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Toxicology Letters 190 (2009) 187–192

Contents lists available at ScienceDirect

Toxicology Letters

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

irect inhibition of ERK1/2 phosphorylation as a possible mechanism for thentiproliferative action of 3,4-diOH-PCB3 in the MCF-7 cell line

nna Ptak, Patrycja Gut, Małgorzata Błachuta, Agnieszka Rak, Ewa Ł. Gregoraszczuk ∗

epartment of Physiology and Toxicology of Reproduction, Institute of Zoology, Jagiellonian University, 30-060 Kraków, Ingardena 6, Poland

r t i c l e i n f o

rticle history:eceived 14 May 2009eceived in revised form 8 July 2009ccepted 10 July 2009vailable online 18 July 2009

eywords:CB3ydroxylated metabolites

a b s t r a c t

Our previously published data showed that 260 h of exposure to 3,4-diOH-PCB3 decreased proliferationin the MCF-7 cell line. In the present study, we sought to determine whether this is due to action on theSHBG/cAMP/PKA system, activation of which can inhibit cell proliferation, or to direct inhibition of ERK1/2phosphorylation. MCF-7 human breast cancer cells were treated for 72 h with 4-monochlorobiphenyl(PCB3), 4′-hydroxy-4-monochlorobiphenyl (4-OH-PCB3) or 3′4′-dihydroxy-4-monochlorobiphenyl (3,4-diOH-PCB3) (300 nM). After the completion of the treatment, cell proliferation was measured with a BrdUincorporation assay. SHBG, cAMP, PKA and ERK1/2 levels in the cells were determined via ELISA.

PCB3 and 4-OH-PCB3 had no effect on extra- or intracellular SHBG levels, while a stimulation ofSHBG intra- but not extracellular levels was noted in cells exposed to 3,4-diOH-PCB3. Both, pre- and

HBG

AMP/PKARK1/2roliferation

co-incubation with SHBG decreased the proliferation of 3,4-diOH-PCB3-treated cells. Neither PCB3 norits metabolite had an effect on the cAMP/PKA pathway. A decrease of both ERK1/2 forms was noted underthe influence of 3,4-diOH-PCB3. In conclusion, the data presented clearly showed that the antiprolifer-ative action of 3,4-diOH-PCB3 is not mediated by activation of the SHBG/AMP/PKA pathway, but many

recephibiti

other plasma membraneinstead is due to direct in

. Introduction

New evidence published during the last decade indicates thatteroid hormones can exert rapid, non-genomic action initiated athe cell surface by binding to steroid-specific membrane receptorsRevelli et al., 1998; Falkenstein et al., 2000; Norman et al., 2004).he results of recent studies suggest that non-genomic steroidctions are susceptible to interference by environmental estrogensThomas and Dong, 2006; Ropero et al., 2006; Watson et al., 2007).ur previously published data showed that PCB3 and its metabolite-OH-PCB3 had no effect on cell proliferation at any time dur-

ng exposure, while 3,4-diOH-PCB3 at the highest concentrationecreased cell proliferation in the MCF-7 cell line (Gregoraszczukt al., 2008).

The sex hormone-binding globulin receptor (SHGB-R) is ateroid-specific membrane receptor that participates in E2 sig-aling from the membrane (Hryb et al., 1990; Porto et al., 1992).

HBG binding inhibits the estradiol-induced proliferation of MCF-7ells (Fortunati et al., 1993, 1996), and this antiproliferative effectf SHBG in MCF-7 cells is mediated by the second messengerAMP and its downstream kinase PKA (Fortunati et al., 1996, 1999;

∗ Corresponding author. Tel.: +48 12 663 26 15; fax: +48 12 634 37 16.E-mail address: ewa.gregoraszczuk@uj.edu.pl (E.Ł. Gregoraszczuk).

378-4274/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.toxlet.2009.07.013

tors seem to be involved in the non-genomic action of 3,4-diOH-PCB3, andon of the ERK1/2 system.

© 2009 Elsevier Ireland Ltd. All rights reserved.

