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Toxicology 253 (2008) 53–61

Contents lists available at ScienceDirect

Toxicology

journa l homepage: www.e lsev ier .com/ locate / tox ico l

,p′-DDE induces mitochondria-mediated apoptosisf cultured rat Sertoli cells

ang Song, Xianmin Liang, Yafei Hu, Yinan Wang, Haige Yu, Kedi Yang ∗

OE Key Laboratory of Environment and Health, Department of Occupational and Environmental Health, School of Public Health,ongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, PR China

r t i c l e i n f o

rticle history:eceived 13 June 2008eceived in revised form 10 August 2008ccepted 18 August 2008vailable online 4 September 2008

a b s t r a c t

p,p′-Dichlorodiphenoxydichloroethylene (p,p′-DDE), the major metabolite of dichlorodiphe-noxytrichloroethane (DDT), is a known persistent organic pollutant and male reproductive toxicant.However, the mechanism underlying male reproductive toxicity of p,p′-DDE remains limited. In thepresent study, Sertoli cells were used to investigate the molecular mechanism involved in p,p′-DDE’smale reproductive toxicity. Results showed that p,p′-DDE exposure at over 30 �M showed induction

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eywords:poptosisax family membersaspaseitochondria

OS,p′-DDE

of apoptotic cell death. p,p -DDE could induce mitochondria-mediated apoptotic changes includingelevation in reactive oxygen species (ROS) generation, decrease in mitochondrial membrane potential(�� m), and release of cytochrome c into the cytosol, which could be blocked by antioxidant agentN-acetyl-l-cysteine (NAC). In addition, elevated ratios of Bax/Bcl-w and Bak/Bcl-w and cleavages ofprocaspase-3 and -9 were induced by p,p′-DDE treatment. All of the results suggested that ROS generationmay play a critical role in the initiation of p,p′-DDE-induced apoptosis by mediation of the disruption of�� , the release of cytochrome c into the cytosol and further the activation of caspase cascade.

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. Introduction

DDT, the first widely used synthetic pesticide, was given creditor having helped one billion people live free from malaria. How-ver, its bioaccumulation, long-range transport, persistence inhe environment and antiandrogenic properties raise the concernbout its possible long-term adverse effects. Though it has beenestricted for use for three decades, DDT could be traced in allumans and in far regions due to its persistence and accumula-ion. It has been calculated that it would take about 10–20 yearsor DDT to disappear from an individual if exposure would totallyease, but its metabolite DDE would possibly persist throughouthe life span (Smith, 1991). Hence, DDT is regarded as one of the 12ersistent organic pollutants in the UNEP Stockholm ConventionUNEP, 2001).

p,p′-DDE, DDT’s major metabolite with the highest persistence,

s the form usually found in human tissues in the highest concen-ration (Walter and Aimin, 2005). It persists in the environment andan be detected in the sera of more than 90% of the northern Ameri-an population (Andreas, 2004). It is also a kind of known endocrine

∗ Corresponding author. Tel.: +86 2783693897; fax: +86 2783692333.E-mail addresses: sygp 0@163.com (Y. Song), yangkd@mails.tjmu.edu.cn

K. Yang).

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300-483X/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.tox.2008.08.013

© 2008 Elsevier Ireland Ltd. All rights reserved.

isruptor chemicals. Kelce and his colleagues have demonstratedhat p,p′-DDE could inhibit the binding of androgen to andro-en receptor. High dose of p,p′-DDE induces a reduction in malenogenital distance, an increase in retention of male nipple andn alternation in expression of the androgen receptor (Kelce etl., 1995). Our previous studies also confirmed that the residuesf organochlorine pesticides (DDT and its metabolites) might pos-ess reproductive and developmental toxicities and play an adverseole in the reproductive endocrine in human beings (Liu and Yang,006).

Cell death by apoptosis is a part of normal development andaintenance of homeostasis (Tebourbi et al., 1998). This death is

hysiologically appropriate, serving distinct functions in differentissues. In the testis, apoptotic death is such a common pro-rammed event that 75% of germ cells are reduced by spontaneouspoptosis (Allan et al., 1992). However, excessive or inadequatepoptosis of testicular cells will result in abnormal spermatoge-esis or testicular tumors (Lin et al., 1997). Recently, DDT and itsetabolite were alleged to induce apoptosis either in vitro or in vivo

xperiments (Tebourbi et al., 1998; Perez-Maldonado et al., 2004).

ut all of those were not aimed at reproductive system.

