Cadmium-induced apoptosis through the mitochondrial pathway in rainbow trout hepatocytes:...

Aquatic Toxicology 69 (2004) 247–258

Cadmium-induced apoptosis through the mitochondrialpathway in rainbow trout hepatocytes:

involvement of oxidative stress

C. Risso-de Faverney∗, N. Orsini, G. de Sousa, R. Rahmani

Laboratoire de Toxicologie Cellulaire, Moléculaire et Génomique, INRA—Centre de Recherches de Sophia-Antipolis,UMR INRA-UNSA 1112, 400 Route des Chappes, BP 167, 06903 Sophia-Antipolis Cedex, France

Received 23 September 2003; received in revised form 5 May 2004; accepted 28 May 2004

Abstract

Cadmium (Cd) induces oxidative stress and apoptosis in trout hepatocytes. We therefore investigated the involvement ofthe mitochondrial pathway in the initiation of apoptosis and the possible role of oxidative stress in that process. This studydemonstrates that hepatocyte exposure to Cd (2, 5 and 10�M) triggers significant caspase-3, but also caspase-8 and -9 activationin a dose-dependent manner. Western-blot analysis of hepatocyte mitochondrial and cytosolic fractions revealed that cytochromec (Cyt c) was released in the cytosol in a dose-dependent manner, whereas the pro-apoptotic protein Bax was redistributed tomitochondria after 24 and 48 h exposure. We also found that the expression of anti-apoptotic protein Bcl-xL, known to be regulatedunder mild oxidative stress to protect cells from apoptosis, did not change after 3 and 6 h exposure to Cd, then increased after 24and 48 h exposure to 10�M Cd. In the second part of this work, two antioxidant agents, 2,2,6,6-tetramethylpiperidinyl-1-oxyl(TEMPO) (100�M) andN-acetylcysteine (NAC, 100�M) were used to determine the involvement of reactive oxygen species(ROS) in Cd-induced apoptosis. Simultaneously exposing trout hepatocytes to Cd and TEMPO or NAC significantly reducedcaspase-3 activation after 48 h and had a suppressive effect on caspase-8 and -9 also, mostly after 24 h. Lastly, the presence ofeither one of these antioxidants in the treatment medium also attenuated Cd-induced Cytc release in cytosol and the level of Baxin the mitochondria after 24 and 48 h, while high Bcl-xL expression was observed. Taken together, these data clearly evidencedthe key role of mitochondria in the cascade of events leading to trout hepatocyte apoptosis in response to Cd and the relationshipthat exists between oxidative stress and cell death.© 2004 Elsevier B.V. All rights reserved.

Keywords: Fish; Hepatocytes; Cadmium; Mitochondria; Oxidative stress; Apoptosis

∗ Corresponding author. Tel.:+33-492386584;fax: +33-492386655.

E-mail addresses: [email protected], [email protected](C. Risso-de Faverney).

1. Introduction

Cadmium (Cd) is an industrial and environmen-tal pollutant that is toxic to a number of tissues likeliver, kidney and thymus (Morselt et al., 1988; Goeringet al., 1995). At the ultrastructural level, the hepa-totoxic effects of Cd include nuclear condensationand mitochondrial swelling. Cd has been shown to

0166-445X/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.aquatox.2004.05.011

248 C. Risso-de Faverney et al. / Aquatic Toxicology 69 (2004) 247–258

induce apoptosis in the epithelial cells of carp (Igeret al., 1994) and trout skin (Lyons-Alcantara et al.,1998). Moreover, this heavy metal-induced apoptosisin mammalian cell lines and a possible mechanismhas been proposed (El-Azzouzi et al., 1994; Kondohet al., 2001). So, apoptosis is thought to be involvedin Cd-induced toxicity although the action mechanismhas yet to be understood.

