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Toxicology Letters

journal homepage: www.elsevier.com/locate/toxlet

Bisphenol A and estradiol impede myoblast differentiation through down-regulating Akt signaling pathway

Ga-Yeon Goa,1, Sang-Jin Leea,1, Ayoung Joa, Jae-Rin Leeb, Jong-Sun Kangb, Mihi Yanga,⁎,Gyu-Un Baea,⁎

a Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women’s University, Seoul 04310, Republic of KoreabDepartment of Molecular Cell Biology, Single Cell Network Research Center, Sungkyunkwan University School of Medicine, Suwon 16419, Republic of Korea

G R A P H I C A L A B S T R A C T

A R T I C L E I N F O

Keywords:Bisphenol AEstradiolMyoblast differentiationAkt signaling

A B S T R A C T

Bisphenol A (BPA), one of the most widespread endocrine disrupting chemicals, is known as an artificial es-trogen, which interacts with estrogen receptor (ER). In this study, we investigated the effects of BPA and es-tradiol on myoblast differentiation and the underlying signaling mechanism. Exposure to BPA (0.01–1 μM) inmouse myoblast C2C12 cells attenuated myogenic differentiation via the reduced expression of muscle-specificgenes, such as myosin heavy chain (MHC), MyoD, and Myogenin, without the alteration of cell proliferation andviability. BPA-exposed C2C12 myoblasts also showed a reduction of Akt phosphorylation ((37–61) %,p < 0.001), a key event for myogenesis. Similarly to BPA, estradiol (0.01–1 μM) reduced the expression ofmuscle-specific proteins and the formation of multinucleated myotubes, and attenuated the muscle differ-entiation-specific phosphorylation of Akt ((42–59) %, p < 0.001). We conclude that BPA and estradiol suppressmyogenic differentiation through the inhibition of Akt signaling.

https://doi.org/10.1016/j.toxlet.2018.04.019Received 18 December 2017; Received in revised form 12 April 2018; Accepted 16 April 2018

⁎ Corresponding authors at: Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women’s University, Cheongpa−ro 47−gil 100, Yongsan−Gu, Seoul 04310,Republic of Korea.

1 These authors contributed equally to this work.E-mail addresses: myang@sookmyung.ac.kr (M. Yang), gbae@sookmyung.ac.kr (G.-U. Bae).

Toxicology Letters 292 (2018) 12–19

Available online 20 April 20180378-4274/ © 2018 Elsevier B.V. All rights reserved.

T

1. Introduction

Estrogenic endocrine disrupting chemicals (EDCs) are a structurallydiverse group of compounds that either mimic or antagonize the effectof endogenous estrogens (Tyler et al., 1998). The biological activities ofestrogens are mediated by two isoforms of estrogen receptors (ERs),namely ERα and ERβ, which are members of the nuclear receptor su-perfamily of ligand-mediated transcriptional factors (Heldring et al.,2007). Recently, ERα and ERβ have been identified in myoblasts andskeletal muscles (Milanesi et al., 2008; Wiik et al., 2009). Therefore,estrogens seem to act on skeletal muscle through ER isoforms (Ogawaet al., 2011).

As one of the most commonly produced synthetic chemicalsworldwide, bisphenol A [BPA, 4,4′-(propane-2,2-diyl) diphenol], whichis known as an endocrine disruptor, has been extensively used inpolycarbonate plastics and epoxy resins that are found in a wide rangeof consumer products, such as food containers, food cans, water bottles,

baby bottles, dental sealants, and water pipes. However, it is releasedafter exposure to elevated temperature (Swedenborg et al., 2009; Yanet al., 2011). BPA mimics the natural hormone estrogen and binds toestrogen receptors (ERs), which causes negative effects on health (Leeet al., 2018; Li et al., 2015; Rubin et al., 2001; Swedenborg et al., 2009;Yang et al., 2009). For example, BPA showed a wide-range of physio-logical toxicities, including anti-thyroid hormone effects or non-clas-sical targeting, such as bones, cardiovascular tissue, pancreas, adiposetissue, and the immune system (Richter et al., 2007; Rubin et al., 2001;Vandenberg et al., 2010). In particular, a recent study showed BPAinduces cardiac fibrosis by activating the ERK1/2 pathway (Hu et al.,2016). Although these BPA toxicities are estimated via the activation ofintracellular signaling pathways associated with ERs (Babiker et al.,2002), the toxic mechanisms of BPA have so far remained unclear,particularly in skeletal muscle.