During the past years, Sertoli cells cultures have been imple-ented by some scientists as in vitro model to study male

eproductive toxicology. In developing reproductive system, Sertoliells play critical roles including structural support, participation

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2.6. ROS determination

2′ ,7′-Dichlorofluorescein diacetate (DCFH-DA, Sigma–Aldrich, St. Louis, MO,USA) was used to detect ROS by flow cytometry. After treatment, cells were

Fig. 1. (A) Effect of p,p′-DDE on the absorbance of Sertoli cells with MTT assay. MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) could be reduced

4 Y. Song et al. / Toxic

n germ cell movement and spermiation, nourishing germ cells byecreting the tubular fluid and numerous factors (Griswold, 1998;riswold et al., 1998). In addition, Sertoli cells make up and regu-

ate the testicular junctional barrier (Griswold, 1998; Griswold etl., 1998). Since the number of Sertoli cells can only determine anite number of spermatozoa in the seminiferous tubules (Russellnd Peterson, 1984), any agent that impairs the viability of Sertoliells may cause serious effect on spermatogenesis. Though thereave been some reports concerning p,p′-DDE’s male reproductiveoxicity, few studies investigated Sertoli cells, particularly no detailsn mitochondria-mediated apoptosis and no measurement of Baxamily members. We have previously examined the effects of p,p′-DE on Sertoli cell (Xiong et al., 2006). That study demonstrated

hat p,p′-DDE could decrease cellular viability and affect the expres-ion of several functional marker genes including transferrin (Tf)nd androgen-binding protein (ABP). Besides, Fas ligand mRNA wasncreased in p,p′-DDE-treated Sertoli cells, which might account forpoptosis (Song and Yang, 2006). In the present study, another crit-cal pathway in apoptosis, mitochondria-mediated pathway wasnvestigated. We investigated the importance of lipid peroxidation,

itochondrial membrane dysfunction, if any, cytochrome c release,aspase activation and the effects of antioxidants in these events in,p′-DDE-induced apoptosis. As Bax family members play an impor-ant role in spermatogenesis and apoptosis, it is also of interest toetermine the regulation of Bax, Bak and Bcl-w in p,p′-DDE-inducedpoptosis.

. Materials and methods

.1. Animals

18-Day-old male Sprague–Dawley (SD) rats were purchased from Tongji Medicalollege Animal Laboratory (Wuhan, China) and kept in accordance with the Guide forhe Care and Use of Laboratory Animals published by Ministry of Health of People’sepublic of China.

.2. Primary culture of Sertoli cells and cell treatments

Sertoli cells cultures were prepared using sequential enzymatic procedures thatave been described previously (Mather et al., 1990) with modifications. Briefly,estes from 18-day-old Sprague–Dawley rats (day of birth = day 0) were collected,xcised rapidly, decapsulated, cut into small fragments, and washed twice in Hanks’alanced salt solution (HBSS). The fragments were then digested sequentially in0 ml of HBSS containing 0.25% trypsin (Amresco, Solon, OH, USA) and 0.1% collage-ase (type I, Invitrogen, Grand Island, NY, USA) in a shaking water bath (35 ◦C, 120ycles/min) for 30 min. Digested cells suspension was washed extensively with no-henol red-Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Grand Island,Y, USA) to remove peritubular cells, followed by filtration through B-D Falcon cell

trainers (nylon mesh size, 70 �m). The final Sertoli cells suspension was supple-ented with 5% fetal bovine serum (Invitrogen, Grand Island, NY, USA) and seeded

n cultured bottle in a humidified atmosphere of 95% air–5% CO2 at 35 ◦C. After 24 h,hese cells were extensively washed twice with HBSS solution to remove unattachedells, then with 20 mM pH 7.4 Tris–HCl for 5 min and with serum starvation for 24 h.he medium was renewed at 2-day interval.

p,p′-DDE (DR Co., Augsburg, Germany) was dissolved in dimethylsulfoxideDMSO, Sigma–Aldrich, St. Louis, MO, USA) as stock solution and diluted with culture

edium to different concentrations before being added to the cells in culture. Thenal DMSO concentration in the medium was not more than 0.3% (v/v) which didot affect the viability of Sertoli cells. Control cells were cultured with 0.3% DMSO.