The potential role of reactive oxygen species (ROS)and free radicals have been highlighted as mediatorsfor apoptotic cell death, suggesting the involvement ofoxidative stress in Cd-induced apoptosis (Buttke andSandström, 1994; Forrest et al., 1994; McConkey andOrrenius, 1994; Stohs et al., 2001). Previous studiesby our team revealed that Cd intoxication of culturedtrout hepatocytes caused apoptosis in vitro (Risso-deFaverney et al., 2001). It was therefore hypothesisedthat Cd-induced apoptosis was dependent on thegeneration of ROS (Risso-de Faverney et al., 2001).However, the mechanisms by which oxidative stresstriggers the apoptotic process were not elucidated inthese studies.

Apoptosis is a spontaneous cell death controlledby an internally encoded suicide program and pro-vides a means for eliminating critically damagedcells without disturbing tissue structure or function.Many investigations have been carried out to try tounderstand the molecular processes involved in apop-totic cell death. Recently, growing attention has beenfocused on the crucial role of mitochondria in celldeath regulation (Green and Reed, 1998; Kroemerand Reed, 2000). Mitochondria display signs of outerand/or inner membrane permeabilization when ex-posed to a variety of pro-poptotic secondary messen-gers. Thus, cytochromec (Cyt c), which is normallyconfined in the mitochondrial intermembrane space,is found in the cytosol of cells undergoing apoptosis(Yang et al., 1997; Kluck et al., 1997). Mitochondrialmembrane permeabilization involves the formationof a dynamic, multiprotein complex on the site ofcontact between the inner and outer mitochondrialmembranes (Crompton, 1999). This process precedesnuclear apoptosis and is inhibited by the presence ofBcl-2 on these organelles (Yang et al., 1997; Klucket al., 1997; Shimizu et al., 1999). Cytosolic Cytcbinds on Apaf-1 and dATP, resulting in recruitmentand activation of pro-caspase-9. Activated caspase-9proteolytically activates caspase-3, so as to orches-

trate the biochemical execution of programmed celldeath (Li et al., 1997; Zou et al., 1997). MitochondrialCyt c release into the cytosol also occurs in apoptosismediated by death receptors (Scaffidi et al., 1999) oroverexpression of pro-apoptotic proteins (Bax, Bid)(Luo et al., 1998; Shimizu et al., 1999). Accord-ingly, Bax translocation from the cytosol (where it isa monomer) to mitochondrial membranes (where itforms a dimer or higher order oligomers) has beenreported in a wide array of apoptosis-inducing cir-cumstances. In contrast, Bcl-xL, an anti-apoptoticmember of the Bcl-2 family, is capable of preventingCyt c release, while also significantly inhibiting celldeath (Finucane et al., 1999; Shimizu et al., 1999).It has been recently reported that cadmium causesmitochondrial dysfunction and caspase-9 activationfollowed by apoptosis (Kondoh et al., 2002; Watjenet al., 2002). However, the role of mitochondria inthis apoptotic cell death process is unclear.

The aim of this work therefore was to investigatethe implication of the mitochondria, the Bcl-2 familymembers and caspase activation in Cd-induced apop-tosis in trout hepatocytes and their possible link to theoxidative stress generated by this heavy metal. Ni-troxide radical 2,2,6,6-tetramethylpiperidinyl-1-oxyl(TEMPO) andN-acetylcysteine (NAC), two antioxi-dants, were used in this study to assess the contribu-tion of ROS in this Cd-induced process.

2. Materials and methods

2.1. Fish

Immature rainbow trout (Oncorhynchus mykiss)with an average weight of 300–400 g were obtainedfrom a local hatchery (Auribeau-sur-Siagne, France).The fish were kept in aerated, charcoal-filtered cir-culating tap water tanks at a temperature of 15◦C;they were fed commercial fish food and acclimatizedto laboratory conditions for a minimum of 2 weeksbefore experimental use.