Zivadinovic et al., 2005). Additionally, it has been reported thatthe cAMP/PKA pathway and the mitogen-activated protein kinase(MAPK, also called extracellular signal-regulated kinase, or ERK)pathways are connected in MCF-7 cells (Lowe et al., 1997; Houslayand Kolch, 2000; Zivadinovic et al., 2005). Notably, ERK can be acti-vated or inhibited by cAMP in a cell-specific manner that dictatesthe growth effect of cAMP (Stork and Schmitt, 2002). Moreover,several studies have demonstrated that xenoestrogens can alsocause the oscillating activation of ERK (Bulayeva and Watson, 2004;Watson et al., 2007; Radice et al., 2008). There are data show-ing that human SHBG binds phytoestrogens (Déchaud et al., 1999;Martin et al., 1996), fatty acids (Martin et al., 1986) and severalpharmaceutical agents (Pugeat et al., 1981). Only one study (Juryet al., 2000) suggests that 4-OH-2′,3′,4′,5′-tetrachlorobiphenyl and4-OH-2,2′,3′,4′,5′-pentachlorobiphenyl might interact with SHBG.

In the present study, we investigated the ability of PCB3 andits hydroxylated metabolites (4-OH-PCB3 and 3,4-diOH-PCB3) tochange SHBG levels and thereby affect cell proliferation. We alsoevaluated their ability to activate the cAMP/PKA pathway and theERK pathway in the MCF-7 cell line.

2. Materials and methods

2.1. Reagents

DMEM medium without phenol red, fetal bovine serum (FBS, heat inacti-vated), insulin-transferrin-sodium (ITS) selenite media supplement, penicillin,

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88 A. Ptak et al. / Toxicolog

treptomycin, trypan blue, PD98059, IBMX (3-isobutyl-1-methylxanthine),harcoal-dextran, and SHBG (sex hormone-binding globulin) were obtained fromigma Chemical Co. (St. Louis, MO, USA). Forskolin (For) was purchased from Cal-iochem (Darmstadt, Germany) and PKI was from BioSource (Camarillo, CA, USA).-Monochlorobiphenyl (PCB3), 4′-hydroxy-4-monochlorobiphenyl (4-OH-PCB3) or′4′-dihydroxy-4-monochlorobiphenyl (3,4-diOH-PCB3) were kindly donated byans-Joachim Lehmler and Larry W. Robertson (Department of Occupational andnvironmental Health, University of Iowa, Iowa City, USA).

.2. Cell culture

MCF-7 human breast cancer cells (ATCC, Manassas, VA, USA) were routinelyultured in DMEM supplemented with 10% heat-inactivated FBS, 100 IU/ml of peni-illin and 100 �g of streptomycin. Forty-eight hours before experiments, the mediumas removed and replaced with DMEM without phenol red supplemented with 5%extran-coated, charcoal treated FBS (5% DC-FBS). Then, cells were plated in the sameedium and allowed to attach overnight. Twenty-two hours before experiments theedium was replaced with phenol red-free DMEM supplemented with ITS (5 �g/ml

nsulin, 5 �g/ml transferrin, 5 ng/ml Na selenite, using the stock preparation fromigma).

.3. SHBG analysis

The cells were seeded in 12-well culture plates at a density of 50 × 104 cells/wellnd then incubated in DMEM supplemented with ITS as a control medium or inedium supplemented with 300 nM PCB3 or its metabolites (4-OH-PCB3 and 3,4-

iOH-PCB3) for 24 h. After 24 h, the medium was collected and the cells were washedith phosphate buffered saline (PBS) and harvested mechanically. The SHBG con-

entrations were determined using the enzyme immunoassay SHBG ELISA (DRGnstruments GmbH, Marburg, Germany). The assay was performed according to the

anufacturer’s instructions. The sensitivity of the assay was 0.2 nmol/l, the intra-ssay variation was 3.0–8.6%, the inter-assay variation was 7.2–11.6%, and the lineareasuring range was 0–260 nmol/l. Absorbance values were measured at 450 nm

sing an ELISA reader (ELx808 BIO-TEK Instruments, USA).To confirm the involvement of SHBG in the action of PCB3 and its metabolites

n cell proliferation, we (1) pre-incubated cells with 1 nM SHBG for 40 min and thenreated them with PCB3, 4-OH-PCB3 or 3,4-diOH-PCB3 for 72 h, and (2) co-incubatedells with 1 nM SHBG and PCB3, 4-OH-PCB3 or 3,4-diOH-PCB3 for 72 h. At the endf the culture, cell proliferation was measured.