Differentiation of skeletal myoblasts is a tightly orchestrated processthat involves myoblast proliferation, cell cycle withdrawal, expression

Fig. 1. BPA inhibits myogenic differentiation. (A) C2C12 myoblasts were treated with the indicated concentration of BPA, and induced to differentiate in DM for2 days, followed by immunostaining for MHC expression (red) and DAPI to visualize nuclei (blue) to reveal myotube formation. Scale bar, 100 μm. (B) Quantificationof myotube formation from data shown in panel (A). Values represent the means of triplicate determinations ± 1 SD. The experiment was repeated three times, withsimilar results. Asterisks indicate significant difference from the control. *P < 0.01. (C) Cell lysates from similar experiments shown in panel (A) were subjected toimmunoblotting with antibodies to MHC, MyoD, Myogenin, and pan-Cadherin as a loading control. The experiment was repeated three times, with similar results.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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of muscle-specific genes, and fusion into multinucleated myofibers(Horsley and Pavlath, 2004; Krauss, 2010). The maintenance of musclemass is important for staving off the risk of metabolic syndrome andage-related muscle loss, which affects the quality of life (Sanchez et al.,2013). Myoblast differentiation is also under the control of severalsignaling pathways, which include Akt and p38MAPK. These signalingpathways play critical roles in early differentiation induction events,such as activation of the MyoD transcription factor through enhancingthe heterodimerization of MyoD with its partner E proteins, activationof Mef2 by phosphorylation, changes in chromatin-remodeling atmuscle-specific genes, and cell survival (Bae et al., 2009, 2010; Serraet al., 2007; Simone et al., 2004).

In the present study, we examined the effects of BPA and estradiol(E2) on the myogenic differentiation of C2C12 myoblasts, and foundthat BPA and estradiol attenuate myoblast differentiation, accompaniedby down-regulation of Akt activation.

2. Materials and methods

2.1. Chemical reagents

BPA was purchased from BD Biosciences (San Jose, CA), and es-tradiol was from Abcam (Cambridge, UK). Fetal bovine serum (FBS),horse serum F-10 medium, and Dulbecco modified Eagle’s medium(DMEM) were purchased from Thermo Scientific (Waltham, MA). Theantibodies used in this study were as follows: MHC (MF-20;

Developmental studies Hybridoma Bank, Iowa, IA), phospho-Akt (CellSignaling Technology, Beverly, MA), Akt, MyoD, Myogenin, and u-tu-bulin from Santa Cruz Biotechnology (Santa Cruz, CA), and pan-Cadherin from Sigma-Aldrich (St. Louis, MO). Completed protease in-hibitor mixture was from GenDepot (Katy, TX). All other chemicalswere obtained from Sigma-Aldrich.

2.2. Cell culture

Primary myoblasts and myoblast C2C12 cells were cultured as pre-viously described (Bae et al., 2010; Kang et al., 2004). To induce thedifferentiation of C2C12 myoblasts, cells at near confluence were switchedfrom DMEM containing 15% FBS (growth medium, GM) to DMEM con-taining 2% horse serum (differentiation medium, DM), and myotube for-mation was observed normally at approximately 2–3 days of differentia-tion. The efficiency of the myotube formation was quantified by atransient differentiation assay as previously described (Lee et al., 2016).For primary myoblast culture, male C57BL/6 mice were purchased fromKOATECH (Pyeongtaek, Korea). All animal experiments were approved bySookmyung Women's University Institutional Animal Care and Use Com-mittee (SMU-IACUC). Primary myoblasts were isolated from the hindlimbs of one-month old C57BL/6 mice, and cultured as previously de-scribed (Kang et al., 2004). Briefly, cells were grown in F-10 mediumcontaining 20% FBS and basic fibroblast growth factor (bFGF: 2.5 ng/mL,Invitrogen, Carlsbad, CA) with daily medium change. To induce differ-entiation, cells were plated with a high cell density, and switched to DM.

Fig. 2. BPA has no direct effect on cell proliferation and growth in C2C12 myoblasts. (A) Cell viability was performed by MTT assay. Data are presented as themean ± standard deviation (SD) of three determinations. The experiment was repeated three independent times, with similar results. **, P < 0.05 ; *, P < 0.01 (B)C2C12 myoblasts were treated with DMSO or 1 μM BPA for 1 day, and were labeled with BrdU for 30min, followed by immunostaining with anti-BrdU and DAPIstaining to visualize nuclei. Scale bar, 100 μm. (C) Quantification of the BrdU-positive cells shown in panel (B). Data are presented as the mean ± SD of threedeterminations. The experiment was repeated three independent times, with similar results. N.S., not significant. (D) Quantification of apoptotic cells by FACSanalysis.

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2.3. Western blot analyses

Western blot analyses were performed as previously described (Baeet al., 2010). Briefly, cells were lysed in extraction buffer (10mM Tris,pH 7.2, 150mM NaCl, 1 mM EGTA, 1% Triton X-100) containingcompleted protease inhibitor mixture. Cell lysates were boiled inLaemni sample buffer for 5min, and then 40 ug proteins were subjectedto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The primary antibodies used were anti-MHC, anti-p-AKT, anti-AKT, anti-MyoD, anti-Myogenin, anti-α-tubulin, and anti-pan-Cadherin.