2.2. Isolation of hepatocytes and culture conditions

Saline buffer containing 10 mM HEPES and0.63 mM EGTA, pH 7.4 at 25◦C was rapidly infusedinto the liver of slaughtered fish to eliminate Ca2+from the extracellular matrix: then a simple HEPES

C. Risso-de Faverney et al. / Aquatic Toxicology 69 (2004) 247–258 249

solution was infused into the liver at 25◦C, followedby a collagenase solution in HEPES (0.5 mg/ml)containing 5 mM CaCl2. After infusion, the liverwas placed in a Petri dish, rinsed with Leibovitz’s(L-15) culture medium supplemented with glutamineand 5% foetal calf serum (FCS), 50 units peni-cillin/streptomycin, 0.1 unit insulin and then mincedwith a stainless steel forceps, so as to disperse thecells in the medium. The resultant cell suspension wasrepeatedly pipetted and filtered through sterile nylon,then centrifuged three times at 50× g for 5 min, atroom temperature. The cell pellet was suspended inthe previous L-15 culture medium but containing 10%FCS. Cells were counted and viability determinedusing the Erythrosin B (0.36%) exclusion test. Eachinfusion yielded about 2× 108 cells and viability ex-ceeded 90%. Isolated trout hepatocytes were seededin collagen type I-coated dishes (2 and 4× 106 cellsper well of 35 and 60 mm diameter, respectively)and cultured in atmospheric air at 15◦C. The cul-ture medium was changed 24 h after cell seeding. Allliquids and glassware were sterilized by filtration orautoclaving before use.

2.3. Exposure of cells

TEMPO stock solution was prepared in DMSO,NAC and Cd in distilled water. TEMPO, NAC or Cdstock solutions were diluted in L-15 culture mediumwithout serum, but supplemented with 2% bovineserum albumin (BSA, v/v). The following increas-ing Cd concentrations were used: 2, 5 and 10�M.After medium removal from the wells, hepatocyteswere exposed to the various compounds at 15◦C fordifferent exposure times according to experiments.Experimental trout hepatocytes were treated with Cdand/or TEMPO (100�M) or NAC (100�M) for 3, 6,24 and 48 h.

2.4. MTT test

Trout hepatocytes in culture were exposed toincreasing Cd alone concentrations (2, 5, 10�M)and/or 100�M TEMPO or 100�M NAC for 3, 6,24 and 48 h, to verify if these exposure conditionswere cytotoxic or not. Cell viability was deter-mined by a colorimetric assay based on the abil-ity of viable cells, but not of dead cells, to reduce3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium

bromide (MTT). This reaction generates a dark blueformazan product. Cells in culture were loaded withMTT solution (0.5 mg/ml) and incubated at 15◦Cfor 2 h. Cell homogenates were used to measure ab-sorbance at 550 nm using a microplate reader (MR7000, Dynatech Laboratories, Inc., USA).

2.5. Analysis of caspase activity

Caspase-3, -8 or -9-like activities were assessed bymeasuring the proteolytic cleavage of fluorometric andspecific substrates Ac-DEVD-AFC, Ac-IETD-AFC orAc-LEHD-AFC, respectively. After treatments as de-scribed above, the cells were scraped and resuspendedin buffer A (25 mM HEPES pH 7.5, 5 mM MgCl2,5 mM EDTA, 5 mM DTT, 2 mM PMSF, 10�g/ml le-upeptin, 10�g/ml pepstatin A). The cell suspensionwas lysed by two freeze-thaw cycles. Protein concen-trations in the homogenates were determined with theBCA Protein Assay Kit (Pierce, Rockford, IL, USA).Equal amounts of homogenates were mixed withthe specific probe (2.5 mM), in buffer B (312 mMHEPES pH 7.5, 31% sucrose, 0.3% CHAPS) at20◦C as described in the manufacturer’s instructions(PROMEGA, CaspACETM Assay System fluoromet-ric, Technical Bulletin, Madison, WI, USA, 1998).The fluorometric assay was performed with a mul-tiwell fluorescence plate reader (Fluorolite 1000,Dynatech Laboratories) at an excitation/emissionwavelength of 390/530 nm. Caspase activities weremeasured in the samples from the appearance ki-netics of free AFC after cleavage by caspase every3 min for 90 min. Fluorescence in these samples wasproportional to the amount of the caspase activity.

