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Reproductive Toxicology 33 (2012) 331– 338

Contents lists available at SciVerse ScienceDirect

Reproductive Toxicology

jo u r n al hom epa ge: ww w.elsev ier .com/ locate / reprotox

hlorpyrifos modifies the expression of genes involved in human placentalunction

agali E. Ridanoa, Ana C. Raccaa, Jésica Flores-Martína, Soledad A. Camolottoa,ladis Magnarelli de Potasb,c, Susana Genti-Raimondia, Graciela M. Panzetta-Dutari a,∗

Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI-CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional deórdoba, Córdoba, ArgentinaInstituto Multidisciplinario de Investigación y Desarrollo de la Patagonia Norte (Universidad Nacional del Comahue-CONICET), Neuquén, ArgentinaFacultad de Ciencias Médicas, Universidad Nacional del Comahue, Cipolletti, Argentina

r t i c l e i n f o

rticle history:eceived 27 October 2011eceived in revised form0 December 2011ccepted 12 January 2012vailable online 21 January 2012

a b s t r a c t

The effects of organophosphate pesticides on human placenta remain poorly investigated although anincreased risk of pregnancy alterations has been reported in women chronically exposed to these pes-ticides. Here, we have addressed whether chlorpyrifos (CPF) modifies the expression of genes relevantfor placental function. Human placental JEG-3 cells were exposed to increasing CPF concentrations up to100 �M for 24 and 48 h and cell viability, mRNA, protein and hormone levels were analyzed. QuantitativeRT-PCR assays revealed that CPF increased the expression of ABCG2, GCM1 and, even more significantly,

eywords:rganophosphate pesticiderophoblastene expressionhCGregnancy

�hCG mRNAs in conditions where cell viability and morphology were not compromised. In addition,�hCG protein synthesis and secretion were time-dependently augmented. Present results may reflect aCPF nocive effect on placenta cells or a placental-defense mechanism to preserve its function. These novelCPF trophoblast target genes should be considered in future studies of pregnancy outcomes associatedwith in vivo exposures.

© 2012 Elsevier Inc. All rights reserved.

. Introduction

The organophosphate (OP) compounds are among the most usedhemical pesticides in agricultural crops and residential applica-ions. Their most studied toxic effects are due to direct inhibition ofhe enzyme acetylcholinesterase, leading to a spectrum of choliner-ic effects and the inhibition of neurotoxic esterase enzyme givingise to the symptoms of delayed neurotoxicity [1]. However, otherechanisms by which OPs can cause a direct toxic action on vari-

us tissues independently of their anti-esterase activity have beeneported [2,3]. For instance, chlorpyrifos (CPF), one of the most

idely used OP, interferes with brain development in part due

o alterations in the activity of transcription factors involved inhe basal machinery of cell replication and differentiation [4]. CPF

Abbreviations: CPF, chlorpyrifos; OP, organophosphate; CTB, cytotrophoblast;hCG, human chorionic gonadotropin �-subunit; GCM1, glial cell missing-1; KLF6,rüpple-like factor 6; StarD7, StAR-related lipid transfer protein 7; ABCG2, ATP-inding cassette sub-family G member 2; DMSO, dimethylsulfoxide.∗ Corresponding author at: CIBICI-CONICET, Departamento de Bioquímica Clínica,

acultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre Medina Allende, Ciudad Universitaria, X5000HUA Córdoba, Argentina.el.: +54 351 4344973x3142; fax: +54 351 4333048x3177.

E-mail address: gpan@fcq.unc.edu.ar (G.M. Panzetta-Dutari).

890-6238/$ – see front matter © 2012 Elsevier Inc. All rights reserved.oi:10.1016/j.reprotox.2012.01.003

induces apoptosis in human immune cells [5] and increases thelevels of kinase ERK1/2, IL-6 and glial fibrillary acidic protein inastrocytes [6]. Recent studies have indicated that OPs may dis-rupt metabolism by altering the expression or function of adenylylcyclase or G-protein-coupled receptors and G-proteins involved inthe adenylyl cyclase signaling cascade in neuronal as well as inperipheral tissues [7].