.3. MTT assay

This assay is dependent on the cellular reduction of MTT (3-(4,5-imethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by the mitochondrialehydrogenase of viable cells to a blue formazan product which can be mea-ured spectrophotometrically. MTT (Sigma–Aldrich, St. Louis, MO, USA) was addednto each treated well with the final concentration of 5 mg/ml for 4 h. Thereafter,00 �l DMSO was added to solve the MTT formazan crystal. Then the culturelate was shaken for 10 min. The optical density (OD) of each well was mea-ured at 490 nm with an ELISA Reader (Bio-Rad instrument Group, Hercules,A, USA).

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253 (2008) 53–61

.4. Lactate dehydrogenase (LDH) leakage

Cellular supernatants were collected after treatment, and LDH leakage wasetermined by an automated procedure using an OLYMPUS AU 1000 automaticiochemical analyzer (Tokyo, Japan). Result was expressed as U/l.

.5. Apoptosis assay with the methods of Hoechst staining and flow cytometricnalysis

Hoechst staining: Sertoli cells were seeded on a cover glass plate in 24-welllate. Following fixation with 4% paraformaldehyde, treated Sertoli cells were incu-ated with Hoechst 33258 (Sigma–Aldrich, St. Louis, MO, USA) and observed underhe inverted fluorescence microscope (IX70, Olympus, Tokyo, Japan). Apoptotic cellsere identified by nuclear condensation and fragmentation. The percentage of apop-

otic cells was calculated from at least 100 cells in at least three different fields.Flow cytometric analysis: Sertoli cells were seeded in 6-well plate, and apopto-

is was tested by apoptosis detection kit (Molecular probes, Eugene, OR) accordingo the instruction. In brief, the single Sertoli cell was collected and incubated in theuffer containing 1 �g/ml PI and 5 �l Annexin V in the dark at room temperature for5 min. Then the stained cells were analyzed by a FACS Calibur flow cytometer (Bec-on Dickinson, San Jose, CA, USA). The data were analyzed with Cellquest software

y the mitochondrial dehydrogenase of viable cells to a blue formazan product whichan be measured spectrophotometrically. Cells were treated with 0.3% DMSO, 10, 30,0 or 70 �M p,p′-DDE for 24 h, and the absorbance was measured by MTT assay. Dataere presented as mean ± S.E.M. of three-independent experiments performed in

riplicate. Significant difference: *P < 0.01, compared with control. (B) Effect of p,p′-DE on lactate dehydrogenase (LDH) release. Sertoli cells were treated with 0.3%MSO, 10, 30 or 50 �M p,p′-DDE for 24 h. Then culture medium was taken, and LDH

elease was then determined in the supernatants using a kit. *P < 0.05, comparedith control.

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oaded with 10 �M DCFH-DA and analyzed in a FACS Calibur flow cytometerBecton–Dickinson, San Jose, CA, USA). A minimum of 10,000 cells per sampleere registered. The intensity of fluorescence is proportional to the amount of ROS

Thannickal and Fanburg, 2000).

.7. Measurement of superoxide dismutase and malondialdehyde

Intracellular ROS could initiate a series of reactions resulting in loss of superoxideismutase and production of lipid peroxidation including malondialdehyde (MDA).reated Sertoli cells were pelleted and lysed in 0.5 ml cell lysis solution (containingmM Na2EDTA, 150 mM NaCl, 10 mM PMSF, 10 mM Tris, 1 mM aprotin) to evalu-te lipid peroxidation following the protocol of SOD and MDA assay kit (Jianchengioengineering Ltd., Nanjing, China).

