1:1000 in TBS + T containing 5% non-fatmilk). Blots were developed for visualization using anenhanced chemiluminescence plus detection kit (Perkin-Elmer, Life Sciences, Boston, MA, USA). The resulting mem-branes were exposed to Fuji medical X-ray Wlm. The proteinbands were followed by densitometry scanning using aChemilmager™ 5500 Fluorescence system equipped withthe analysis software AlphaEase FC™ (Alpha InnotechCorporation, San Leandro, CA 94577, USA). To test for theamount of protein loading, the blots were stripped and re-probed with antibody (diluted 1:1000) against �-tubulin.

2.8. Determination of protein content and statistical analysis

Protein content was determined according to Bradford(1976) using serum albumin as the calibration standard. Theexperiments were performed at least three times on diVerent

hepatocyte preparations. Statistical analyses were performedwith the two-tailed Student t-test for unpaired data on smallsamples using InStat software (GraphPad Software). Statis-tical signiWcance was assessed at the p < 0.05 level.

3. Results

3.1. Concentration and time-dependent decrease in MTT activity

We Wrst characterized the concentration-dependentcytotoxic eVect of Cd on primary cultures of rat hepato-cytes as a function of time. As shown in Fig. 1, cell exposureto 1 �M Cd for up to 24 h only slightly aVected cell viabilityas revealed by MTT measurements compared to controlvalues estimated in untreated cells. At higher concentra-tions (5 and 10 �M), Cd signiWcantly decreased MTT activ-ity with a concentration-dependent eVect observed at 24 h:at this time, MTT activity was reduced by 39% and 67%compared to control values, respectively (black columns).As expected, cell treatment with 2 ng/ml TGF-� for 24 hresulted in a signiWcant 38% decrease in cell viability. Fromthese data, 1 and 5 �M (Cd concentrations with moderatetoxic impact) were chosen for subsequent experiments onCd-induced apoptosis.

3.2. Cadmium-induced apoptosis

Microscopy observation of hepatocytes exposed to 1 or5�M Cd for 24 h revealed marked morphological changes.Whereas the size of control cells appeared normal withround and homogenous nuclei, Cd-treated cells exhibitedtypical features of apoptosis such as plasma membraneblebbing and cell shrinkage (data not shown). In accordancewith these morphological changes, apoptosis was furtherconWrmed by the observation that Cd also induced bothchromatin condensation and DNA fragmentation. The

Fig. 1. Cd cytotoxicity as measured by the MTT assay. Rat hepatocyteswere exposed to 1, 5 or 10 �M Cd for 4 h (open columns), 12 h (gray col-umns) or 24 h (black columns). A 24-h exposure to TGF-� (2 ng/ml) wasalso performed for comparison. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was performed as described in Sec-tion 2. Data are expressed relative to control values measured in untreatedcells (100% viability). Data shown are means § SD evaluated for fourdiVerent cell preparations. (¤) SigniWcant diVerence (p 6 0.05) comparedwith control values.


Xuorescence intensity of cell staining with Hoechst 33258increased as early as 3 h following exposure to either 1 and5 �M Cd, respectively, compared to untreated control cells,showing Cd-induced condensation of nuclear chromatin(Fig. 2). The highest Xuorescence intensity was observed incells exposed to 5�M for 24 h. As for a 24-h treatment with2 ng/ml TGF-�, a 24-h and 12-h exposure to 1 and 5�M Cd,respectively, markedly increased the number of apoptoticcells containing condensed nuclear fragments and irregularaggregation of chromatin. In comparison, untreated cellsexhibited low levels of Xuorescence with unaltered chroma-tin, which was uniformly spread throughout the nuclei.