Additionally, many reports on in utero OP pesticide expo-sures suggest adverse effects on pregnancy outcomes such asfetal growth, increased risks for preterm delivery, spontaneousabortions [8–12] and impairment in neurodevelopment and psy-chomotor indices [13–15]. Furthermore, a significant inverseassociation between birth weight and length and levels of CPFin umbilical cord plasma has been documented [16]. However,the impact of OP pesticides on gestation remains controversial[3]. Although epidemiology studies are required for risk assess-ment purposes, they present some limitations. For instance, it maybe difficult to eliminate exposure to confounder substances, todemonstrate causality, or to obtain accurate measures of individ-ual exposure to the toxic in question [17]. Therefore, in order to

complement those studies and to identify cellular pathways andbiochemical processes possibly involved in the placental responseto OP exposure, it is relevant to investigate the effects that OPscause on in vitro trophoblast cell models. The trophoblasts are

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he main functional cells of the placenta and their differentia-ion and function are intimately related to fetal development and

aternal health. Trophoblasts mediate implantation, give rise toost of the extraembryonic tissues, and display essential functions

uring pregnancy. Extravillous trophoblasts participate in preg-ancy immunotolerance, angiogenesis and maternal blood effluxontrol, among other important functions. On the other hand, vil-ous trophoblasts regulate nutrient, gases and metabolic wastexchange between the mother and fetus. They differentiate form-ng the syncytium layer which acts as a placental barrier protectinghe fetus from endogenous and exogenous toxic metabolites thatrrive through the maternal blood [18]. Nevertheless, it is largelynown that many drugs and environmental chemicals may alterrophoblast function or may cross the placenta leading to unwantedffects on the developing fetus [12]. In addition, accumulating evi-ence suggests that a correct placenta development and gestationalnvironment have a profound and permanent impact not only onhe fetus but also on the individual health in adult life [19,20]. Someeports have addressed the effects that OPs cause on placental athe molecular level. Thus, it has been reported that altered placen-al acetylcholinesterase and catalase activities are associated withrenatal exposure to OP pesticides [21]. Azinphos-methyl, phosmetnd CPF modify placental phosphoinositide metabolism and PI-4inase activity [22], and CPF induces apoptosis in the trophoblastic-erived JAR cell line, through a signaling mechanism not dependentn FAS/TNF, activation of caspases or inhibition of cholinesterase23]. However, information about pesticide effects on gene expres-ion and protein levels on trophoblast cell systems is still scarce.ere, we employed the placental-derived JEG-3 cell line, consid-red a useful model for examining placental toxicity [24], to testhe hypothesis that CPF affects the expression of relevant genesnvolved in the maintenance of a healthy pregnancy.

. Materials and methods

.1. Chemicals

CPF (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate) (CAS#2921-88-) with purity of 99.5% was purchased from the Sigma Chemical Company (St. Louis,O, USA).

.2. Cell culture and CPF treatment

The choriocarcinoma-derived JEG-3 cell line was grown in Dulbecco’s modifiedagle’s medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL peni-illin and 0.1 mg/mL streptomycin. CPF was prepared as a 0.25 M stock solution inimethylsulfoxide (DMSO). Immediately before use it was diluted to the workingoncentration with media and added to cell cultures so that final DMSO concentra-ions did not exceed 0.04%. Treatments were performed under 1% of FBS to avoidhe interaction of the toxic with serum proteins [23]. Previous experiments con-rmed that cell viability was not compromised when cells were grown in 1% FBSnd that the observed effects were not due to the DMSO concentrations used (dataot shown). Complete medium, including CPF was changed every 24 h and incuba-ions were performed for up to 72 h. Twenty-four hours before treatment, 5.5 × 105

EG-3 cells were plated on each well of 6-well plates in 2 mL of culture medium, or × 104 cells on each well of 96-well plates in 100 �L of culture medium.

.3. Cell viability/cytotoxicity assay

After determining the optimum cell number to carry out these experiments, × 104 JEG-3 cells were plated in each well of 96-well plates. They were treatedith CPF (0, 5, 50, 75, 100 �M) for 24 or 48 h and cell viability was evaluated using

he metabolic dye 3-(4,5-dimethlthiazol-2-yl)-2,5-diphenyl-tetrazolium bromideMTT) assay. Briefly, MTT solution (5 mg/mL) was added to the culture medium1:10) and incubated for 2.5 h at 37 ◦C. After the incubation, the medium wasemoved and the precipitated dye was dissolved in 100 �L of DMSO. Absorbance wasead at 540 nm, and the results were expressed as percentage cell viability relativeo control. Four independent experiments were conducted in quadruplicates.