.4. cAMP analysis

Cells were plated at a density of 0.5 × 106 cells/well in 24-well plates and thenncubated in DMEM supplemented ITS as a control medium or in medium supple-

ented with 300 nM PCB3 or its metabolites (4-OH-PCB3 and 3,4-diOH-PCB3) for4 h. After 24 h, cells were pretreated for 30 min with 1 mM IBMX and then treatedor the next 30 min with PCB3 or its metabolites and forskolin (10 �M). At the end ofhe incubation, the cells were lysed with 0.1 M HCl, and cAMP levels were evaluatedith the Direct Cyclic AMP Enzyme Immunoassay Kit (Assay Desings, Inc., Ann Arbor,I, USA). The assay was performed according to the manufacturer’s instructions.

he sensitivity of the assay was 0.037 pmol/ml, and the linear measuring range was–20 pmol/ml. Absorbance values were measured at 405 nm using an ELISA readerELx808 BIO-TEK Instruments, USA).

To confirm the involvement of cAMP in the action of PCB3 and its metabolites inell proliferation, we pre-incubated cells with 10 �M forskolin for 1.5 h, then treatedhem with PCB3, 4-OH-PCB3 or 3,4-diOH-PCB3 for 72 h. At the end of the cultureeriod, cell proliferation was measured.

.5. PKA analysis

Cells were plated at a density of 0.5 × 106 cells/well in 24-well plates and thenncubated in DMEM supplemented ITS as a control medium or in medium supple-

ented with 300 nM PCB3 or its metabolites (4-OH-PCB3 and 3,4-diOH-PCB3) for4 h. After 24 h, cells were pretreated for 30 min with 1 mM IBMX. Cells were thenreated with PKI (1.5 �M) for 90 min followed by PCB3 or its metabolites for 30 min.t the end of the incubation, the cells were lysed with lysis buffer (20 mM MOPS,0 mM �-glycerolphosphate, 50 mM sodium fluoride, 1 mM sodium vanadate, 5 mMGTA, 2 mM EDTA, 1% NP40, 1 mM dithiothreitol (DTT), 1 mM benzamidine, 1 mMhenylmethane-sulphonylfluoride (PMSF) and 10 �g/ml each of leupeptin and apro-inin), and PKA activity was evaluated with the PKA Kinase Activity Assay KitAssay Desings, Inc., Ann Arbor, MI, USA). The assay was performed according tohe manufacturer’s instructions. The intra-assay coefficient of variation was <10%,he inter-assay coefficient of variation was ≤12%, and the linear measuring range

as 0–300 ng/assay. The protein concentration was determined using the Bradfordethod. Absorbance values were measured at 405 nm using an ELISA reader (ELx808

IO-TEK Instruments, USA).To confirm the involvement of PKA in the action of PCB3 and/or its metabolites

n cell proliferation, cells were pre-incubated with 1.5 �M PKI for 1.5 h, and thenreated with PCB3, 4-OH-PCB3 or 3,4-diOH-PCB3 for 72 h. At the end of the cultureeriod, cell proliferation was measured.

rs 190 (2009) 187–192

2.6. Phospho- and total-ERK1/2 analysis

Cells were plated at a density of 5 × 104 cells/well in 96-well plates and thenincubated in DMEM supplemented with ITS as a control medium or in medium sup-plemented with 300 nM PCB3 or its metabolites (4-OH-PCB3 and 3,4-diOH-PCB3)for 24 h. After 24 h, cells were incubated for 1 min, 5 min or 15 min for measure-ment of phospho- and total-ERK1/2 levels in control cells. 5 min incubation waschosen for further experiments with PCB3 or its metabolites. The levels of phospho-and total-ERK1/2 were determined by EIA using a commercially available ELISA Kit(RayBio®Cell-Based Erk1/2(Thr202/Tyr204), USA). All samples were run in duplicatein the same assay. The kit protocol was followed per the manufacturer’s instructions.The inter- and intra-run precision had coefficients of variation of 3.0% and 3.5%,respectively. Absorbance values were measured at 450 nm using an ELISA reader(ELx808 BIO-TEK Instruments, USA).