2.6. Western-blot analysis

For cytochromec and Bax immunoblot analysis,cytosolic and mitochondrial extracts from trout hep-atocytes were obtained as described byHerrera et al.(2001). Western-blot analysis of Hsp60 was performedto verify the purity of the mitochondria isolate prepa-ration (as described below). Bcl-xL protein levelswere detected after lysing cells in hypotonic isolationbuffer (25 mM HEPES pH 7.5, 5 mM MgCl2, 5 mMEDTA, 5 mM DTT, 2 mM PMSF, 10�g/ml leupeptin,10�g/ml pepstatin A). Protein content was estimatedusing the Coomassie Plus Protein Assay Reagent, withbovine serum albumin as a standard. Fifty microgram


of total protein were deposited onto 12% sodiumdodecylsulfate-polyacrylamide gels and electrophoret-ically transferred onto PVDF membranes (AmershamLife Science, Buckinghamshire, UK). After blockingwith 6% non-fat skimmed milk in TTBS (10 mmol/lTris–HCl [pH 7.5], 140 mmol/l NaCl, 0.01% Tween20) overnight at 4◦C, the membrane was incubatedwith primary antibody in TTBS containing 3% BSAand then washed with TTBS. Primary antibodies weremurine monoclonal anti-cytochromec (0.5�g/ml,clone 2H8.2C12, Zymed Laboratories Inc., SanFrancisco, CA, USA), rabbit polyclonal anti-Bax(0.5�g/ml, clone SC-526, Santa Cruz Biotechnol-ogy Inc., CA, USA), rabbit polyclonal anti-Bcl-xL(0.2�g/ml, Laboratory Transduction, Lexington, KY,USA), murine monoclonal anti-�-tubulin (0.1�g/ml,clone 2.1, Sigma–Aldrich, St. Louis, MO, USA)and rabbit polyclonal anti-Hsp60 (0.1�g/ml, cloneSC-526, Santa Cruz Biotechnology Inc., CA, USA).The membrane was then incubated for 1 h at roomtemperature with horseradish peroxidase-conjugatedsecondary antibodies (anti-mouse immunoglobin Gor anti-rabbit immunoglobin G, Promega, Madison,WI, USA). Immunodetection was performed by usingchemiluminescence (ECL®, Enhanced ChemiLumi-nescence, Amersham Life Science, Burkinghamshire,UK) and BIOMAX ML film (Eastman Kodak,Rochester, NY, USA). The films were scanned andthe intensity of each immunoreactive band was esti-mated by a densitometric quantification using ScionImaging Software.

2.7. Statistical analysis

The experiments were carried out with five trout.Each result of caspase activity represented the arith-metic mean of enzyme activities for five trout (n = 5)and was expressed as the percentage of enzymeactivity measured in control cultures. Statisticalanalysis was performed using the non-parametricMann–WhitneyU-test. The levels of probability arenoted *P < 0.05 or **P < 0.01.

3. Results

Before measuring the effects of treatment with in-creasing Cd concentrations (2, 5 and 10�M) and/or

100�M TEMPO or NAC for 3, 6, 24 and 48 h ontrout hepatocytes, it has been demonstrated by usingthe MTT test that these exposure conditions were notcytotoxic (data not shown).

Because mitochondrial dysfunction secondary toCyt c release into the cytosol is followed by caspase-9and -3 activation and partially initiated by caspase-8activation, we further analysed, in more detail, themechanisms whereby trout hepatocyte intoxicationby Cd triggers caspase activation. To determine thelevel of protease activity following apoptosis induc-tion, caspase activity was quantified in cells treatedwith 2, 5 and 10�M Cd for 24 and 48 h and com-pared with that obtained in untreated hepatocytes.As shown inFig. 1a, caspase-3 activity was inducedsignificantly in a dose-dependent manner after 24 hexposure to reach at 10�M Cd more than 101%above control level (P < 0.01). When the treatmentwas prolonged to 48 h, this enzyme activity signifi-cantly increased to 88, 113 and 195% above controllevel with 2, 5 and 10�M Cd, respectively (P < 0.01for control versus Cd) (Fig. 1a). Fig. 1b illustratesthe time-course of caspase-9 activity changes. A sig-nificant, dose-dependent increase in caspase activitywas observed 24 h after Cd treatment (140, 445 and1125% on control with 2, 5 and 10�M Cd, respec-tively, P < 0.01) whereas no significant change wasdetected after 48 h of exposure. The contribution ofcaspase-8 in this process was investigated further. Asseen inFig. 1c, caspase-8 activity assays gave pos-itive results at 24 h (140, 420 and 940% on controlwith 2, 5 and 10�M Cd, respectively,P < 0.01) asdid those performed with caspase-9, suggesting thatCd-induced trout hepatocyte apoptosis is mediated bythe activation of caspase-9, -3 and -8.