.4. Nuclear morphology and immunofluorescence assays

JEG-3 cells were cultured on cover-slips using supplemented medium asescribed above containing 0, 50 or 100 �M CPF. The cover slips were washed three

xicology 33 (2012) 331– 338

times with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4; pH7.4) and cells were fixed 10 min in methanol at −20 ◦C or 20 min in 3% paraformalde-hyde at room temperature, according to the antibody used. Immediately after,cells were incubated 10 min with 10 mM ammonium chloride to inhibit quench-ing, washed with PBS three times and permeabilized for 7 min with 0.1% TritonX-100 in PBS or 15 min with 0.2% Triton-X100 in PBS, conforming to the anti-body employed. Cells were rinsed with PBS three times, blocked with 2.5% normalgoat serum in 0.2% Tween-20 in PBS (PBST) and with 0.4% fish skin gelatin inPBST. Then they were incubated with the following primary antibodies: mouseanti-desmosomal protein (0.045 mg/mL, ZK-31, Sigma Chemical Co.; 1:400), rabbitpolyclonal anti-GCM1 (AB2, Sigma–Aldrich; 1:50), rabbit polyclonal anti-� humanchorionic gonadotropin (hCG, A0231, Dako, 1:500) and rabbit anti-acetyl-histone H3(06-599, Upstate; 1:500). Cells were washed with PBST, blocked as described above,and incubated with the appropriate species-specific secondary antibodies, eitherred Alexa Fluor 594-conjugated goat anti-mouse IgG (Fab) or green Alexa Fluor488-conjugated donkey anti-rabbit IgG (Fab) (Molecular Probes) in a 1:720 finaldilution. All antibody incubations were carried out in a humidity chamber for 1 hat 37 ◦C. Nuclei were counterstained with Höechst 33342 dye (2 �g/mL) (MolecularProbes, Cat. H-21492). Slides were rinsed, mounted in Aqueous Mounting Mediumwith fluorescence tracers (Fluor Safe, Calbiochem, Cat. 345789) and were visualizedon a Nikon optical microscope (Nikon eclipse TE2000-U, USA). Cells with normal andabnormal nuclear morphology (condensed/fragmented chromatin) were scored. Foreach preparation at least five random fields with a total of more than 500 cells inthree independent experiments were counted and the results were expressed aspercentage of normal/abnormal nuclear morphology.

2.5. Western blotting

Whole protein extracts from JEG-3 were prepared in 5× Laemmli buffer con-taining 60 mM Tris–HCl pH 6.8, 10% glycerol, 2% sodium dodecyl sulphate, 1%2-�-mercaptoethanol and 0.002% bromophenol blue. Total protein samples wereseparated on a 10% SDS-PAGE, and proteins were transferred to a nitrocelluloseHybond-ECL (Amersham Bioscience). The membranes were blocked in 5% non-fat milk in TBS (20 mM Tris–HCl, 150 mM NaCl pH 7.8), supplemented with 0.2%Tween-20 (TBS-T) 1 h at room temperature or overnight at 4 ◦C and blots wereincubated with primary antibodies diluted in TBS-T for 1 h at room temperatureor overnight at 4 ◦C, in both cases according to the antibody. The following anti-bodies were used: rabbit polyclonal anti-� human chorionic gonadotropin (hCGA0231, Dako; 1:1000), rabbit polyclonal anti-GCM1 (AB2, Sigma–Aldrich; 1:750),mouse monoclonal anti-human ABCG2 (BXP-21 sc-58222, Santa Cruz; 1:300) andmouse monoclonal anti-�-actin (Sigma–Aldrich; 1:2000). After washing, the blotswere incubated with horseradish peroxidase-conjugated donkey anti-rabbit orsheep anti-mouse IgG secondary antibodies (Amersham Bioscience; 1:5000) inTBS-T, at room temperature for 1 h. Protein-antibody complexes were visualizedusing an enhanced chemiluminescence detection system (SuperSignalWest Pico;Pierce) and exposed to Kodak T-Mat G/RA film. Ponceau staining (0.2% Ponceau, 3%trichloroacetic acid, 3% sulfosalicylic acid) was used to verify protein transferencefrom gel to nitrocellulose membrane.