To confirm the involvement of ERK1/2 in the action of PCB3 and/or its metabolitesin cell proliferation, we used a known inhibitor of this kinase, PD098059. Cells werepre-incubated with 30 �M PD098059 for 1 h, then treated with PCB3, 4-OH-PCB3or 3,4-diOH-PCB3 for 72 h. At the end of the culture period, cell proliferation wasmeasured.

2.7. Cell proliferation

DNA synthesis in proliferating cells was determined by measuring BrdU incor-poration with the commercial Cell Proliferation ELISA System (Roche MolecularBiochemicals, Mannheim, Germany). The cells were seeded in 96-well culture platesat a density of 2.5 × 104 cells/well and then incubated in DMEM supplemented withITS as a control medium or with PCB3 or its metabolites for 72 h. After 72 h, mediumwas removed and the cells were incubated for 2 h with a BrdU labeling solution (pro-vided by the kit) containing 10 �M BrdU. The assay was performed according to themanufacturer’s instructions. Absorbance values were measured at 450 nm using anELISA reader (ELx808 BIO-TEK Instruments, USA). Culture medium alone was usedas a control for nonspecific binding.

2.8. Statistical analysis

Each treatment was repeated three times (n = 3), and each repetition was run inquadruplicate. The average of the four values was used for statistical calculations.Statistical analysis was performed using Statistica 6.0. Data were analyzed by 1-wayanalysis of variance (ANOVA) followed by Tukey’s Honestly Significant Differences(HSD) multiple range test. Groups that are significantly different from each other(p < 0.05) are indicated in the figures with different letters. Data points with thesame letters are not significantly different.

3. Results

3.1. Involvement of SHBG in the action of PCB3, 4-OH-PCB3 and3,4-diOH-PCB3 on cell proliferation

PCB3 and 4-OH-PCB3 had no effect on extracellular or intra-cellular SHBG levels. Stimulatory action on intracellular but notextracellular SHBG levels was noted in cells exposed to 3,4-diOH-PCB3 (5.47 ± 0.06 nmol/l vs. 4.55 ± 0.07 nmol/l for the untreatedcells; Fig. 1a).

MCF-7 cell growth was not affected by the use of SHBG alone.PCB3 and 4-OH-PCB3 both failed to affect the proliferation ofcells either pre-incubated and co-incubated with SHBG, while 3,4-diOH-PCB3 decreased cell proliferation similarly in both cultureconditions (Fig. 1b).

3.2. Involvement of the cAMP/PKA second messenger system inthe action of PCB3, 4-OH-PCB3 and 3,4-diOH-PCB3 on cellproliferation

Forskolin, a known stimulator of cAMP, increased cAMP levels1.6-fold (Fig. 2a) and decreased MCF-7 cell line proliferation to 70%that of control cells (Fig. 2b). Neither PCB3 nor its hydroxylatedmetabolites increased basal cAMP levels. In concomitant treat-ments with forskolin, cAMP levels increased to the level observed

under the influence forskolin alone (Fig. 2a), and cell proliferationdecreased to the level observed under the influence of forskolinalone (Fig. 2b).

PKI (a PKA inhibitor) reduced basal PKA levels 2.4-fold (Fig. 3a)and increased cell proliferation 1.2-fold (Fig. 3b). Neither PCB3 nor

A. Ptak et al. / Toxicology Letters 190 (2009) 187–192 189

Fig. 1. The effect of PCB3, 4-OH-PCB3 and 3,4-di-OH-PCB3 on (a) intracellularamsa

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Fig. 2. The effect of PCB3, 4-OH-PCB3 and 3,4-di-OH-PCB3 on (a) cAMP levels, and

nd extracellular SHBG levels, and (b) cell proliferation. Each point represents theean ± S.E.M. of results from three independent experiments, each of which con-

isted of four replicates per treatment group. All means marked with (*) (p < 0.05)nd different letters are significantly different from the control.

ts hydroxylated metabolites increased basal PKA levels. The reduc-ion of PKA levels in cells co-treated with PKI and the reagents was ofhe same level as was noted under the influence PKI alone (Fig. 3a).either PCB3 nor its hydroxylated metabolites had a statistically

ignificant effect on PKI-stimulated proliferation (Fig. 3b).