Typical formation of DNA» 200 bp fragments, whichshow up as ladders on agarose gels, was also observed inCd-treated cells in a time-dependent manner, which is thebiochemical hallmark of apoptosis (Fig. 3). Kinetic analysisof DNA fragmentation revealed that formation ofDNA» 200 bp fragments in rat hepatocytes became appar-ent at 12–24 h and 6 h under exposure conditions with 1 and5 �M Cd, respectively. As shown in Fig. 3B, large quantitiesof DNA fragments were detected following a 24-h exposureto 5 �M. Under these experimental conditions, DNA frag-mentation was even more pronounced compared to celltreatment with TGF-�. Thus, a good relationship betweenCd-induced chromatin condensation and DNA fragmenta-tion was observed. Taken together, all these results revealthat Cd-induced apoptosis in rat hepatocytes is time- andconcentration-dependent.

3.3. Caspase activation

The involvement of caspases in Cd-induced apoptosiswas tested using speciWc Xuorogenic assays for caspase-3,

-8, and -9. Results of these studies are shown in Fig. 4 whereit can be seen that exposure to Cd led to a signiWcant time-dependent enhancement in caspase-3 (Fig. 4A) and -9(Fig. 4C) activity up to 2.5-fold at 24 h, compared with con-trol values measured in untreated hepatocytes. Stimulationof caspase activity was also concentration-dependent: sig-niWcant activation of caspase-3 could be detected as early as6 h and 12 h under exposure conditions with 5 and 1 �MCd, respectively. There was a concomitant increase incaspase-3 and -9 activities (Fig. 4A and C). Cadmium alsomodiWed caspase-8 activity, but the stimulatory eVect wasmuch lower compared to caspase-3 and -9, and the time-and concentration-dependent pattern was diVerent(Fig. 4B). Note that caspase-8 was also found to be muchless sensitive to TGF-� than caspase-3 and -9.

The activation of caspase-3 was further supported byWestern blot analysis demonstrating the time- and concen-tration-dependent processing of pro-caspase as revealed bythe signiWcant decrease in the level of the inactive form (a32 kDa) in the presence of 1 �M (Fig. 6A and B) but morepronouncedly with 5�M Cd (Fig. 7A and B) with diVerenttime-course relationship depending on the Cd concentra-tion. For comparison, high levels of the pro-form weredetected in the cytosol of control cells and contents of pro-caspase 3 did not decrease as much as a function of time inculture, although basal apoptotic processes are likely tooccur in primary cultures of hepatocytes (Fig. 5).

3.4. Cytochrome c release

One of the apoptogenic proteins involved in the mito-chondria-induced activation of caspases is cytochrome c.When it is released in the cytosol in the presence of suYcient

Fig. 2. Cd-induced nuclear chromatin condensation. Rat hepatocytes stained with Hoechst 33258 were exposed to 1 or 5 �M Cd for 3 h, 6 h, 12 h, or 24 h.Photograph series (320£ magniWcation) is representative of four diVerent experiments. Untreated control cells and cells exposed to TGF-� (2 ng/ml) for24 h were used as negative and positive control samples, respectively.


ATP, cytochrome c forms the apoptosome with Apaf-1 andthe signaling procaspase-9 (Hengartner, 2000). This apop-tosome then activates caspase-9, which in turn can activatethe executioner caspase-3. Therefore, to get insights intoapoptotic signaling upstream of Cd-induced caspase-3 acti-vation, the kinetics of cytochrome c appearance in the cyto-sol was studied by Western blot analysis. As shown in Figs.6 and 7A and C, cell exposure to Cd resulted in a signiWcanttime-dependent increase in the level of cytosolic cyto-chrome c as early as 3 h with a 2-fold and a 3-fold increaseobserved at 24 h with 1 and 5�M Cd, respectively. In con-trast, cyctochrome c detection in control cell cultures wasvery weak (Fig. 5). These results suggest that release ofmitochondrial cytochrome c may be responsible for caspase-3activation. Note that the early Cd-induced increase in the

Fig. 3. Oligonucleosomal DNA fragmentation. Cell cultures were exposedto 1 �M (A) or 5 �M (B) Cd for the indicated times. DNA was extractedfrom cell samples and DNA fragments were assessed on a 1.5% agarosegel containing ethidium bromide as described in Section 2. M: 100 bp lad-der standard marker. The data shown are representative of three diVerentexperiments. DNA from TGF-�-treated cells (2 ng/ml for 24 h) was alsoused for comparison.

cytosolic levels of cytochrome c was similar for both 1 and5 �M Cd, whereas a concentration-dependent relationshipwas more pronounced for longer exposure times with 5 �MCd.