2.6. Hormone secretion

Culture supernatants of cells incubated with CPF (50, 100 �M) or with vehiclealone were collected and stored at −80 ◦C. Secreted �hCG was quantified throughan automated immunochemiluminometric assay (Immulite 2000 HCG, Siemens)according to the manufacture protocol. Electrochemiluminescence immunoassays(ECLIA, Roche) were used for quantification of total progesterone and estradiol con-centrations.

2.7. qRT-PCR

Total RNA was extracted from cultured cells using Trizol Reagent (Invitrogen).One microgram of total RNA was reverse-transcribed in a total volume of 20 �L usingrandom primers (Invitrogen) and 50 U of M-MLV reverse transcriptase (PromegaCorp.). The PCR primers listed in Table 1 were employed to quantify the ABCG2,StarD7, �hCG, GCM1 and KLF6 transcripts. Results were normalized to cyclophilinA (PPIA). Primer sequences were compared against the human genomic and thetranscript data base with the BLAST program [25] at the NCBI Web site to assessin silico PCR specificity. Transcripts were quantified by real time qRT-PCR (ABI7500 Sequence Detection System, Applied Biosystems) using the Sequence Detec-tion Software v1.4. Experiments were performed using 1× SYBR Green PCR MasterMix (Applied Biosystems). The cycling conditions included a hot start at 95 ◦C for10 min, followed by 40 cycles at 95 ◦C for 15 s and 60 ◦C for 1 min. Specificity wasverified by melting curve analysis and agarose gel electrophoresis. Relative geneexpression was calculated according to the 2−��Ct method. Each sample was ana-lyzed in triplicate. No amplification was observed in PCRs using as template wateror RNA samples incubated without reverse transcriptase during the cDNA synthesis.

2.8. Statistical analysis

Statistical analyses were performed using the GraphPad Prism 5.0 soft-ware. One-way Analysis of Variance (ANOVA) followed by Dunnett’s Multiple

M.E. Ridano et al. / Reproductive To

Table 1Primer sequences and concentrations used in qRT-PCR assays.

Gene Primer name Sequence (5′–3′) nM

ˇhCG ˇhCG-539 For GCT ACT GCC CCA CCA TGA CC 300ˇhCG-632 Rev ATG GAC TCG AAG CGC ACA TC 300

KLF6 KLF6-882 For CAC CAA AAG CTC CCA CTT GAA 200KLF6-960 Rev CAC ACC CTT CCC ATG AGC AT 200

StarD7 StarD7 For GGT AAT CAA GCT GGA GGT GAT TG 100StarD7 Rev GAG TAC ATT GGA TAA GGA AAA TGG GT 100

ABCG2 ABCG2-F CAA TGG GAT CAT GAA ACC TG 100ABCG2-R CAT TTA TCA GAA CAT CTC CAG A 100

GCM1 GCM1 For GAG GCA CGA CGG ACG CTT TAT ATT CAA 250

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GCM1 Rev TTG GAC GCC TTC CTG GAA A 250PPIA cyclo A For GTC AAC CCC ACC GTG TTC TT 100

cyclo A Rev CTG CTG TCT TTG GGA CCT TGT 100

omparison post-test was used to determine statistical significance. A p-value <0.05as considered statistically significant.

. Results

.1. JEG-3 cells viability and morphology

Viability of JEG-3 cells cultured in the presence of 5–100 �Mf CPF for 24 or 48 h remained statistically undistinguishablerom the control condition (Fig. 1). Cell morphology was ana-yzed by desmosomal protein immunodetection and chromatinuorescence staining. Most JEG-3 cells showed normal nuclearorphology and cell shape after cultured in the presence of up

o 100 �M of CPF for 24 and 48 h compared to control cultures.ells remained mononucleated with defined cell borders stained bynti-desmoplakin. In addition, no major morphological alterationsere observed in cells treated with 50 �M of CPF for 72 h (Fig. 2A).