.8. Measurement of mitochondrial membrane potential

The fluorescent probe JC-1 (5,5′ ,6,6′-tetrachloro-1,1′ ,3,3′-tetraethylbenzi-idazole carbocyanide iodide; Molecular Probes, Eugene, OR) was used to measure

he mitochondrial membrane potential of Sertoli cells. JC-1 is a cationic dye thatccumulated in the mitochondria depending upon the mitochondrial membraneotential. It fluoresces in red range (590 nm) under normal conditions. If theitochondrial membrane potential is lost, red fluorescence is decreased. Followingmethod described in reference (Salvioli et al., 1997), Sertoli cells (5 × 105) were

ncubated with 1.0 �g/ml JC-1 for 15 min at 37 ◦C and analyzed immediately byow cytometric analysis using a BD FACS AriaTM flow cytometer and BD FACSDiVaoftware (Becton Dickinson, Mountain View, CA). In each experiment, at least0,000 events were analyzed. The values of relative monomer (green) fluorescencentensity were used for data presentation.

.9. Western blotting

Sertoli cells (5 × 106 cells) were lysed in 100 �l lysing buffer (20 mM Tris, pH.4, 10 mM EDTA, 2 mM EGTA, 250 mM sucrose, 1% Triton X-100, and 1 mM phenyl-ethylsulfonyl chloride) and scraped from the culture plate to detect Bax familyembers and caspase proteins. Each protein sample was measured by a Bio-RadC kit (Bio-Rad, Hercules, CA). Cell extracts were separated in SDS-polyacrylamideels and transferred electrophoretically onto a PVDF membrane. The membraneas blocked in PBS containing 5% non-fat dry milk, and then incubated at 4 ◦C

vernight with anti-Bak (G-23, Santa Cruz Biotechnology, Inc., Santa Cruz, CA),nti-Bax (e Bioscience, CA, USA), anti-Bcl-w (StressGen Biotechnologies Corp., Vic-

oria, British Columbia, Canada) or anti-�-actin (Santa Cruz Biotechnology, Inc.,anta Cruz, CA) at 1:200 dilution, with anti-procaspase-3 (Santa Cruz Biotechnol-gy, Inc., Santa Cruz, CA) or anti-procaspase-9 (wako, Saitama, Japan) at 1:1000ilution. Then the membranes were incubated at 37 ◦C for 2 h with the secondaryntibody conjugated with horseradish peroxidase (Amersham Pharmacia, Bucking-amshire, UK) diluted at 1:5000. And immune-reactive proteins were detected

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ig. 2. Effect of NAC on p,p′-DDE-induced morphologic changes of Sertoli cells. Cultured S,p′-DDE (D). In other experiment, Sertoli cells were preincubated with 300 �M NAC forere fixed, stained with Hoechst 33258 and observed under a fluorescence microscope.

ells including blebbing cells, chromatin condensation or fragmentation. Representative i

253 (2008) 53–61 55

ith ECL Western blotting detection system (Pierce Biotechnology Inc., Rockford,L, USA). Densitometric analysis of immunoblots was performed with Gel pro 3.0oftware.

Cytosolic cytochrome c content was analyzed according to Kluck et al. (1997).riefly, the cytosolic protein was separated in SDS-polyacrylamide gels and trans-

erred electrophoretically onto a PVDF membrane after pelleting the membranes12,000 × g) and filtering the supernatant through 0.2 and 0.1 mm Ultrafree-MC fil-ers (Millipore Trading Co., Ltd., USA). Then the blot was probed with a monoclonalntibody to mouse cytochrome c peptide (Santa Cruz Biotechnology, Inc., Santa Cruz,A) at a dilution of 1:500. A secondary probe with horseradish peroxidase-labeledntibodies (Amersham Pharacia, Buckinghamshire, UK) was detected by ECL (Pierceiotechnology Inc., Rockford, IL, USA).

.10. Statistical analysis

Results were represented statistically as mean ± S.E.M. Significance wasssessed by ANOVA following appropriate transformation to normalized data andqualized variance where necessary. Mean values were compared by subsequenttudent–Newman–Keuls (SNK) with the SPSS statistical package 12.0 (SPSS Inc.,hicago, IL, USA). A difference at P < 0.05 was considered statistically significant.ll assays were performed in triplicate.

. Results

MTT assay and LDH leakage: Sertoli cells were treated with 10,0, 50 or 70 �M p,p′-DDE for 24 h and analyzed with MTT assay. Ashown in Fig. 1A, the absorbance of Sertoli cells was reduced afterreatment with 30, 50 or 70 �M p,p′-DDE (P < 0.05). In 70 �M p,p′-DE-treated group, absorbance was about 50% of that in DMSO-

reated group, representing cellular viability. Based on this result,,p′-DDE at concentration of 10, 30, 50 �M was used in subsequentxperiments.