.3. Involvement of ERK1/2 kinase in the action of PCB3,-OH-PCB3 and 3,4-diOH-PCB3 on cell proliferation

Taking into consideration the data of Migliaccio et al. (1996) bothotal and phospho-ERK1/2 kinase levels were measured in controlultures after 1, 5 and 15 min of incubation. The maximum levelsf both total and phospho-ERK1/2 were noted after 5 min of incu-ation, and this time was chosen for further experiments (Fig. 4).CB3 and 4-OH-PCB3 had no significant effect on the levels of eitherRK1/2 forms, while a decrease in the levels of both ERK1/2 formso 60% those of control cells was noted under the influence of 3,4-iOH-PCB3 (Fig. 5a).

PD98059 (a selective ERK1/2 inhibitor) decreased basal cell pro-iferation to 80% of that of control cells. A further 30% reduction inell proliferation (to 50% that of control cells) was noted in cellsreated with both PD98059 and PCB3 metabolites (Fig. 5b).

. Discussion

It is well known that MCF-7 breast cancer cells express a spe-ific binding site for SHBG (Fortunati et al., 1993; Porto et al., 1992).ore importantly, the interaction of SHBG with the membrane-

(b) cell proliferation. Each point represents the mean ± S.E.M. of results from threeindependent experiments, each of which consisted of four replicates per treatmentgroup. Statistically significant differences between points in each graph are indicatedwith different letters (p < 0.05).

binding side inhibits estradiol-induced proliferation, and this actionis mediated by cAMP and PKA (Fortunati et al., 1996; Zivadinovicet al., 2005). Our previously published data showed that PCB3and its 4-OH-PCB3 metabolite had no effect on cell proliferationat any time point during exposure, while 3,4-diOH-PCB3 (at thehighest concentration) decreased proliferation in the MCF-7 cellline (Gregoraszczuk et al., 2008). The question arises whether theobserved antiproliferative action of 3,4-diOH-PCB3 is mediated byactivation of SHBG and the cAMP/PKA pathway. To our knowledgethere are no data showing changes in SHBG levels due to the influ-ence of xenoestrogens or their metabolites. However, it is wellknown that endogenous estrogens, both in vivo (Odlind et al., 1982)and in vitro (Loukovaara et al., 1995; Lee et al., 1987), act on SHBGsynthesis.

Our data indicate that neither PCB3 nor 4-OH-PCB3 affect eitherextra- or intracellular levels of SHBG or cell proliferation. However,an increase in intracellular SHBG levels was noted under the influ-ence of 3,4-diOH-PCB3. Moreover, neither pre- nor co-incubationwith SHBG altered the antiproliferative effect of 3,4-diOH-PCB3.According to Hryb et al. (1990) the interaction of SHBG withits membrane-binding site requires the occurrence of a precisesequence of events if it is to be biologically relevant. First, SHBGmust bind to membranes (pre-incubation), and second, a ligandmust interact with the SHBG bound to membranes. It is only atthis point that the biological effect is elicited. If the ligand binds to

SHBG before the protein binds to the membrane (co-incubation) itis blocked from interacting with membranes.