3.5. EVect of caspase inhibitors on Cd-induced apoptosis

Although caspase cascade has been shown to play a crit-ical role in apoptosis, caspase activation is not the onlypathway leading to apoptosis (Zamzami and Kroemer,1999). To test whether caspase-independent pathways may

Fig. 4. Kinetics of Cd-induced caspase activity. Caspase-3 (A), caspase-8(B), and caspase-9 (C) activities were measured following a 3-h (open col-umns), 6-h (dashed columns), 12-h (gray columns) or 24-h (black col-umns) exposure to 1 or 5 �M Cd using the Xuorometric speciWc substratesAc-DEVD-AMC, Ac-IETD-AFC and Ac-LEHD-AMC, respectively.Caspase activities in TGF-�-treated cells (2 ng/ml for 24 h) were also mea-sured for comparison. Data are expressed as x-fold over control valuesestimated in untreated cells. Data shown are means § SD evaluated forthree diVerent cell preparations. (¤) SigniWcant diVerence (p 6 0.05) com-pared with control values.


be involved in Cd-induced apoptosis in rat hepatocytes,cells were incubated with 100 �M Z-VAD-fmk, a broadspectrum caspase inhibitor, or 20 �M Ac-DEVD-CHO, ainhibitor of caspase-3 (-like) proteases, 1 h prior to expo-sure to Cd. As shown in Fig. 8, neither of the two inhibitorssigniWcantly reduced the level of Hoechst staining in pri-

Fig. 5. Pro-caspase-3 processing as well as cytochrome c and AIF releasein untreated control cells as a function of time in culture. Cytosol proteinswere separated on a 12% SDS–polyacrylamide gel, transferred to a PVDFmembrane, and immunoblotted with the corresponding antibodies. West-ern blots of cytochrome c (15 kDa), AIF (57 kDa), and pro-caspase-3(32 kDa) proteins are representative of three diVerent experiments.

Fig. 6. Kinetics of pro-caspase-3 processing and cytochrome c release incells exposed to 1 �M Cd. Following hepatocyte exposure to 1 �M Cd forthe indicated times, cytosol proteins were separated on a 12% SDS–poly-acrylamide gel, transferred to a PVDF membrane, and immunoblottedwith the corresponding antibodies. Representative Western blots (A) anddensitometric analysis of pro-caspase-3 (32 kDa) (B) and cytochrome c(15 kDa) (C) proteins. Data shown are means § SD evaluated for threediVerent cell preparations. (¤) SigniWcant diVerence (p 6 0.05) comparedwith control values.

mary cultures following a 24-h exposure to 1 or 5 �M Cd,although Z-VAD-fmk succeeded in preventing apoptosisinduced by a 24 h-exposure to TGF-�. Similarly, thecaspase inhibitors failed to prevent Cd-induced DNA frag-mentation at 1�M (Fig. 9A) as for 5 �M (Fig. 9B), suggest-ing the presence of caspase-independent pathways.

3.6. Release of apoptosis-inducing factor

AIF is a mitochondrial protein that can be translocatedinto the cytosol as well as the nuclei, where it elicits DNAfragmentation independently of caspase activation. Toinvestigate its possible involvement in Cd-induced apopto-sis in rat hepatocytes, Western blot analyses were con-ducted on the cellular cytosolic protein fraction to studythe kinetics of AIF release in the cytosol. As shown in Figs.10 and 11, cytosolic AIF proteins were detected as early as3 h following exposure to either 1 or 5�M Cd. Cadmium-induced mitochondrial AIF release was time- and concen-tration-dependent. In contrast, cytosolic levels of AIF were

Fig. 7. Kinetics of pro-caspase-3 processing and cytochrome c release incells exposed to 5 �M Cd. Following hepatocyte exposure to 5 �M Cd forthe indicated times, cytosol proteins were separated on a 12% SDS–poly-acrylamide gel, transferred to a PVDF membrane, and immunoblottedwith the corresponding antibodies. Representative Western blots (A) anddensitometric analysis of pro-caspase-3 (32 kDa) (B) and cytochrome c(15 kDa) (C) proteins. Data shown are means § SD evaluated for threediVerent cell preparations. (¤) SigniWcant diVerence (p 6 0.05) comparedwith control values.


low and constant as a function of time in culture in controlcells (Fig. 5).