2.4. Immunocytochemistry and confocal microscopy

Immunostaining for MHC expression was performed as previouslydescribed (Bae et al., 2010; Lee et al., 2016). Briefly, C2C12 myoblastswere fixed with 4% paraformaldehyde, permeabilized with 0.5% TritonX-100 in phosphate-buffered saline (PBS), blocked, and stained withanti-MHC, followed by an Alexa Fluor 594-conjugated secondary anti-body (Molecular Probes, Eugene, OR). Images were captured by Nikon

ECLIPSE TE-2000U microscopy and processed with NIS-Elements Fsoftware (Nikon, Tokyo, Japan). In detail, the extent of myotube for-mation was quantified and MHC-positive cells was scored as mono-nucleate, containing two to five nuclei, or containing six or more nuclei.Generally 10 different fields and at least 200 cells/field were quantifiedand experiments were repeated at least three times with similar results.Quantification of MHC positive cells is plotted as percentile.

To observe the effects of BPA and estradiol on Akt phosphorylation inC2C12 myoblasts, these cells were treated with 1 μM of BPA or estradiolfor 2 days, and were fixed with 4% paraformaldehyde. Cultures weresubsequently permeabilized with 0.5% Triton X-100 in PBS, blocked in 5%horse serum in 0.1% Triton X-100 in PBS, and incubated with anti-p-Akt,followed by incubation with Alexa Fluor 594-conjugated secondary anti-body. Nuclei were counterstained with DAPI. We scored the intensity ofthe immunofluorescent p-Akt signal in DMSO-treated cells versus BPA-treated cells with the average p-Akt signal in DMSO-treated cells set to 1.0.Images were obtained by Zeiss LSM-510 Meta confocal microscopy (CarlZeiss AG, Oberkochen, Germany) and the p-Akt fluorescent signal wasquantified with Image Gauge software version 4.0.

Fig. 3. BPA attenuates the activation of Akt in myoblast differentiation. (A) C2C12 myoblasts were treated with BPA, and induced to differentiate in DM for 2 days.Cell lysates were subjected to immunoblotting with antibodies to p-Akt and Akt. The experiment was repeated three times, with similar results. (B) Quantification ofblots from three experiments similarly performed to that shown in panel (A). The relative signal intensity of p-Akt proteins to total Akt proteins was determined. Thevalues from DMSO-treated control cells were set to 1.0. Values represent the means of triplicate determinations ± 1 SD. Significant difference from control,*P < 0.01. (C) C2C12 myoblasts were treated with 1 μM BPA in DM for (0–3) day of differentiation. Cell lysates were subjected to immunoblotting with antibodies toMHC, MyoD, p-Akt, and Akt. The experiment was repeated three times, with similar results. (D) BPA-treated C2C12 myoblasts were induced to differentiate for 2 day,fixed and immunostained with antibodies against p-Akt (Green), followed by DAPI staining to visualize nuclei (blue). Scale bar, 10 μm. (E) Quantification of therelative signal intensity of p-Akt-positive cells shown in panel (D). Values are determinants of 10 fields, and experiments were repeated three times, with similarresults. Significant difference from control, *P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version ofthis article.)

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2.5. Bromodeoxyuridine (BrdU) incorporation assay

BrdU incorporation assay was performed as previously described(Bae et al., 2009). Briefly, C2C12 myoblasts were cultured in 6-wellplates, and treated with control DMSO or 1 μM of BPA in GM at 37 °C.After 24 h or the treatment, 10 μM of BrdU was added to the controlDMSO- or BPA-treated C2C12 cells, and they were incubated for 30minat 37 °C. The cells were fixed and immunostained with anti-BrdU an-tibody (Santa Cruz Biotechnology) overnight at 4 °C, followed by in-cubation for 2 h at room temperature with fluorescein isothiocyanate-conjugated secondary antibody (Invitrogen). Images were captured byNikon ECLIPSE TE-2000U microscopy, and processed with NIS-Ele-ments F software.

2.6. Annexin V-FITC/PI assay

Annexin V/PI (propidium iodide) double staining was carried outwith the Annexin V-FITC apoptosis detection kit (BD Biosciences), ac-cording to the manufacturer’s protocol, and samples were analyzed byFACSCalibur flow cytometry (BD Biosciences) and Cell Quest™ ProSoftware (BD Biosciences).