n this condition, the same acetyl histone H3 chromatin stain-ng pattern was observed in treated and untreated cells (Fig. 2B),uggesting that this pesticide does not produce a gross modifi-ation on chromatin acetylation as has been reported for otheroxics like ethanol [26]. Quantification of nuclei with condensed orragmented chromatin showed only a slight increase in abnormaluclear morphology that was dependent on the dose and incuba-ion time (Fig. 2C). In sum, cell viability and morphology was notignificantly modified when cells were treated with CPF concen-rations of up to 100 �M for 24 h, and they were slightly affectedhen treatment was extended to 48 h suggesting that, in this con-

ext, CPF does not induce a marked damage of JEG-3 cells. Thereforee used these culture conditions in order to test the hypothesis

hat CPF alters the expression of molecules relevant to placentalhysiology.

ig. 1. Viability of JEG-3 cells exposed to CFP. Cells were cultured in the presencef the indicated concentrations of CFP or vehicle alone (CPF 0, control condition) for4 h (grey bars) or 48 h (black bars). Cell viability was assessed by the MTT reductionssay. Results represent the mean ± SEM of four independent experiments per-ormed in quadruplicate, and are presented as percentage of control. No significantifference (p < 0.05) compared to the control condition was detected.

xicology 33 (2012) 331– 338 333

3.2. CPF modifies the mRNA level of functional importantplacental genes

mRNA levels of the following genes were evaluated by qRT-PCR:the � subunit of the key hCG hormone implicated in pregnancymaintenance; the GCM1 transcription factor gene which is a centralregulator of trophoblast differentiation [27]; StarD7 whose expres-sion increases during differentiation of CTBs [28]; the membranemultidrug transporter ABCG2 implicated in protecting the fetusagainst the potential toxicity of drugs, xenobiotics, and metabo-lites [29]; and the trophoblastic-enriched KLF6 transcription factorinvolved in transcriptional regulation of placental genes during tro-phoblast differentiation [30,31].

�hCG mRNA level was clearly augmented in cells grown in thepresence of 50 or 100 �M of CPF during 24 h and further increasedto more than 15-folds after 48 h compared with cells cultured inthe presence of vehicle alone (Fig. 3). CPF treatment also inducedthe expression of GCM1 at both concentration and time exposure,although to a less extent. In addition, ABCG2 transcript level wasincreased in cells treated for 24 h, remaining augmented only in thepresence of 100 �M of CPF after 48 h. By contrast, the expression ofStarD7 and KLF6 mRNAs were not significantly modified in any ofthe assayed conditions (Fig. 3).

3.3. ˇhCG synthesis is markedly increased in cells exposed to CPF

Then, we analyzed whether the increase in mRNA levelswas translated into a higher protein expression. Western blotassays demonstrated that intracellular �hCG level was markedlyincreased in CPF-treated cells compared with control cells in a doseand time dependent manner (Fig. 4A and B). Induction of �hCGexpression was further established by immunofluorescence assays.JEG-3 cells cultured 48 h in the presence of CPF showed an intensegreen signal corresponding to immunodetected �hCG which wasnot observed under the same light exposure and image capturecondition in control cultures (Fig. 4C).

3.4. ABCG2 but not GCM1 protein levels are modified in cellsexposed to CPF

In addition, the amount of the ABCG2 ATP-dependent xenobi-otic transporter protein was increased after 48 h of CPF treatment.However, GCM1 protein level remained unmodified in all testedconditions (Fig. 5A and B). Immunofluorescence assays furtherrevealed that GCM1 protein distribution and fluorescence intensitywas not markedly altered in JEG-3 cells cultured in the presenceof CPF for 24 h (data not shown). The absence of an absolutecorrelation between the induction of GCM1 mRNA level and itscorresponding protein level might reflect differences in mRNA andprotein half life, or may be the consequence of a complex expressioncontrol mechanism.