4. Discussion

Cadmium has been identiWed as a Group I carcinogen inhumans by the International Agency for Research on Can-cer (IARC, 1993). Its dispersion in the environment hasincreased steadily over the past decades due to its wide-spread use in industries and other anthropogenic activities.Environmental pollution by Cd is a worldwide problem, par-ticularly because of its long biological half-life estimated tobe 10–30 years in humans (Goyer, 1997); there is no eVectivetherapy for Cd poisoning. At low concentrations, Cd stimu-lates DNA synthesis and cell proliferation (Beyersmann andHechtenberg, 1997). This stimulatory eVect could be relatedto the induction of transcription of speciWc genes, such asp53, c-jun and c-myc, as well as interference with the mito-gen-activated protein kinase (MAPK) signaling pathwayswhich regulate proliferation. On the other hand, dependingon the exposure conditions and the model used, Cd may alsoinduce either necrosis or apoptosis in mammalian cells(Hamada et al., 1997; Robertson and Orrenius, 2000; Ishidoet al., 2002; Lemarié et al., 2004). Apoptosis might contributeto the hepatotoxicity of Cd in vivo (Habeebu et al., 1998;Tzirogiannis et al., 2003). The present study demonstrate

that Cd induces apoptosis in primary cultures of rat hepato-cytes in a time- and concentration-dependent manner (Figs.2 and 3). This phenomenon occurred under exposure condi-tions where Cd only slightly aVected mitochondria function(Fig. 1). These results are consistent with in vivo data show-ing liver apoptosis in mice 9–14 h following i.p. injection with5–60�mol Cd/kg, whereas necrosis was predominant 14–48 hafter Cd injection (Habeebu et al., 1998).

Caspases, a family of cysteine-dependent aspartate-directed proteases, play critical roles in the initiation andexecution of apoptosis (Budihardjo et al., 1999). Caspasesare synthesized as pro-enzymes, which are cleaved (execu-tioner caspases) or which dimerize (initiator caspases) toform active enzymes. The subcellular localization of the pro-and the active forms of caspases has been established(Zhivotovsky et al., 1999). The initiator caspases (e.g.,caspase-2, -8, -9, -10 and -12) respond to various stimuli ofthe extrinsic or intrinsic pathways, and subsequently cata-lyze the activation of more abundant and catalyticallyrobust eVector caspases (e.g., caspase-3, -6 and -7). The latterare mainly responsible for the proteolytic cleavage eventsleading to the typical apoptotic phenotype. It has been pre-viously demonstrated that Cd induces apoptosis in diVerentcell types by a pathway dependent on caspase activation(Kim et al., 2000; Li et al., 2000; Kondoh et al., 2002; Gen-nari et al., 2003). In these cases, Cd strongly activated the

Fig. 8. EVects of caspase inhibitors on Cd-induced apoptosis. Rat hepatocytes stained with Hoechst 33258 were exposed to 1 or 5 �M Cd for 24 h. TGF-�-treated cells (2 ng/ml) for 24 h were also used for comparison. In some cases, cells were pretreated for 1 h with a pan-caspase inhibitor (Z-VAD-fmk,100 �M) or with an inhibitor of caspase-3-like protease (Ac-DEVD-CHO, 20 �M) before exposure to Cd. Photograph series (320£ magniWcation) is repre-sentative of three diVerent experiments.