2.7. Statistics

All of experiments were performed in triplicate. After normalitytests (the Shapiro-Wilk test), we analyzed differences between twogroups with Student’s t-test. The P value<0.05 was considered

Fig. 4. Estradiol has antagonistic effect on myogenic differentiation. (A) C2C12 myoblasts were treated with the indicated concentration of estradiol, and induced todifferentiate in DM for 2 days, followed by immunostaining for MHC expression (red) and DAPI to visualize nuclei (blue) to reveal myotube formation. Scale bar,100 μm. (B) Quantification of myotube formation from data shown in panel (A). Values represent means of triplicate determinations ± 1 SD. The experiment wasrepeated three times, with similar results. Asterisks indicate significant difference from the control. *P < 0.01. (C) Cell lysates from similar experiments shown inpanel (A) were subjected to immunoblotting with antibodies to MHC, MyoD, Myogenin, and pan-Cadherin as a loading control. The experiment was repeated threetimes, with similar results. (D) Cell lysates from similar experiments shown in panel (A) were subjected to immunoblotting with antibodies to p-Akt and Akt. Theexperiment was repeated three times, with similar results. (E) Quantification of three blots from similar experiments to that shown in panel (D). The signal intensityof p-Akt was quantified, and the relative values were normalized to Akt. The values of control sample were set to 1.0. Values represent the means of triplicatedeterminations ± 1 SD. Significant difference from control, *P < 0.01. (F) C2C12 myoblasts were treated with 1 μM estradiol in DM for (0 to 3) day of differ-entiation. Cell lysates were subjected to immunoblotting with antibodies to MHC, MyoD, p-Akt, and Akt. The experiment was repeated three times, with similarresults. (G) Estradiol-treated C2C12 myoblasts were induced to differentiate for 2 day, fixed and immunostained with antibodies against p-Akt (Green), followed byDAPI staining to visualize nuclei (blue). Scale bar, 10 μm. (H) Quantification of the relative signal intensity of the p-Akt-positive cells shown in panel (G). Values aredeterminants of 10 fields, and experiments were repeated three times, with similar results. Significant difference from control, *P < 0.01. (For interpretation of thereferences to colour in this figure legend, the reader is referred to the web version of this article.)

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significant. All statistical analyses were performed with JMP v.4.02(SAS Institute, Cary, NC).

3. Results

3.1. BPA suppresses myogenic differentiation

To examine the effect of BPA on myoblast differentiation, C2C12myoblasts were induced to differentiate for 2 day in the presence ofvehicle DMSO or the indicated concentration of BPA (0.01, 0.1, 0.5 and1 μM), followed by MHC immunostaining. We found C2C12 myoblaststreated with BPA exhibited impaired differentiation, with smallermyotubes formation and fewer nuclei, in a dose-dependent manner,compared to the DMSO-treated control cells (Fig. 1A). As a result, BPAreduced the proportion of myotubes containing six or more nuclei,while it substantially elevated the proportion of mononucleate myo-cytes in a dose-dependent manner (Fig. 1B). In particular, BPA-exposedC2C12 cells exhibited a drastically increased proportion of mono-nucleate cells from 44 to 76%, and a substantial reduction of largermyotubes containing six or more nuclei from 16 to 6% at 1 μM of BPA.In agreement with immunostaining data, BPA-treated C2C12 myoblastswere starkly reduced in the expression of muscle-specific protein in-cluding MHC, MyoD, and Myogenin, in a dose-dependent manner

(Fig. 1C). These results suggest that BPA inhibits C2C12 myoblast dif-ferentiation at a morphological as well as biochemical level.

3.2. BPA is not related to proliferation and cell death in C2C12 cells

We studied whether the inhibitory effect of BPA on myoblast dif-ferentiation was due to enhanced cell proliferation or cytotoxicity. Forthis purpose, C2C12 cells were treated with the indicated concentrationof BPA in growth medium or serum-free medium, and incubated for24 h. Low concentration of BPA (< 50 μΜ) had no cytotoxic effects,while cell viability was dramatically decreased at a high dose of BPA(≥50 μM), when cells were cultured in serum-free medium (Fig. 2A). Incontrast, no cytotoxicity was observed in growth medium culturestreated with 0.1–50 μM of BPA, while cell viability was gradually de-creased from 5 μM of BPA. Thus, the fact that a low concentration ofBPA inhibits myoblast differentiation may not be due to cytotoxicity.

To determine the effect of BPA on myoblast proliferation, C2C12cells were subjected to a BrdU incorporation assay. We performed ex-periments to find proper concentration and chose the highest con-centration (1 μM of BPA and E2) to show no effect on cell death orproliferation and a more specific effect on myoblast differentiation.Treatment with 1 μM of BPA did not overtly alter the proliferative ca-pacity of C2C12 myoblasts, compared to DMSO-treated cells (Fig. 2B

Fig. 5. BPA and estradiol induce the impaired myoblast differentiation. (A) Primary myoblasts were treated with 1 μM BPA or estradiol, and induced to differentiatein DM for 4 day, followed by immunostaining for MHC expression (red) and DAPI to visualize nuclei (blue) to reveal myotube formation. Scale bar, 100 μm. (B)Quantification of myotube formation from data shown in panel (A). Values represent means of triplicate determinations ± 1 SD. The experiment was repeated threetimes, with similar results. Asterisks indicate significant difference from the control. *P < 0.01. (For interpretation of the references to colour in this figure legend,the reader is referred to the web version of this article.)