The leakage of LDH was notably enhanced in 30 or 50 �M p,p′-DE-treated group (P < 0.05) (Fig. 1B).

Effect of NAC on p,p′-DDE-induced apoptosis in Sertoli cells: Ser-oli cells were incubated in various concentrations of p,p′-DDE (10,

0, 50 �M) for 24 h. In other experiment, cells were preincubatedith 300 �M NAC for 1 h followed by incubation with 50 �M p,p′-DE for 24 h. Then apoptosis was examined by Hoechst stainingnd flow cytometric analysis. Morphologically, Sertoli cells with 30r 50 �M p,p′-DDE exhibited specific apoptotic characters such as

ertoli cells were exposed to 0.3% DMSO (A), 10 or 30 �M p,p′-DDE (B and C), 50 �M1 h and followed by incubation with 50 �M p,p′-DDE for 24 h (E). Thereafter, cellsArrows in these pictures indicated morphological changes in the nuclei of Sertolillustrations were shown for at 400× magnification.

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uclear condensation, nuclear shrinkage and fragmentation, whichould be blocked by preincubation with NAC (Fig. 2).

Flow cytometric analysis (Fig. 3) showed that p,p′-DDE causedpoptotic cell death in a dose-dependent manner. After treatment

f 50 �M p,p′-DDE, apoptosis reached a peak value of 30%, whichould be attenuated by NAC preincubation.

ROS production, SOD activity and MDA level: ROS generationas expressed as the fluorescence intensity measurement in ana-

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ig. 3. Representative plots of PI-Annexin staining of Sertoli cells with p,p′-DDE with or10, 30 or 50 �M) for 24 h. In other experiment, Sertoli cells were preincubated with 300 �poptosis was tested by apoptosis detection kit. A–E represented as treatment of 0.3% DMnd E). (F) The apoptosis percentage showed that 30 or 50 �M p,p′-DDE could induce apohree-independent experiments performed in triplicate. Significant difference: *P < 0.05, c

253 (2008) 53–61

yzed cells. Representative results of ROS were shown in Fig. 4A.t could be observed that 50 �M p,p′-DDE induced a significantncreasing level of ROS production (P < 0.05), which could be neu-ralized by preincubation with NAC.

The SOD activity in all p,p′-DDE groups was significantly lowerhan that in the vehicle group (P < 0.05). The MDA level in 30 or0 �M p,p′-DDE-treated group was remarkably higher than that inontrol group (P < 0.05)(Fig. 4B).

without NAC. Sertoli cells were incubated with various concentrations of p,p′-DDEM NAC for 1 h and followed by incubation with 50 �M p,p′-DDE for 24 h. Then cellSO (A), 10 or 30 �M p,p′-DDE (B and C), 50 �M p,p′-DDE without or with NAC (D

ptotic Sertoli cells death blocked by NAC. Data were presented as mean ± S.E.M. ofompared with control. #P < 0.05, compared with 50 �M p,p′-DDE group.

Y. Song et al. / Toxicology

Fig. 4. (A) p,p′-DDE’s effect on ROS generation neutralized by NAC. 2′ , 7′-dichlorofluorescein diacetate (DCFH-DA, Sigma–Aldrich, St. Louis, MO, USA) wasused to detect ROS by flow cytometry. Thus, the amount of ROS is proportionalto the intensity of DCF which is formed by deesterification of DCFH-DA to DCFHand oxidation in cells (Thannickal and Fanburg, 2000). Sertoli cells were incubatedwith various concentrations of p,p′-DDE (10, 30 or 50 �M). In other experiment, cellswere preincubated with 300 �M NAC for 1 h and followed by incubation with 50 �Mp,p′-DDE for 24 h. Then ROS production was determined as indicated methods. Datawere given as the mean ± S.E.M. of three-independent experiments performed intriplicate. (B) Effect of p,p′-DDE on SOD activity and MDA content in Sertoli cells.After treatment with various concentrations of p,p′-DDE (10, 30 or 50 �M), Sertolicells were lysed to detect the SOD activity and MDA content by the commercial testkp

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Loss of mitochondrial membrane potential following treat-ent with p,p′-DDE. Based on Fig. 5, loss of mitochondrial

otential was induced after treatment with 10, 30 or 50 �M,p′-DDE (P < 0.05), suggesting the damage to the mitochondria.reincubation with 300 �M NAC for 1 h attenuated this damageignificantly.