Bcl-xL is known to exhibit anti-apoptotic effects,whereas Bax and Cytc act as inducers of apoptosis andthe right balance between these molecules will mod-ulate apoptotic processes (Nagata, 1997; Reed, 1997;Chao and Korsmeyer, 1998; Herrera et al., 2001). So,quantification of these anti- and pro-apoptotic proteinsin trout hepatocytes was done by Western blotting toprovide information concerning the effects of Cd onthe expression of these molecules as well as its rolein apoptosis. In the first time, we analyzed Bcl-xL ex-pression in trout hepatocytes after 3, 6, 24 and 48 hexposure to Cd. Its presence was detected in totalcell extracts, corresponding to a major 28 kDa band


Fig. 1. Dose- and time-dependent effects of increasing Cd concentrations on caspase-3 (a), -9 (b) and -8 (c) activity in the absence andpresence of TEMPO or NAC in trout hepatocytes. Cells were incubated for 24 and 48 h with 2, 5 and 10�M Cd alone (�) or with bothCd (same concentrations) and 100�M TEMPO (�) or 100�M NAC ( ). Each bar represents the mean of caspase-3, -9 or -8 activities forfive different experiments expressed as the percentage of enzyme activity found in control cultures. Asterisks indicate significant differencefrom control (*P < 0.05; **P < 0.01; Mann–WhitneyU-test).

(Fig. 2a). The expression levels of this Bcl-2 familyprotein did not significantly change after 3 and 6 h ex-posure to Cd, but increased in the presence of 10�MCd, after 24 and 48 h (2.5-fold control) (Fig. 2a).

We next investigated whether Cytc was released inCd-treated cells after 24 and 48 h exposure (Fig. 3a).The antibody used in this experiment recognized the12 kDa protein corresponding to Cytc. In addition,a positive control, the Cytc from horse heart typeIII confirmed the specificity of the bands (data notshown). Immunoblot analysis revealed a time- andconcentration-dependent increase in Cytc level inthe cytosol and concomitant decrease in the mito-

chondria (Fig. 3a). Massive Cytc release into thecytosol was observed after 24 and 48 h exposure to5 and 10�M Cd (nine-fold on control,P < 0.05)(Fig. 3a). In contrast, tubulin protein contents (usedas control for loading) remained unchanged and mi-tochondrial matrix protein Hsp60 (used as a positivemitochondria control) was undetectable in cytosolicfractions (Fig. 3a and b). To investigate the possiblerole of Bax in Cd-induced apoptosis in trout hepato-cytes, we analyzed the effect of that heavy metal onpro-apoptotic protein Bax translocation from cytosolto the mitochondrial membrane. The expression levelof Bax protein, corresponding to a major 23 kDa band,

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Fig. 2. Cd-induced decrease in Bcl-xL expression levels (a) and role of (b) TEMPO or (c) NAC in the upregulation of Bcl-xL in trout hepatocytes. Trout hepatocytes werecultured with (a) increasing Cd alone concentrations (2, 5 and 10�M) or with both Cd (at the same concentrations) and (b) 100�M TEMPO or (c) 100�M NAC for 3,6, 24 and 48 h. Equal amount of total protein lysates were loaded on a 12% SDS-polyacrilamide gel, and transferred to a Hybond-P membrane. The blot was then probedwith anti-Bcl-xL (0.2�g/ml) and anti-tubulin (0.1�g/ml) antibodies and processed by ECL as described inSection 2. Detection of tubulin proteins has been included as acontrol for loading and membrane transfer. Fold inductions of Bcl-xL (determined by using a densitometer) are given (X) with one corresponding to the level of Bcl-xL incontrol cells. The position of the molecular mass markers (MW), are indicated in thousands at the left of panels. Each immunoblot shown here represents the typical resultfrom several independent experiments.