Fig. 6. Potential toxic mechanisms of BPA on differ-entiating myoblasts. The representative promyogenickinases, p38MAPK and Akt play critical roles in myo-blast differentiation. Activated p38MAPK phosphor-ylates E proteins, leading to enhancement of hetero-dimerization with myogenic basic helix-loop-helix(bHLH) factors during myogenic differentiation. Theseheterodimers can induce gene expression required formyogenic differentiation. Akt signaling up-regulatesheterodimerization with MyoD and E proteins and al-ters in chromatin remodeling complex to myogenicloci. Both p38MAPK and Akt signalings are requiredfor activating and reinforcing each other’s activity tolead to efficient myogenesis. BPA impedes myoblastdifferentiation resulting in inhibiting Akt signaling.

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and C). Furthermore, the treatment with 1 μM of BPA did not affect cellsurvival, as assessed by Annexin V/PI staining using FACS analysis(Fig. 2D). These results suggest that BPA treatment with a concentrationbelow 1 μM did not elicit any discernable effect on cytotoxicity, cellproliferation, or death of C2C12 myoblasts. Therefore, the inhibitoryeffects of BPA on myoblast differentiation are likely due to the sup-pression of promyogenic pathways.

3.3. BPA inhibits Akt phosphorylation in myoblast differentiation

As Akt, a promyogenic kinase, has been known to play an importantrole in enhancing myoblast differentiation (Bae et al., 2010; Serra et al.,2007), we studied the regulatory pathways, including the phosphor-ylation of Akt for BPA-inhibited myoblast differentiation. First, C2C12myoblasts were exposed to various concentrations of BPA, and assessedfor the activation status of Akt by immunoblotting analysis with anantibody to the active phosphorylated form of Akt (p-Akt). Fig. 3A andB shows that BPA-exposure decreased the phosphorylation levels of Aktin a dose-dependent manner, relative to control. However, the total Aktlevels stayed relatively constant. For detailed analyses of the expressionof muscle-specific proteins and Akt phosphorylation during differ-entiation, C2C12 myoblasts were exposed to a final concentration of1 μM of BPA, and at 2 days of differentiation exhibited dramaticallysuppressed expression of muscle-specific proteins, such as MHC, MyoD,and p-Akt, relative to DMSO-treated C2C12 cells (Fig. 3C). To confirmwhether BPA treatment suppressed Akt phosphorylation, C2C12 myo-blasts were induced to differentiate for 2 days. Fig. 3D shows that BPA-exposed C2C12 myoblasts revealed a decreased number of cells positivefor p-Akt signals, compared with DMSO-treated cells. As a result, BPA-exposed C2C12 myoblasts displayed a 0.38-fold decrease in the p-Aktsignal intensity (Fig. 3E). These data suggest that the BPA treatmentattenuates Akt phosphorylation and down-regulates the expression le-vels of muscle-specific proteins, leading to the inhibition of myogenicdifferentiation.

3.4. Estradiol blocks myogenic differentiation

To investigate whether estradiol could suppress myogenic differ-entiation, C2C12 myoblasts were induced to differentiate for 2 days inthe presence of vehicle DMSO or indicated concentration of estradiol(0.01, 0.1, 0.5 and 1 μM), followed by MHC immunostaining. In similarmanner to the negative effect of BPA, estradiol treatment impairedmyoblast differentiation with smaller myotube formation in a dose-dependent manner, whereas control, i.e. DMSO-treated C2C12 myo-blasts formed bigger MHC-positive myotubes with more nuclei(Fig. 4A). The second negative control without vehicle (DMSO) was notdifferent with the first negative control with DMSO in the differentia-tion. For the quantification of MHC-positive myotube formation, theextent of myotube formation was quantified and MHC-positive cellswere scored as mononucleate, containing two to five nuclei, or con-taining six or more nuclei. Generally 10 different fields and at least 200cells/field were quantified and experiments were repeated at least threetimes with similar results. Quantification of MHC positive cells isplotted as percentile. Estradiol treatment reduced the proportion ofmyotubes containing six or more nuclei, while it substantially elevatedthe proportion of mononucleate myocytes in a dose-dependent manner(Fig. 4B). In detail, C2C12 cells exhibited dramatically increased pro-portion of mononucleate cells from 42 to 85%, and a substantial re-duction of myotubes containing six or more nuclei from 22 to 2% at1 μM estradiol. In agreement with MHC staining, estradiol-treatedC2C12 myoblasts displayed a reduced expression of MHC, MyoD, andMyogenin (Fig. 4C). We further investigated Akt activation in the es-tradiol-exposed cells. As a result, estradiol gradually down-regulatedAkt phosphorylation, compared to control (Fig. 4D and E). In addition,estradiol treated C2C12 myoblasts displayed dramatically reduced ex-pression of MHC and MyoD, and abrogated Akt phosphorylation,

relative to DMSO-treated C2C12 cells (Fig. 4F) in a day-dependentmanner. In addition, treatment with low concentrations of estradiol (1to 10 nM) also impeded the formation of MHC-positive multinucleatedmyotube and reduced the expression of myogenic-specific proteins andthe phosphorylation of Akt (Supplementary Fig. 1A and B).