Our results indicate that 3,4-diOH-PCB3 does not inter-act with SHBG, either through the membrane-binding site ordirectly. There is only limited information available regarding

190 A. Ptak et al. / Toxicology Letters 190 (2009) 187–192

Fig. 3. The effect of PCB3, 4-OH-PCB3 and 3,4-di-OH-PCB3 on (a) PKA levels, and(igw

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Fig. 5. The effect of PCB3, 4-OH-PCB3 and 3,4-di-OH-PCB3 on (a) phospho- and total-ERK1/2 levels, and (b) cell proliferation. Each point represents the mean ± S.E.M. of

b) cell proliferation. Each point represents the mean ± S.E.M. of results from threendependent experiments, each of which consisted of four replicates per treatmentroup. Statistically significant differences between points in each graph are indicatedith different letters (p < 0.05).

he ability of xenoestrogens to interact with SHBG. Jury etl. (2000) examined a panel of nine hydroxy-polychlorinatediphenyls (HO-PCBs) with a screening assay, and found thatlthough the PCBs had little or no ability to interact with SHBG,nly 4-OH-2′,3′,4′,5′-tetrachlorobiphenyl and 4-OH-2,2′,3′,4′,5′-entachlorobiphenyl bound SHBG in undiluted serum. Seven otherydroxylated PCBs (4-OH-2,3,5,3′,4′,5′-hexaCB; 4-OH-2,3,5,3′,4′-enta-CB; 4-OH-3,5,2′,3′,4′-penta-CB; 4-OH-2,3,5,2′,3′,4′-hexaCB;-OH-2,4,5,2′,3′,4′,5′-heptaCB; 4-OH-2,3,5,2′,3′,4′,5′-hepta-CB; 4-H-2,3,5,6,2′,4′,5′-hepta-CB) were only active in diluted serum.

sed by us 3,4-diOH-4′-monoCB are low chlorinated biphenyls inomparison with high chlorinated CB used by Jury et al. (2000).t is necessary to mentions that binding to SHBG, does not nec-ssarily have to correlate with activation of the cascade of MAP

ig. 4. Time-dependent total and phospho-ERK1/2 levels in the MCF-7 cell line.

results from three independent experiments, each of which consisted of four repli-cates per treatment group. All means marked with (*) (p < 0.05) and different lettersare significantly different from the control.

kinase. Thomas and Dong (2006) showed that 2,2′,5-PCB-4-OHelicits its responses via nontraditional estrogen actions mediatedthrough G-protein-coupled receptor 30 (GPR30). GPR30 is a seven-transmembrane receptor which activates second messengers suchas adenylate cyclase and MAP kinase in response to ligand (Filardoand Thomas, 2005). Thus, another signal transduction pathway,other than SHBG, may be responsible for action of 3,4-diOH-PCB3.However, further studies are necessary to confirm this hypothesis.

The next interesting finding of this study is that 3,4-diOH-PCB3is ineffective for the activation of cAMP. Moreover, using a PKAinhibitor, we showed that neither PCB3 nor its metabolites actthrough PKA. No information is available in the literature aboutthe interaction of xenoestrogens and even phytoestrogens withthe cAMP/PKA pathway. We decided to undertake this experi-ment because of the fact that PCB3 and especially its hydroxylatedmetabolites are potent estrogens in the ovary (Ptak et al., 2005).From our (unpublished data) and the data of Fortunati et al. (1999),it is known that E2 has no detectable effect on cAMP when cellsare maintained in 10% SHBG-FREE/DCC-FCS medium, though it canact via the cAMP system in MCF-7 cells maintained in 10% FCSmedium, suggesting that it is the action of FCS that modulates E2activity. In our study, we used serum-free DMEM supplementedwith ITS, which is estrogen-free and SHBG-free. The lack of aneffect on cAMP/PKA in our culture conditions further confirmedour hypothesis that the action of 3,4-diOH-PCB3 is not mediated bySHBG.

Our most interesting finding is that direct inhibition of ERK1/2

underlies the antiproliferative action of 3,4-diOH-PCB3 in the MCF-7 cell line. It must also be pointed out that 4-OH-PCB3 reducedERK1/2 level although not significantly. Thus it seems that 4-OH-PCB3 has a similar activity as 3,4-diOH-PCB3, only so much weaker,

A. Ptak et al. / Toxicology Lette

Fig. 6. Proposed mechanism of antiproliferative action of 3,4-di-OH-PCB3 in MCF-7cells concerning absence of the activation of SHBG/cAMP/PKA pathway, and involve-ment of ERK1/2 on MCF-7 proliferation. (Scheme of signaling pathways is takenfaAs