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Fig. 3. Cd-induced (a) cytochromec release in cytosol from mitochondria and (b) Bax translocation to mitochondria in trout hepatocytes. Trout hepatocytes were cultured withincreasing Cd alone concentrations (2, 5 and 10�M) for 24 and 48 h. Cytosolic and mitochondrial extracts were separated by 12% SDS-polyacrilamide gel electrophoresis,analyzed by immunoblotting with (a) anti-cytochromec (0.5�g/ml) and (b) anti-Bax (0.5�g/ml) antibodies, then processed by ECL as described inSection 2. As a control,filters were also analyzed by immunoblotting with anti-tubulin (0.1�g/ml) and with anti-Hsp60 (0.1�g/ml) antibodies. Fold inductions of Cytc or Bax (determined by usinga densitometer) are given (X) with one corresponding to the level of cytochromec or Bax in control cells. The position of the molecular mass markers (MW), are indicatedin thousands at the left of panels. Each immunoblot shown here represents the typical result from several independent experiments.

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Fig. 4. Role of antioxidants (TEMPO or NAC) in (a–b) Cytc release and (c–d) Bax translocation to mitochondria, induced by Cd in trout hepatocytes. Trout hepatocytes werecultured with both increasing Cd concentrations (2, 5 and 10�M) and (a and c) 100�M TEMPO or (b and d) 100�M NAC for 24 and 48 h. Cytosolic and mitochondrialextracts were separated by 12% SDS-polyacrilamide gel electrophoresis, analyzed by immunoblotting with (a–b) anti-cytochromec (0.5�g/ml) and (c–d) anti-Bax (0.5�g/ml)antibodies, then processed by ECL as described inSection 2. As a control, filters were also analyzed by immunoblotting with anti-tubulin (0.1�g/ml) and with anti-Hsp60(0.1�g/ml) antibodies. Fold inductions of Cytc or Bax (determined by using a densitometer) are given (X) with one corresponding to the level of cytochromec or Bax incontrol cells. The position of the molecular mass markers (MW), are indicated in thousands at the left of panels. Each immunoblot shown here represents the typical resultfrom several independent experiments.


was modified by Cd exposure for 24 and 48 h (Fig. 3b).In contrast, and consistently with the above results,mitochondrial Bax levels considerably increased in adose- and time-dependent manner in Cd-treated cellsto maximum expression after 24 and 48 h exposure to10�M Cd (10-fold on control,P < 0.05); concomi-tant decrease in Bax was observed in cytosol (Fig. 3b).These effects were specific because the levels of tubu-lin and mitochondrial matrix protein Hsp60 were un-changed in mitochondrial extracts (Fig. 3b).

To evaluate the relative contribution of the oxida-tive pathway to the Cd-mediated induction of apopto-sis, the same experiments were performed as describedabove, but with trout hepatocytes in culture exposedsimultaneously to Cd (2, 5 and 10�M) and 100�MTEMPO or NAC. We examined whether the presenceof either one of these antioxidants (TEMPO or NAC),could have a suppressive effect on the Cd-related ac-tivation of caspases.Fig. 1a–cshows that the pres-ence of TEMPO or NAC significantly attenuated theCd-induced increase in caspase-3, -9 and -8 activity(P < 0.01 for Cd versus Cd plus TEMPO or NAC).These data therefore strongly suggest that Cd-inducedcaspase activation is mediated by oxidative stress.

In parallel experiments, Western blotting next wasapplied to determine the effects of antioxidant agents(TEMPO or NAC) on the Cd-related expression ofsome anti- and pro-apoptotic proteins. As shown inFig. 2b and c, the presence of one of these molecules,TEMPO or NAC, in the culture medium mostly up-regulated Bcl-xL after 24 and 48 h, unlike in untreatedcells or cells exposed to Cd alone. In contrast, im-munoblot analysis revealed that the presence of antiox-idant significantly reduced the Cd-induced expressionof cytosolic Bax and mitochondrial Cytc (P < 0.01for Cd versus Cd plus TEMPO or NAC) (Fig. 4a–d).