Moreover, estradiol-exposed C2C12 myoblasts had fewer number ofcells positive for p-Akt signals, compared with DMSO-treated cells(Fig. 4G). To quantify these results, we scored the intensity of the im-munofluorescent p-Akt signal in DMSO-treated cells versus estradiol-treated cells, with the average p-Akt signal in DMSO-treated cells set to1.0. As a result, estradiol-exposed C2C12 myoblasts exhibited 0.32-folddecreased relative p-Akt signal intensity (Fig. 4H). These data suggestthat the estradiol treatment attenuated the induction of muscle-specificgene expression through inhibiting Akt phosphorylation. Taken to-gether, these data demonstrate the negative effect of BPA and estradiolon muscle differentiation.

3.5. BPA and estradiol attenuate the primary myoblast differentiation

To examine whether BPA and estradiol affect the myogenic differ-entiation in primary myoblasts as well as C2C12 myoblasts, we haveisolated primary myoblasts from hind limbs of one-month mice, andinduced differentiation for 4 days in DM, followed by immunostainingfor MHC expression. Consistent with the results of BPA and estradiol,BPA- or estradiol-treated primary myoblasts formed more single nucleicontaining myocytes, and less MHC expression than control myoblasts(Fig. 5A). Exposure to BPA or estradiol dramatically reduced the pro-portion of myotubes containing six or more nuclei, while it elevated theproportion of mononucleate myocytes (Fig. 5B). These data suggest thatBPA and estradiol result in impaired primary myoblast differentiation(Fig. 6).

4. Discussion

Endogenous estrogens are well recognized as playing importantroles in the regulation and maintenance of sex-specific myogenic dif-ferentiation. However, BPA or its metabolites are well known obeso-gens that induce adipogenesis (Boucher et al., 2015), and little is knownabout their myogenic effects, and their interactions with endogenousestrogens. In addition, skeletal muscle is expected to be a target tissuefor estrogens, because two ER isoforms are expressed in skeletal muscle(Wiik et al., 2009). BPA has been reported to bind to hormone re-ceptors, and influence multiple endocrine pathways (Deutschmannet al., 2013; Matsushima et al., 2008; Riu et al., 2011). In particular,BPA reduces Akt phosphorylation in skeletal muscle, and alters serumadipocytokine levels, thereby inducing glucose intolerance (Moon et al.,2015). In this study, we investigated the effects and molecular me-chanism of BPA on myoblast differentiation, and found BPA has noeffects on the proliferation or apoptosis of C2C12 myoblasts; however,it influences the capacity for myoblast differentiation at low con-centration (less than 1 μM) (Fig. 1A). In addition, we found that BPAand estradiol inhibit myoblast differentiation with reduced muscle geneexpression and myotube formation (Figs. 1A, 4A and 5A). Like the ef-fect of BPA on glucose intolerance (Boucher et al., 2015), in the presentstudy BPA exposure reduced Akt phosphorylation, which plays a criticalrole in myoblast differentiation. As there were no overt signs that BPA-induced cell death or cytotoxicity was associated with differentiation,BPA-inhibitory effect on Akt activation is specific to the differentiationprogram. The binding of estrogen (17ß-estradiol) to the ligand-bindingdomain of ERα exerts genomic effects on DNA sequences called es-trogen response elements (EREs), which regulate the transcription oftarget genes. However, 17ß-estradiol decreases the levels of myogenicregulatory factors (MRFs) during skeletal muscle formation, and acts torepress myogenesis in mouse C2C12 myoblast and mouse satellite cells(Ogawa et al., 2011). Thus, it is conceivable that BPA might also sup-press MRFs, which can be observed in higher dose of BPA (Fig. 1C).

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Efficient myoblast differentiation requires not only Akt but also p38MAPK activation, and it appears that regulates cell cycle control, MyoDdimerization with E proteins, and Mef2 transcriptional activity inmyoblast differentiation (Bae et al., 2009; Krauss, 2010). However, theexposure of BPA in myoblasts did not affect p38MAPK activities onmyogenesis (data not shown). Previous studies have shown that APPL1/Akt might form different pools of signaling complexes of JLP/p38MAPKin myoblast differentiation (Bae et al., 2010). The suggestion that BPAinhibits mechanisms for the activation of Akt in differentiating myo-blasts might include signaling by APPL1, an interacting partner of Akt(graphic highlight).