executioner caspase-3, the inhibition of which preventedapoptosis. Our kinetic studies on caspase activation revealeda signiWcant and concomitant increase in caspase-9 andcaspase-3 activities in Cd-treated compared to untreatedcells (Fig. 4A and C). A 24-h exposure to 1 or 5�M Cd ledto similar or even higher levels of caspase induction com-pared to treatment with TGF-�, a well known inducer ofapoptosis in hepatocytes (for review, see Bissell, 2001). Notethat some studies have provided evidence of the involve-ment of reactive oxygen species (ROS) as well as cyto-chrome c release and activation of the Apaf-1 apoptosomecomplex in the mitochondrial-dependent apoptosis inducedby TGF-� (Thannickal and Fanburg, 1995; Freathy et al.,2000; Herrera et al., 2001). Elevated intracellular ROSwould lead to the disruption of mitochondrial membranepotential (�m), which in turn results in cytochrome c release,and caspase-9 followed by caspase-3 activation. Our results

Fig. 9. EVect of caspase inhibitors on Cd-induced DNA fragmentation.Cells were exposed to 1 �M (A) or 5 �M (B) Cd for 24 h. In some cases,cells were pretreated for 1 h with a pan-caspase inhibitor (Z-VAD-fmk,100 �M) or an inhibitor of caspase-3 (Ac-DEVD-CHO, 20 �M) beforeexposure to Cd. DNA was extracted from cell samples, and DNA frag-ments were assessed on a 1.5% agarose gel containing ethidium bromideas described in Section 2. M: 100 bp ladder standard marker. The datashown are representative of three diVerent experiments.

also conWrm the involvement of this pathway in primarycultures of rat hepatocytes, as TGF-� had little eVect oncaspase-8 activity compared to caspase-9 and -3 (Fig. 4).Furthermore, a time-dependent processing of pro-caspase-3(32 kDa) was clearly observed in Cd-treated cells with

Fig. 10. Kinetics of AIF release in the cytosol of cells exposed to 1 �M Cd.Following hepatocyte exposure to 1 �M Cd for the indicated times, cyto-sol proteins were separated on a 12% SDS–polyacrylamide gel, trans-ferred to a PVDF membrane, and immunoblotted with the correspondingantibodies. Representative Western blots (A) and densitometric analysisof AIF (57 kDa) (B) proteins. Data shown are means § SD evaluated forthree diVerent cell preparations. (¤) SigniWcant diVerence (p 6 0.05) com-pared with control values.

Fig. 11. Kinetics of AIF release in the cytosol of cells exposed to 5 �M Cd.Following hepatocyte exposure to 5 �M Cd for the indicated times, cyto-sol proteins were separated on a 12% SDS–polyacrylamide gel, trans-ferred to a PVDF membrane, and immunoblotted with the correspondingantibodies. Representative Western blots (A) and densitometric analysisof AIF (57 kDa) (B) proteins. Data shown are means § SD evaluated forthree diVerent cell preparations. (¤) SigniWcant diVerence (p 6 0.05) com-pared with control values.


higher levels of disappearance of the pro-form with 5�Mcompared to 1�M Cd (Figs. 6 and 7). On the other hand, Cdhas little eVect on caspase-8 activity (Fig. 4B), suggestingthat the death-receptor pathway might not contributeappreciably to the observed Cd-induced apoptosis. Similarresults have been obtained for Cd-induced hepatotoxicity inTNF-�-null mice (Harstad and Klaassen, 2002), whereasTNF-� can be directly cytotoxic to hepatocytes (Adamsonand Billings, 1992; Tartaglia et al., 1993).