3.5. CPF induces ˇhCG, but not, progesterone and estradiolsecretion

We also investigated the consequence of CPF treatment on�hCG, as well as progesterone and estradiol, secretion. After 48 h oftreatment with 50 or 100 �M CPF, hormone quantification assaysrevealed a clear increase in �hCG concentration detected in thesupernatant corresponding to the last 24 h of culture (Fig. 6). In con-trast, the concentration of secreted estradiol remained unmodified

and a small increase in progesterone, although statistically non-significant, was detected. Altogether, these results indicate thatCPF is able to induce �hCG transcription, synthesis and secretion inthe placental-derived JEG-3 cell line. In addition, they suggest that

334 M.E. Ridano et al. / Reproductive Toxicology 33 (2012) 331– 338

Fig. 2. Effect of CPF on JEG-3 cell morphology. Cells were exposed to 0.04% DMSO (control), 50 �M or 100 �M of CPF for 24, 48 or 72 h. Then cells were fixed and analyzedby immunofluorescence microscopy assays. (A) Assays performed with anti-desmoplakin antibody (red) and nuclear staining by Höechst dye (blue). Arrow: an abnormalnucleus; arrowhead: a mitotic nucleus. (B) Assay performed with an anti-acetylated histone H3 antibody (green). (A and B) Final magnification 400×; scale bar = 10 �m. (C)Normal, black bar, and abnormal (condensed/fragmented nuclei), grey bar, cells were counted in at least five random fields in each coverslip. More than 500 nuclei in eachculture condition in three independent experiments were scored and results are expressed as percentage of total nuclei. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of the article.)

Fig. 3. Effect of CPF on the expression of �hCG, GCM1, ABCG2, KLF6 and StarD7 transcripts. mRNA expression of the indicated genes was quantified by qRT-PCR (ABI 7500,Applied Biosystems) in JEG-3 cells cultured during 24 h (grey bars) or 48 h (black bars) in the presence of the indicated CPF concentrations or vehicle alone (0). Results werenormalized to cyclophilin A and expressed according to the 2−��Ct method using as calibrator the mRNA level obtained from the control condition (0). Data are presentedas mean ± SEM of at least three independent experiments performed in triplicates. *Significantly different from the corresponding control (0) group (p < 0.05).

M.E. Ridano et al. / Reproductive Toxicology 33 (2012) 331– 338 335

Fig. 4. CPF increases the expression of �hCG protein. (A) Western blot analysis of protein extracts prepared from JEG-3 cells cultured in the presence or not of CPF for 24 or 48 h.Assays were performed using anti-�hCG and �-actin antibodies as described in Section 2. Representative blots are shown. (B) Densitometric quantification of �hCG/�-actinratio of at least three independent experiments expressed as mean ± SEM. *p < 0.05 compared to control. (C) Immunofluorescence of �hCG (green), desmoplakin (red) andn icle a4 (For it

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uclear stain with Höechst (blue) in JEG-3 cells after 48 h of culture with CPF or veh00×. Right panels: merge image, original magnification 1000×. Scale bar = 10 �m.o the web version of the article.)

PF exposure does not affect the production of other placental hor-ones produced by the trophoblast as progesterone and estradiol.

. Discussion

Placental function and development requires the interac-ion between endogenous programs and external signals whichonverge on the regulation of gene expression mediated by tran-cription factors. This process may be adversely modulated bynvironmental toxic substances, as demonstrated in different cellodels. CPF is one of the most widely used organophosphorus pes-

icides in agriculture and several reports have indicated that it maynfluence cell physiology, replication or differentiation [3]. Here,

e have investigated the effects of CPF on important functionalene expression in the trophoblast-derived JEG-3 cell line, used as aodel of human trophoblast cells. Employing treatment conditionshere cell morphology and viability was not seriously compro-ised, we found that �hCG gene transcript and protein, as well

s, protein secretion was markedly up-regulated by CPF. In addi-ion, CPF modified the expression of ABCG2 and GCM1 genes, whilehe expression of the KLF6 transcription factor and the lipid trans-ort StarD7 mRNAs remained unchanged. It has been previouslyeported that CPF affects pro-apoptotic gene expression in JARrophoblast-like cell line [23], but as far as we know, present studys the first report showing that CPF dysregulates the expression ofenes specifically related to trophoblast function and differentia-ion.