To compare the toxicity of BPA to that of E2, we used somewhathigh concentration, 1 μM. As human daily exposure to BPA is approx.1/228-1/45 μM (Yang et al., 2014), we can inversely apply uncertaintyfactor, 100, i.e., 1/2-2.2 μM to mice. for For real exposure issues, wetried to match animal exposure levels, 1 μM of BPA and E2, to humanreal exposure levels

5. Conclusion

Our results suggest that both BPA and estradiol block myoblastdifferentiation, through suppression of Akt signaling and muscle-spe-cific gene expression. The inhibitory effect of BPA on myoblast differ-entiation might contribute to worsening metabolic health, in additionto its adipogenic effects.

Conflict of interest

The author declare no conflict of interest.

Acknowledgements

This work was supported by National Research Foundation of Korea(NRF) grants funded by the Korea government (MSIP) (NRF-2017R1D1A1B03032839) to Sang-Jin Lee, (NRF-2016R1A2B4014868)to Gyu-un Bae, and (NRF-2011-0030074) to Mihi Yang and Gyu-un Bae.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in theonline version, at https://doi.org/10.1016/j.toxlet.2018.04.019.

References

Babiker, F.A., De Windt, L.J., van Eickels, M., Grohe, C., Meyer, R., Doevendans, P.A.,2002. Estrogenic hormone action in the heart: regulatory network and function.Cardiovasc. Res. 53, 709–719.

Bae, G.U., Kim, B.G., Lee, H.J., Oh, J.E., Lee, S.J., Zhang, W., Krauss, R.S., Kang, J.S.,2009. Cdo binds Abl to promote p38alpha/beta mitogen-activated protein kinaseactivity and myogenic differentiation. Mol. Cell. Biol. 29, 4130–4143.

Bae, G.U., Lee, J.R., Kim, B.G., Han, J.W., Leem, Y.E., Lee, H.J., Ho, S.M., Hahn, M.J.,Kang, J.S., 2010. Cdo interacts with APPL1 and activates Akt in myoblast differ-entiation. Mol. Biol. Cell 21, 2399–2411.

Boucher, J.G., Boudreau, A., Ahmed, S., Atlas, E., 2015. In vitro effects of bisphenol Abeta-D-glucuronide (BPA-G) on adipogenesis in human and murine preadipocytes.Environ. Health Perspect. 123, 1287–1293.

Deutschmann, A., Hans, M., Meyer, R., Haberlein, H., Swandulla, D., 2013. Bisphenol Ainhibits voltage-activated Ca(2+) channels in vitro: mechanisms and structural re-quirements. Mol. Pharmacol. 83, 501–511.

Heldring, N., Pike, A., Andersson, S., Matthews, J., Cheng, G., Hartman, J., Tujague, M.,Strom, A., Treuter, E., Warner, M., Gustafsson, J.A., 2007. Estrogen receptors: how dothey signal and what are their targets. Physiol. Rev. 87, 905–931.

Horsley, V., Pavlath, G.K., 2004. Forming a multinucleated cell: molecules that regulatemyoblast fusion. Cells Tissues Organs 176, 67–78.

Hu, Y., Zhang, L., Wu, X., Hou, L., Li, Z., Ju, J., Li, Q., Qin, W., Li, J., Zhang, Q., Zhou, T.,Zhang, L., Xu, C., Fang, Z., Zhang, Y., 2016. Bisphenol A an environmental estrogen-like toxic chemical, induces cardiac fibrosis by activating the ERK1/2 pathway.Toxicol. Lett. 250–251, 1–9.

Kang, J.S., Yi, M.J., Zhang, W., Feinleib, J.L., Cole, F., Krauss, R.S., 2004. Netrins andneogenin promote myotube formation. J. Cell Biol. 167, 493–504.

Krauss, R.S., 2010. Regulation of promyogenic signal transduction by cell–cell contactand adhesion. Exp. Cell Res. 316, 3042–3049.

Lee, S.J., Hwang, J., Jeong, H.J., Yoo, M., Go, G.Y., Lee, J.R., Leem, Y.E., Park, J.W., Seo,D.W., Kim, Y.K., Hahn, M.J., Han, J.W., Kang, J.S., Bae, G.U., 2016. PKN2 and Cdointeract to activate AKT and promote myoblast differentiation. Cell. Death. Dis. 7,e2431.

Lee, H.S., Kang, Y., Tae, K., Bae, G.U., Park, J.Y., Cho, Y.H., Yang, M., 2018. Proteomicbiomarkers for bisphenol A-early exposure and women's thyroid cancer. Cancer Res.Treat. 50, 111–117.

Li, L., Wang, Q., Zhang, Y., Niu, Y., Yao, X., Liu, H., 2015. The molecular mechanism ofbisphenol A (BPA) as an endocrine disruptor by interacting with nuclear receptors:insights from molecular dynamics (MD) simulations. PLoS One 10, e0120330.