4. Discussion

Cd is toxic to a number of tissues and induces apop-tosis in vitro and in vivo (Lohmann and Beyersmann,1994; Ishido et al., 1995, 1998; Habeebu et al., 1998;Tsangaris and Tzortzatou-Stathopoulou, 1998; Guoet al., 1998). However, the exact mechanism of apop-tosis induction remains to be elucidated. The apop-tosis pathway can be either mitochondria-dependentor mitochondria-independent and Cytc release from

mitochondria is in many cases the trigger for apop-tosis induction (Reed, 1997; Green, 1998; Green andKroemer, 1998; Green and Reed, 1998).

The main findings of this study relate to the cellu-lar mechanisms of Cd-induced apoptosis in primarycultured trout hepatocytes. The results show that (1)Cyt c release, Bax translocation and caspase activa-tion occur during Cd-induced apoptosis and (2) thoseapoptotic processes are dependent on oxidative stress.We had previously noted that Cd activates caspase-3and induces DNA fragmentation in trout hepatocytes,suggesting that this heavy metal induces cellularapoptosis (Risso-de Faverney et al., 2001). The datareported in this paper show that Cd induces caspase-3,-9 and -8 activation and Cytc release from mito-chondria. This is the first report of Cd-inducing Cytc release from mitochondria in trout hepatocytes. Themitochondrial release of Cytc appears to play a keyrole in the activation of Apaf-1, which in turn cleavesand processes downstream caspases (Green, 1998;Green and Kroemer, 1998; Thornberry and Lazebnick,1998). Therefore, Cytc release from mitochondriamight be partly involved in Cd-induced apoptosis.Further investigations are needed for in-depth anal-ysis of the mode of action of mitochondrial Cytcrelease by Cd. We also found that in Cd-treated trouthepatocytes caspase-8 and -9 are first activated, fol-lowed by caspase-3. Therefore, we hypothesize thatcaspase-8 and -9 are probably the most apical caspasein Cd-induced apoptosis, and finally signal convergesto caspase-3. According to certain reports (Li et al.,2000), caspase-8 not only directly cleaves and acti-vates caspase-3 (Muzio et al., 1998) but also indi-rectly activates caspase-3 by inducing Cytc release(Bossy-Wetzel and Green, 1999). Although no reportshave been published on Cd-induced caspase-8 activa-tion, it is speculated that Cd may activate caspase-8 byenhancing Fas ligand expression. This is not only be-cause caspase-8 is a key component of the Fas/APO 1death receptor-triggered apoptosis pathway (Li et al.,1998), but also because many other apoptotic path-ways initiated by distinct stimuli require Fas involve-ment. Of course, we cannot rule out the possibilitythat Cd activates caspase-8 via a Fas L-independentpathway like activated Lck (Belka et al., 1999). Fur-ther studies are needed to clarify that mechanism.

The Bcl-2-related protein family constitutes one ofthe most biologically important classes of apoptosis


regulatory gene products (Adams and Cory, 1998;Fadeel et al., 1999). We showed that in trout hep-atocytes, Cd does not modify the baseline level ofBcl-xL, an anti-apoptotic member of the Bcl-2 familycapable of preventing Cytc release (Shimizu et al.,1999; Finucane et al., 1999). In contrast, there is anincrease in Bax expression and/or translocation fromcytosol to mitochondria in Cd-exposed trout hepato-cytes. Bax up-regulation occurs in a dose-dependentmanner and time-course analysis of the process indi-cates that the increase in mitochondrial protein levelscorrelates with Cytc release as well as caspase-9 and-3 activation. Taken together, these results indicatethat Bax and Bcl-xL are possibly involved in thecaspase-dependent pathway of Cd-induced apoptosis,resulting in Cyt c release and enhancement of theapoptotic process.