The effect of p,p′-DDE on Bax family members of Sertoli cells.s shown in Fig. 6, significant increases of Bax and Bak werebserved in Sertoli cells treated with 30 or 50 �M p,p′-DDE. NACad little effect on these changes. Bcl-w protein level declined sig-ificantly in 50 �M p,p′-DDE group, which could be rescued byAC.

Cytochrome c translocation, procaspase-9 and -3 cleavage toertoli cells apoptosis. As indicated in Fig. 7A, 30 or 50 �M p,p′-

DE treatment induced cytochrome c translocation to cytosol.reincubation with NAC could rescue this translocation effec-ively (P < 0.05). A significant decrease of procaspase-9 and -3 wasbserved over 30 �M p,p′-DDE treatment (Fig. 7B and C), indicatinghe caspase activation, respectively.

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253 (2008) 53–61 57

. Discussion

In the present study, p,p′-DDE could induce apoptosis of Sertoliells via activation of mitochondria-mediated pathway includinglevation of ROS, decrease in �� m along with the cytochrome celease from mitochondria into the cytosol and activation of theaspase-9 and -3. Importantly, the antioxidant NAC could attenuateost of these changes. It is concluded that ROS elevation is an early

vent in p,p′-DDE-induced apoptosis cascade.Apoptosis is an important process to eliminate unwanted or

efective cells through an orderly process of cellular disintegrationAmeisen, 1996). In terms of Sertoli cells’ critical role in spermato-enesis, any agent that impairs the viability of Sertoli cells mayause serious effect on spermatogenesis. The present study showedhat p,p′-DDE could induce apoptotic Sertoli cells death verifiedrom morphological observations and flow cytometric assessment.uch an observation is compatible with recent report from Perez-aldonado. His findings showed that apoptosis could be observed

n peripheral blood mononuclear cells (PBMC) with p,p′-DDE, and,p′-DDD (Perez-Maldonado et al., 2004). In addition, evidence toupport this include that apoptosis of Sertoli cells could be inducedy some other known endocrine disruptors (Iida et al., 2003; Qiant al., 2006). From all of the above reports, apoptosis of Sertoli cellsay be another molecular mechanism of endocrine disruptors inale reproductive toxicology.In this study, 10 �M p,p′-DDE did not have a statistically signif-

cant effect on the viability of cultured Sertoli cells. The cellulariability was decreased significantly by 30 or 50 �M p,p′-DDE,robably because of apoptotic cell death. Tebourbi et al. (1998)as reported that the apoptosis of rat thymocytes was induced by�g/ml (20 �M) p,p′-DDD. Perez-Maldonado reported that apop-

osis was observed in peripheral blood mononuclear cells (PBMC)ith 20 �g/ml p,p′-DDE and p,p′-DDD (Perez-Maldonado et al.,

004). To summarize, p,p′-DDE at doses over 10 �M may be harm-ul to the cells in vitro probably because of apoptosis induction,hile the concentration lower than 10 �M may have only slight

oxic effects.The role of ROS in apoptosis has been suspected for more than

0 years. Originally, ROS was suggested to induce apoptosis viairect damage to cellular components. Recent literature suggestshat ROS as a signaling molecule could induce downstream apop-otic processes. Our present study showed that p,p′-DDE couldnterfere with normal metabolism of oxygen, and additional ROS

ere created. SOD as the ROS scavengers was depleted, and MDAs a product of lipid peroxidation was accumulated. In the ratestes, equivalent SOD activity is expressed as primary antioxidantBauché et al., 1994). Destroyed antioxidative status in a cell formsxidative stress, which could be associated with previous findingshat DDT induced DNA single-strand breaks (Hassoum et al., 1993)nd lipid peroxidation (Barros et al., 1994). In our previous stud-es, p,p′-DDE was also found to induce DNA damage in Sertoli cellsSong and Yang), which might account for subsequent developmentf apoptosis.