Matsushima, A., Teramoto, T., Okada, H., Liu, X., Tokunaga, T., Kakuta, Y., Shimohigashi,Y., 2008. ERRgamma tethers strongly bisphenol A and 4-alpha-cumylphenol in aninduced-fit manner. Biochem. Biophys. Res. Commun. 373, 408–413.

Milanesi, L., Russo de Boland, A., Boland, R., 2008. Expression and localization of es-trogen receptor alpha in the C2C12 murine skeletal muscle cell line. J. Cell. Biochem.104, 1254–1273.

Moon, M.K., Jeong, I.K., Jung Oh, T., Ahn, H.Y., Kim, H.H., Park, Y.J., Jang, H.C., Park,K.S., 2015. Long-term oral exposure to bisphenol A induces glucose intolerance andinsulin resistance. J. Endocrinol. 226, 35–42.

Ogawa, M., Yamaji, R., Higashimura, Y., Harada, N., Ashida, H., Nakano, Y., Inui, H.,2011. 17beta-estradiol represses myogenic differentiation by increasing ubiquitin-specific peptidase 19 through estrogen receptor alpha. J. Biol. Chem. 286,41455–41465.

Richter, C.A., Birnbaum, L.S., Farabollini, F., Newbold, R.R., Rubin, B.S., Talsness, C.E.,Vandenbergh, J.G., Walser-Kuntz, D.R., vom Saal, F.S., 2007. In vivo effects of bi-sphenol A in laboratory rodent studies. Reprod. Toxicol. 24, 199–224.

Riu, A., Grimaldi, M., le Maire, A., Bey, G., Phillips, K., Boulahtouf, A., Perdu, E., Zalko,D., Bourguet, W., Balaguer, P., 2011. Peroxisome proliferator-activated receptorgamma is a target for halogenated analogs of bisphenol A. Environ. Health Perspect.119, 1227–1232.

Rubin, B.S., Murray, M.K., Damassa, D.A., King, J.C., Soto, A.M., 2001. Perinatal exposureto low doses of bisphenol A affects body weight, patterns of estrous cyclicity, andplasma LH levels. Environ. Health Perspect. 109, 675–680.

Sanchez, A.M., Csibi, A., Raibon, A., Docquier, A., Lagirand-Cantaloube, J., Leibovitch,M.P., Leibovitch, S.A., Bernardi, H., 2013. eIF3f: a central regulator of the antag-onism atrophy/hypertrophy in skeletal muscle. Int. J. Biochem. Cell Biol. 45,2158–2162.

Serra, C., Palacios, D., Mozzetta, C., Forcales, S.V., Morantte, I., Ripani, M., Jones, D.R.,Du, K., Jhala, U.S., Simone, C., Puri, P.L., 2007. Functional interdependence at thechromatin level between the MKK6/p38 and IGF1/PI3K/AKT pathways duringmuscle differentiation. Mol. Cell 28, 200–213.

Simone, C., Forcales, S.V., Hill, D.A., Imbalzano, A.N., Latella, L., Puri, P.L., 2004. p38pathway targets SWI-SNF chromatin-remodeling complex to muscle-specific loci. Nat.Genet. 36, 738–743.

Swedenborg, E., Ruegg, J., Makela, S., Pongratz, I., 2009. Endocrine disruptive chemicals:mechanisms of action and involvement in metabolic disorders. J. Mol. Endocrinol.43, 1–10.

Tyler, C.R., Jobling, S., Sumpter, J.P., 1998. Endocrine disruption in wildlife: a criticalreview of the evidence. Crit. Rev. Toxicol. 28, 319–361.

Vandenberg, L.N., Chahoud, I., Padmanabhan, V., Paumgartten, F.J., Schoenfelder, G.,2010. Biomonitoring studies should be used by regulatory agencies to assess humanexposure levels and safety of bisphenol A. Environ. Health Perspect. 118, 1051–1054.

Wiik, A., Ekman, M., Johansson, O., Jansson, E., Esbjornsson, M., 2009. Expression ofboth oestrogen receptor alpha and beta in human skeletal muscle tissue. Histochem.Cell Biol. 131, 181–189.

Yan, S., Chen, Y., Dong, M., Song, W., Belcher, S.M., Wang, H.S., 2011. Bisphenol A and17beta-estradiol promote arrhythmia in the female heart via alteration of calciumhandling. PLoS One 6, e25455.

Yang, M., Ryu, J.H., Jeon, R., Kang, D., Yoo, K.Y., 2009. Effects of bisphenol A on breastcancer and its risk factors. Arch. Toxicol. 83, 281–285.

Yang, M., Lee, H.S., Hwang, M.W., Jin, M., 2014. Effects of Korean red ginseng (PanaxGinseng Meyer) on bisphenol A exposure and gynecologic complaints: single blind,randomized clinical trial of efficacy and safety. BMC Complement. Altern. Med. 25(14), 265.

G.-Y. Go et al. Toxicology Letters 292 (2018) 12–19

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