The most striking effect of CPF on trophoblastic cell genexpression was the induction of �hCG. Preliminary observationsuggest that this effect is not restricted to JEG-3 cells becauseimilar results were obtained with BeWo, a different trophoblast-ell line (data not shown). The fact that CPF has an effect on

hCG is quite significant since expression of the hCG hormone

s tightly associated with normal or altered trophoblast func-ion [32]. Higher than average third trimester serum hCG levelsave been reported in pregnant women with preeclampsia and

lone (control). Left panels: only the green channel is shown, original magnificationnterpretation of the references to color in this figure legend, the reader is referred

superimposed preeclampsia with chronic hypertension [33]. More-over, elevated levels of maternal serum hCG measured at 15–20weeks gestation may result from premature accelerated differ-entiation of the villous cytotrophoblasts, leading to subsequentpathologic alterations in syncytiotrophoblast morphology whichincrease the risk of severe preeclampsia and intra-uterine growthrestriction [34]. Therefore, the capability of CPF to induce �hCGsynthesis and secretion could pose an increased risk of placentaldisorders due to an altered trophoblast function or differentiation.Nevertheless, whether the observed modification in gene expres-sion reflects a nocive effect of CFP on placenta cells or a defensemechanism in order to preserve placental function even in thepresence of the toxic is not known at present. Interestingly, a pro-tective role of hCG in arsenic-mediated ovarian and uterine toxicity[35], as well as in apoptosis induced by oxidative stress in decidu-alizing human endometrial stromal cells [36], have been recentlyproposed.

The increase in ABCG2 protein expression may also reflect theactivation of trophoblastic cell defense mechanisms induced by thepresence of CPF. ATP-binding cassette efflux transporters, includ-ing the ABCG2 protein, are highly expressed in placental tissuesand are believed to contribute to the placenta’s ability to reduce thepassage of therapeutic or toxic compounds to the fetus [37]. More-over, ABCG2 has been reported to enhance hypoxic cell resistancevia removal of toxic products of heme metabolism from cytoplasm[38], a function that may be important for trophoblast survival insome pregnancy complication.

GCM1 is a critical placental transcription factor which promotessyncytiotrophoblast formation and placental vasculogenesis. Itsexpression is highly regulated at both the transcriptional and trans-lational level including ubiquitination and proteasome-mediateddegradation providing a precise and complex control of GCM1

activity [39,40]. This complex regulation may explain the absenceof a direct correlation between the effects induced by CPF on GCM1mRNA and protein expression. Nevertheless, we cannot discard thepossibility of a small modification in its expression level which

336 M.E. Ridano et al. / Reproductive Toxicology 33 (2012) 331– 338

Fig. 5. ABCG2 and GCM1 protein expression in cells treated with CPF. Proteinextracts prepared from JEG-3 cells exposed or not to CPF for 24 or 48 h weresubjected to western blot analysis using anti-ABCG2 (A) or anti-GCM1 (B) and anti-�-actin antibodies. Representative blots are shown. The bar graphs represent thedp

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Fig. 6. Quantification of �hCG, estradiol and progesterone hormones secreted intothe culture media of JEG-3 cells exposed to CPF during 48 h. Results are expressedas hormone concentration secreted into the culture medium in the last 24 h of

induced effects in in vitro culture conditions to in vivo exposure

ensitometric quantification of ABCG2/�-actin or GCM1/�-actin ratio of three inde-endent experiments expressed as mean ± SEM. * p < 0.05 compared to control.

ould be undetectable by immunodetection assays. While this ishe first report of a toxic compound affecting GCM1 mRNA expres-ion, some reports have described an augmented hCG expression inrophoblast cells exposed to different environmental toxics such asisphenol A, DDT, and trialkyltin compounds [41–43]. These data,ogether with the increase of hCG in pregnancy diseases of placen-al origin suggest that dysregulation in hCG expression is a markerf placental injury or toxicity.

The specific molecular or cellular mechanisms by which CPFauses the observed changes in �hCG, GCM1 and ABCG2 expressionre unknown at present, but several possibilities may be envi-ioned. One is that the expression of these proteins is modified as aonsequence of an oxidative stress provoked by CPF in trophoblastells. In fact, CPF is known to produce oxidative stress in several

ell types [44,45], and serum hCG increase has been proposed as aarker of oxidative stress during pregnancy [46]. Another hypothe-

is is that CPF activates the cAMP response element binding protein

treatment relative to the control condition (0) defined as 1. Data is representedas mean ± SEM of at least four independent experiments. * p < 0.05 compared tocontrol.

as has been reported in neurons [47,48] or some components ofthe signal transduction through the adenylyl cyclase cascade asreported to occur in brain during a discrete developmental periodin late gestation [49]. This is an attractive alternative since adeny-lyl cyclase pathway is indeed involved in the expression of �hCG,GCM1 [50] and ABCG2 genes [51].