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rom Zivadinovic et al., 2005 represents proposed signaling pathways of estradiolction.) The down arrow indicates a ERK1/2 decrease and proliferations decrease.C, adenylyl cyclase; Ras-Raf, mitogen-activated protein kinase; ERK, extracellularignal-regulated kinase.

hat we can hypothesis that it could be probably seen as syner-ism with other inhibitors. We showed previously, that PCB3 andoth of its hydroxylated metabolites had no effect on caspase-8 andaspase-9 activity when cells were grown in medium deprived ofstrogen, but reduced caspase-9 activity when cells were grownn medium supplemented with serum containing estradiol. Inter-stingly, a decrease of DNA fragmentation was observed uponreatment with 3,4-diOH-PCB3 in both culture conditions, suggest-ng that 3,4-diOH-PCB3 affects a caspase-independent pathwayf cell death (Ptak et al., 2009). There are reports that xenoe-trogens can cause oscillating activation of ERKs. Bulayeva and

atson (2004) tested a phytoestrogen (coumestrol), organochlori-ated pesticides and their metabolites (endosulfan, dieldrin, DDE)nd detergents (p-nonylphenol, bisphenol A). These xenoestrogensexcept bisphenol A) produced rapid (3–30 min after application),oncentration-dependent ERK1/2 phosphorylation. Also, Radicet al. (2008) demonstrated that the mitogenic effect of PCB153as ERK1/2-mediated. Moreover, they showed that inhibition of

RK1/2 with PD98059 completely blocks the mitogenic effect ofCBs. To better understand the inhibition of ERK1/2 by 3,4-diOH-CB3, we also used a selective inhibitor of ERK1/2 (PD98059) thatrevents phosphorylation and therefore activation of MAPK andRK1/2 without acting directly on MAPK (Alessi et al., 1995). Ourndings demonstrated that the inhibition of ERK1/2 by PD98059ecreases cell proliferation, and 3,4-diOH-PCB3 magnifies thisffect.

In conclusion, the data presented here clearly show that (1)he antiproliferative action of 3,4-diOH-PCB3 is not due to inhi-ition of the ERK1/2 pathway by the SHBG/AMP/PKA pathwayZivadinovic et al., 2005), but rather is due to direct inhibitoryction on the ERK1/2 system (Fig. 6). However, it is necessary tooint that in the presented data we excluded that is the SHBG-eceptor, but that many other plasma membrane receptors seem toe involved in the non-genomic action of xenoestrogens suggestedy Thomas and Dong (2006), (2) indicate that 3,4-diOH-PCB3 are

nti-carcinogens with respect to the mammary gland as observedn rat experiments by Ramamoorthy et al. (1999) for 3,3′,4,4′-etrachlorobiphenyl. However, the mechanism of antiproliferativection of this two PCBs congeners is different. A time-series with.0 ng/ml PCB3 or metabolites showed estrogenic action at each test

rs 190 (2009) 187–192 191

interval (24, 48, 72, and 96 h) and established a rank order potencyof 3,4-OH-PCB > 4-OH-PCB3 ≥ PCB3 (Ptak et al., 2005). Moreover ithas been showed by us that PCB3 acts as an agonist of ER� but notthe Ah receptor in the ovarian follicles (Ptak et al., 2008). 3,3′,4,4′-etrachlorocb used by Ramamoorthy et al. (1999) does not exhibited

ER agonist activity and exhibit a broad spectrum of antiestrogenicresponses consistent with ligand-mediated AhR-ER crosstalk.

Conflict of interest

The authors declare that they have no proprietary, financial orother personal interest of any nature or kinds in any product, serviceand/or company.

Acknowledgements

This work was supported by the Polish Committee for ScientificResearch from 2006 to 2009 as a project 1929/PO1/06/31. AgnieszkaRak is the scholar of Foundation for Polish Science. The authorsthank Hans-Joachim Lehmler and Larry W. Robertson (Departmentof Occupational and Environmental Health, University of Iowa, IowaCity, USA) for the kind gift of PCBs.

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