Our data also show that Cd induces mitochondrial cyto-chrome c release, a critical step in the formation of apopto-somes, which ultimately lead to caspase-3 activation.Western blot analysis revealed signiWcant levels of cytosoliccytochrome c as early as 3 h following exposure to Cd (Figs.6 and 7). Thus, the release of cytochrome c seems to occurprior to caspase-3 activation. All these results show theinvolvement of a mitochondrial-mediated pathway inapoptosis induced by Cd. Mitochondrial release of cyto-chrome c, caspase-3 activation and DNA fragmentation allare hallmarks of caspase-mediated programmed cell death(Budd et al., 2000). Although Cd is not a metal that cata-lyzes the Fenton reaction, Cd toxicity is believed to berelated, at least in part, to the generation of ROS (Koizumiet al., 1996; Hart et al., 1999; Robertson and Orrenius,2000; Shih et al., 2003; Gennari et al., 2003). Mitochondria,one of the major ROS-producing organelles, are alsobelieved to play a critical role as mediator in the initiationof cell death via necrotic vs. apoptotic pathways. Usinginhibitors of the electron transport chain and the mito-chondrial permeability transition pore, Shih et al. (2004)demonstrated that apoptogenic activity of Cd in the humanfetal lung Wbroblastic cells MRC-5 is partially related tooxidative stress. In this study, collapse of �m was observedduring the early apoptotic process. Cadmium may alsodirectly inhibit the electron transfer chain and induce reac-tive oxygen species in isolated mitochondria of pig liver,brain, and heart (Wang et al., 2004). Note that Cd-induced�m dissipation has also been shown not to necessarilyrequire ROS (Bolduc et al., 2004).

AIF is a mitochondrial protein whose translocation inthe cytosol as well as in the nucleus mediates caspase-inde-pendent apoptosis in a number of model systems (Susinet al., 1999; Daugas et al., 2000; Joza et al., 2001; Yu et al.,2002; Zhang et al., 2002). AIF is a »67 kDa Xavoproteinlocated in the intermembrane space of the mitochondria. Inresponse to apoptogenic stimuli, AIF can be processed(cleaved) to its mature »57 kDa form and release into thecytosol. Subsequent translocation of AIF to the nucleusinduces high molecular weight DNA fragmentation (50 Kb)and chromatin condensation (Daugas et al., 2000; Zhanget al., 2002). The mechanism underlying these AIF-mediatedevents leading to cell death is unclear (AIF itself does nothave endonuclease activity), but AIF might bind to DNAand recruit proteases and nucleases responsible for chroma-tin condensation (Hong et al., 2004). AIF has recently beenshown to be released into the cytoplasm of cells exposed toanticancer drugs or environmental contaminants including

Cd (Cande et al., 2002; Shih et al., 2003; Lemarié et al.,2004). Shih et al. (2003, 2004) have demonstrated in lungMRC-5 cells that Cd-induced ROS formation leads to �mdissipation and AIF translocation in the nucleus within 4 hfollowing exposure to 25–150�M Cd. In addition, the mito-chondria-derived proteins, endonuclease G (Endo G) andHtrA2/Omi, have been identiWed as potential caspase-inde-pendent apoptotic mediators (Suzuki et al., 2001). In thehuman hepatoma cell line Hep3B, Cd-induced apoptosiswas related to a time-dependent loss in �m followed by therelease of both Endo G and AIF in the cytosol (Lemariéet al., 2004). Our own data reveal that inhibition of caspases,particularly caspase-3, does not prevent apoptosis (Figs. 8and 9) and that Cd levels as low as 1�M promote IAFrelease in the cytosol (Figs. 10 and 11). Interestingly, parallelkinetic studies on cytochrome c and IAF have also revealeda concomitant release of both mitochondrial-associatedproteins. All together these results suggest the presence ofmitochondrial-mediated caspase-independent pathways inCd-induced apoptosis in primary cultures of rat hepatocytes.

In conclusion, our results demonstrate that low levels ofCd induce apoptosis in rat hepatocytes in a time- and con-centration-dependent manner. Our Wndings suggest that themitochondria play a critical role in the early apoptoticevent, triggering the release of both cytochrome c and AIFproteins. The respective contribution of caspase-dependentand caspase-independent pathways in Cd-induced apopto-sis remains to be estimated; especially that caspase activa-tion does not necessarily lead to apoptosis, and could, insome cases, represent a sign rather than a mechanism of celldeath (Kroemer and Martin, 2005).

Acknowledgements

This work was supported by the Natural Sciences &Engineering Research Council of Canada, NSERC (Col-laborative Health Research project, grant CHR238001-00;and Discovery grant, C.J., RGPIN-203202).

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