In the experimental conditions used, viability of JEG-3 andBeWo cells was not severely compromised with CPF concentra-tions of up to 100 �M, suggesting that trophoblast cells are quiteresistant to CPF. These results are somehow different from thosereported by Saulsbury et al. [23]. They found that CPF caused a dose-dependent reduction in JAR cellular viability with an IC50 value of59.1 ± 1.19 �M at 24 h. Although we cannot rule out that the differ-ence observed between both reports is because different methodswere used to asses cell viability, we should emphasize that chori-ocarcinoma derived JEG-3, BeWo and JAR (the most accepted andwidely used cell lines for the study of trophoblast function) dif-fer in several characteristics such as proliferative activity, degreeof differentiation [52] and toxic metabolism [53]. Therefore, theobserved discrepancies in cell viability may be due to their differentgene expression profiles.

Some limitations preclude the direct extrapolation of presentfindings to the actual in vivo effects of CPF on placenta duringpregnancy. First, the study was performed using a transformedtrophoblast cell line and, as in all in vitro studies, a major limi-tation is that cells are not in their normal environment. There areno neighboring cells or tissues to interact with and to supply poten-tially important factors which could modify cell susceptibility to thetoxic. Second, studies were performed using the CPF parent com-pound. This may not completely reflect the situation with in vivoexposure where CPF is metabolically activated, mainly in the liver,to its more toxic oxon-derivative, responsible of cholinesteraseinhibition. However, contributions of noncholinergic mechanismto the net adverse effect on brain development [54] and JAR tro-phoblast cell line [23] have been reported. In addition, studies ofour laboratory showed that in the experimental conditions used,JEG-3 cell acetycholinesterase activity decreased suggesting thatCPF-oxon was indeed produced (Chiapella et al., personal com-munication). A third issue related to current human exposurelevels and CPF concentrations used in our experiments should beconsidered. The problems associated with the translation of OP-

doses, as well as the inherent difficulties associated with expo-sure assessment to nonpersistent OP pesticides, as CPF, are largelyrecognized [55,56]. Many in vitro studies have employed similar

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oncentrations to analyze OP effects on neuronal and non-neuronalell models [57–59]. Whyatt et al. [60] evaluated residential expo-ure to OP and showed that CPF maternal plasma concentrationsere in the order of pg/g. These values differ widely from thosesed in our experiments. However, it has been stated that concen-rations in the micromolar range, although high, are still within theange of potential human exposure [61,44]. Measurements of OP ineconium, which yield a longer-term dosimeter of prenatal expo-

ure [62], suggest that the fetus and placenta may be exposed toigh CPF doses in agricultural communities [63].

Despite the limitations discussed above, present findings are rel-vant because they allowed the identification of novel biomarkersf CPF effects on trophoblast cells. They point to the human pla-enta as a possible target organ and encourage further in vitro andn vivo studies to address the involvement of these molecules inregnant women response to CPF exposure.

In sum this study reveals that, in conditions where JEG-3 cellsemain viable, CPF is able to alter the expression of �hCG, GCM1nd ABCG2 molecules which are relevant for the maintenance of aealthy pregnancy.

unding

This work was supported by Consejo Nacional de Investiga-iones Científicas y Técnicas (CONICET: PIP No 6352, PIP No 919),rgentina. Agencia Nacional de Promoción Ciencia y Tecnológica

FONCyT: PICT-2006-00147). Ministerio de Ciencia y Tecnologíae la Provincia de Córdoba. Secretaría de Ciencia y Tecnologíae la Universidad Nacional de Córdoba, Argentina (SECyT-UNC:5/C479). SACyT, Comisión Nacional Salud, Ciencia y Tecnología.he funders had no role in study design, data collection and analysis,ecision to publish, or preparation of the manuscript.

onflicts of interest

The authors declare that there are no actual or potential conflictsf interest.

cknowledgements

We gratefully acknowledge Dra Miriam Virgolini for her sug-estions and review of the manuscript. SG-R and GMP-D are Careernvestigators of CONICET. MER, ACR, JFM and SC thank CONICET forheir fellowships.

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