In order to demonstrate a direct association between apopto-is induction and increased levels of intracellular ROS, antioxidantgent N-acetylcysteine (NAC) was used. NAC, a glutathione inducerMeister and Anderson, 1983), could antagonize the potentiallydverse effects of ROS. In our conditions, pretreatment with NACould attenuate oxidative stress and block p,p′-DDE-induced apop-osis, suggesting a functional role of ROS during this process. Our

ndings are in agreement with those reported previously. For

nstance, ROS production and apoptosis have also been reportedn p,p′-DDE-treated human peripheral blood mononuclear cellsPérez-Maldonado et al., 2005). Apoptosis by endosulfan in auman T-cell leukemic line was probably due to excessive ROS

58 Y. Song et al. / Toxicology 253 (2008) 53–61

Fig. 5. Representative dot plots of stained Sertoli cells by flow cytometric analysis. Sertoli cells were incubated with various concentrations of p,p′-DDE (10, 30, 50 �M).In other experiment, cells were preincubated with 300 �M NAC for 1 h and followed by incubation with 50 �M p,p′-DDE. Then mitochondrial membrane potential (�� m)was determined with JC-1 by flow cytometric analysis. A–E represented as treatment of 0.3% DMSO (A), 10 or 30 �M p,p′-DDE (B and C), 50 �M p,p′-DDE without or withNAC (D and E). Lower right quadrant showed apoptotic Sertoli cells exhibiting green fluorescing monomers. (F) FCM analyzed the percentage of cells containing polarized ordepolarized mitochondria by plots analysis of the ratio of green fluorescence intensities. The data were given as mean ± S.E.M. of three-independent experiments performedin triplicate. *P < 0.05, compared with the control group. #P < 0.05, compared with 50 �M p,p′-DDE group.

Y. Song et al. / Toxicology 253 (2008) 53–61 59

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ig. 6. (A) Effect of NAC on p,p′-DDE-induced changes of Bax family members. Sertolell lysates was used in Western blotting for Bax, Bak, Bcl-w and beta-actin detectioells treated with p,p′-DDE with or without NAC. Results were expressed as ratio oith the control group. #P < 0.05, compared with 50 �M p,p′-DDE group.

roduction (Kannan et al., 2000). However, little is known aboutow ROS promote apoptosis in Sertoli cell. ROS may act as sig-aling molecules that initiate apoptosis by activating downstreameath effectors rather than by direct damage to cellular compo-ents (Sarafian and Bredesen, 1994). The primary source of ROS isitochondrial electron transport chain. A central role for mitochon-

ria in the apoptotic death of many types of cells is also indicatedy extensive evidence (Thannickal and Fanburg, 2000). Theoreti-ally, as a consequence of ROS generation in cells, mitochondrialysfunction should occur (Ling et al., 2003). As was expected, sig-ificant dissipation of mitochondrial membrane potential (Fig. 5)as observed in the current study, indicative of mitochondrial dys-

unction. However, we have no direct evidence to state how theOS do damage to mitochondria. Ferreira et al. (1997) confirmedhat DDE could interact with succinate dehydrogenase (complex II),ecreasing respiration and membrane potential. Whether it func-

ions in Sertoli cells needs to be further validated with inhibitors of

itochondrial electron transport chain in the following studies.The elimination of intracellular superoxidant state by N-

cetylcysteine could block p,p′-DDE-induced �� m decrease,

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ig. 7. (A) Effect of NAC on p,p′-DDE-induced cytochrome c translocation to the cytosol. Seytosol was used in Western blotting for cytochrome c detection. (B and C) Procaspase-3 aysates was used in Western blotting for procaspase-3 and -9 detection. (D and E) QuantitP < 0.05, compared with the control group. #P < 0.05, compared with 50 �M p,p′-DDE gro

were treated with p,p′-DDE preincubated with or without NAC. Protein from wholeQuantitative analysis of the immunoreactive Bax, Bak and Bcl-w proteins in Sertolial density presents in Bax, Bak and Bcl-w vs. beta-actin band. *P < 0.05, compared

ytochrome c release, and apoptotic death, suggesting that ROSffected a late event in the apoptotic cascade. The main determin-ng factor for death commitment in these cells is the cytochromerelease from mitochondria into the cytoplasm (Deshmukh and