Recent studies in different systems suggest that ox-idative stress and consequent generation of ROS mayserve as a common mediator in apoptosis (Kane et al.,1993; Buttke and Sandström, 1994; Hartwig, 1994;Beyersmann and Hechtenberg, 1997; Lyons-Alcantaraet al., 1998; Herrera et al., 2001). Our results indi-cate that the presence such antioxidants as spin probeTEMPO or glutathione precursor NAC abolish the ac-tivation of caspase-3, -9 and -8, mitochondrial Cytc release and Bax translocation to the mitochondrialmembrane of trout hepatocytes, thus confirming thatthose antioxidants can prevent heavy metal-inducedapoptosis. So, the present results indicate that oxida-tive stress may be involved in Cd-induced apoptosis introut hepatocytes. This hypothesis is further supportedby the fact that Cd is known to induce oxidative stressby depleting intracellular stores of glutathione andprotein-bound sulphydryl groups, resulting in the pro-duction of reactive oxygen species (Stohs and Baghi,1995). Results from studies using cultured cells havedemonstrated Cd-induced formation of superoxide an-ion radicals (Amoruso et al., 1982) and implicatedsuperoxide anions in Cd-induced DNA single-strandscission (Ochi et al., 1983). Cd inhibits superoxidedismutase in vivo, resulting in elevated superoxidelevels (Shukla et al., 1987). By increasing lipid per-oxidation, Cd could therefore be a source of activeoxygen species (Goering et al., 1995). Interestingly,antioxidant-inhibited apoptosis is accompanied by anincrease in the expression of Bcl-xL proteins in trouthepatocytes. It has been shown that overexpression of

Bcl-2 and Bcl-xL proteins prevents a wide variety ofcells from undergoing apoptosis induced by apoptoticstimuli (White, 1996; Ibrado et al., 1997; Adams andCory, 1998; Fadeel et al., 1999). The enhanced Bcl-xLprotein levels in trout hepatocytes appear to representa part of the underlying mechanism of protective ef-fect of TEMPO or NAC. The anti-apoptotic effects ofantioxidants in Cd-treated cells may thus be relatedin part to the modulation of Bcl-xL expression. Theseresults suggest that ROS and Bcl-xL levels play an es-sential role in the mitochondrial-dependent apoptosisinduced by Cd in trout hepatocytes. In fact, the regula-tion of gene expression by oxidants, antioxidants andthe redox state has emerged as a novel subdisciplinein molecular biology. So, at least three well-definedtranscription factors—nuclear factor kappa B (NF�B),activator protein-1 (AP-1), and signal transducers andactivators of transcription—have been identified as be-ing regulated by the intracellular redox state (Sen andPacker, 1996; Simon et al., 1998). Bcl-xL expressionappears to be regulated by the three transcription fac-tor families (Jacobs-Helber et al., 1998; Silva et al.,1999; Chen et al., 2000). Further work will be nec-essary to fully understand the molecular mechanismwhereby Cd—through oxidative stress—for one part,and antioxidants TEMPO or NAC, for the other part,regulate Bcl-xL expression in trout hepatocytes.

5. Conclusion

In conclusion, the results presented in this reportdemonstrate that Cd intoxication induces apoptosis inprimary cultured trout hepatocytes via mitochondrialCyt c release and a caspase-dependent pathway andthat the process is reduced by loading cells with an-tioxidants NAC or TEMPO. Our data also suggestthat Bcl-xL up-regulation by TEMPO or NAC pro-vides an anti-apoptotic protection for trout hepatocytesagainst Cd-mediated stress. We therefore hypothesisethat Cd toxicity is linked to the generation of reac-tive oxygen species, which induces apoptosis. Eluci-dating the mechanisms involved in the induction ofCyt c release and in caspase activation by Cd will re-quire further investigations. Further studies will alsobe needed to investigate the implication of several tran-scription factors in trout liver cells, such as NF�B,AP-1, STATs and Ets, in the regulation of cellular


defense or apoptotic death gene(s) and the involvementof oxidative stress in this phenomenon. Moreover, ad-ditional research is needed to understand the exact roleof unrepaired DNA damage in the Cd-induced apop-totic process generated by the oxidative stress and thatof the tumor suppressor protein p53 in the regulationof apoptosis.

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