ohnson, 1998). Once in the cytoplasm, cytochrome c interactsith apoptosis activating factor 1 (Apaf-1) and caspase-9, caus-

ng formation of a complex known as the apoptosome (Adrian andartin, 2001). The apoptosome activates caspase-9, which then

leaves and activates other caspases that degrade many proteinubstrates, leading to cell death (Li et al., 1997). The findings ofhis study demonstrated that expression of procaspase-3 and -9as decreased, indicating the caspases activating. However, Wój-

owicz and his colleagues have reported decreases in caspase-3fter DDE in JEG-3 cells (Wójtowicz et al., 2007). There may be aifference between cell lines. More importantly, they used much

ower concentrations of DDT and the cells were cultured in medium

ontaining serum. The differences in culture conditions and con-entration of chemicals might be the best explanation.

The initial investigations of Bcl-2 family members have shownhat they play an important role in regulating mitochondria-

rtoli cells were treated with p,p′-DDE pretreated with or without NAC. Protein fromnd -9 cleavage in Sertoli cells exposed to p,p′-DDE for 24 h. Protein from whole cellative analysis of the immunoreactive cytochrome c, procaspase-3 and -9 cleavage.up.

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ediated apoptosis (Korsmeyer et al., 1995). Bcl-2 family membershare one or more Bcl-2 homology (BH) domains and are dividednto two main groups according to whether they promote or inhibitpoptosis (Yang et al., 1995). They may either promote cell survivalBcl-2, Bcl-xL, Bcl-w, Mcl-1, A1/Bfl-1) or encourage cell demise (Bax,ak, Bcl-Xs, Bad, Bid, Bik, Hrk, Bok) (Zamzami et al., 1998; Adamsnd Cory, 1998; Evan and Littlewood, 1998). Because the membersf those opposing factions can associate and seemingly neutral-ze mutual function, their relative abundance in a particular cellype may determine its threshold for apoptosis (Oltvai et al., 1993).his paper showed an increase in Bax, Bak and a decrease in Bcl-

protein level, leading to an increase in Bax/Bcl-w and Bak/Bcl-watio. So far, Bcl-w appears to be a critical pro-survival protein ofhe Bcl-2 family in the Sertoli cells. In Bcl-w-deficient mice, lossf Sertoli cells was observed when they are adult (Russell et al.,001). Yan et al. (2000) reports that the ratios of Bax/Bcl-w andak/Bcl-w may be decisive for the survival of Sertoli cells. NAC couldeverse Bcl-w’s change interestingly, but it had little effect on Baxr Bak. These findings indicate that ROS may be an early event inecreased Bcl-w in p,p′-DDE-treated Sertoli cells. The mechanismtill remains unknown. Furthermore, Bax and Bak are essential toitochondrial outer membrane permeabilization during apoptosis

Antignani and Youle, 2006). Bax or Bak affects the permeabiliza-ion of the outer mitochondrial membrane, allowing proteins inhe mitochondrial intermembrane space, such as cytochrome c, toscape into the cytosol where they can induce the caspases activa-ion and cell death (Green and Kroemer, 2004).

The present data indicated that p,p′-DDE could induce apopto-is in Sertoli cells through mitochondria-mediated pathway, whichight elucidate our previous studies that p,p′-DDE could decrease

ertoli cell viability (Xiong et al., 2006). Furthermore, the majorityf the current study was focused on the effects of only one chemicalt a time, whereas organism is daily exposed to mixtures of variousroducts in the environment. Hence it is noted that toxicity studiesocusing on mixtures to low concentration for long time should beegarded as priority in the next studies.

In summary, our findings demonstrate that ROS generation isn early event in p,p′-DDE-induced apoptotic cascade, becausebrogation of ROS generation could inhibit the decrease in �� m

nd cytochrome c release from mitochondria as well as apoptosis.n view of the present results, we conclude that p,p′-DDE couldctivate the apoptosis machinery in a pro-oxidant, mitochondria-ependent manner by activating of the intrinsic programmed celleath pathway. The study herein has provided preliminary but

mportant data for further study of male reproductive toxicity of,p′-DDE.

onflict of interest

None.

cknowledgement

This work was supported by grants from the National Naturalcience Foundation of China (30671734).

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