Cigarette smoke induces proteasomal-mediated degradation of DNA methyltransferases and methyl...

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Reproductive Toxicology 58 (2015) 140–148

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Reproductive Toxicology

j ourna l ho me pa g e: www.elsev ier .com/ locate / reprotox

igarette smoke induces proteasomal-mediated degradation of DNAethyltransferases and methyl CpG-/CpG domain-binding proteins in

mbryonic orofacial cells

artha Mukhopadhyay, Robert M. Greene ∗, M. Michele Pisanoniversity of Louisville Birth Defects Center, Department of Molecular, Cellular and Craniofacial Biology, ULSD, University of Louisville, Louisville, KY 40202,nited States

r t i c l e i n f o

rticle history:eceived 24 November 2014eceived in revised form 18 August 2015ccepted 12 October 2015

eywords:igarette smokeleft lip/palate

a b s t r a c t

Orofacial clefts, the most prevalent of developmental anomalies, occur with a frequency of 1 in 700 livebirths. Maternal cigarette smoking during pregnancy represents a risk factor for having a child with acleft lip and/or cleft palate. Using primary cultures of first branchial arch-derived cells (1-BA cells), whichcontribute to the formation of the lip and palate, the present study addressed the hypothesis that com-ponents of cigarette smoke alter global DNA methylation, and/or expression of DNA methyltransferases(Dnmts) and various methyl CpG-binding proteins. Primary cultures of 1-BA cells, exposed to 80 �g/mLcigarette smoke extract (CSE) for 24 h, exhibited a >13% decline in global DNA methylation and triggered

ouseranchial archroteasomeNA methylationNA methyltransferasespG-binding proteins

proteasomal-mediated degradation of Dnmts (DNMT-1 and -3a), methyl CpG binding protein 2 (MeCP2)and methyl-CpG binding domain protein 3 (MBD-3). Pretreatment of 1-BA cells with the proteasomalinhibitor MG-132 completely reversed such degradation. Collectively, these data allow the suggestionof a potential epigenetic mechanism underlying maternal cigarette smoke exposure-induced orofacialclefting.

. Introduction

Orofacial clefting (cleft lip with or without cleft palate, CL/P,r cleft palate only, CPO), is currently the most prevalent of con-enital anomalies, with a frequency of 1 in 700 live births [1,2].uring week four of human gestation, the upper lip forms via

usion of the maxillary process (MxP) of the first branchial archith the medial nasal process (MNP) [3]. Between weeks 7 and 10

f gestation, the secondary palate develops from the oral aspectf each maxillary process and eventually gives rise to the rooff the oral cavity [4]. Thus, both the upper lip and the palateevelop from cells derived from the first branchial arch. While

wide range of genetic and environmental factors can adverselyffect formation of the lip or palate [5], maternal cigarette smok-

∗ Corresponding author at: Department of Molecular, Cellular and Craniofa-ial Biology, University of Louisville, ULSD, 501 South Preston Street, Suite 350,ouisville, KY 40202, United States. Fax: +1 502 852 4702.

E-mail address: dr.bob.greene@gmail.com (R.M. Greene).

following prenatal cigarette smoke exposure [14]. This is of notesince certain abnormalities of the mandible, also derived fromthe first branchial arch, can secondarily cause clefts of the palate.Precise genetic and/or epigenetic mechanisms underlying in uterocigarette smoke exposure-induced orofacial clefting remain, how-ever, largely undefined. Recent studies demonstrating prenatalcigarette smoke exposure-induced alterations in DNA methylationin offspring [15–18], suggest that teratogenic effects of cigarettesmoke could be partly mediated by alteration of gene expressionthrough modulation of DNA methylation.

Methylation of mammalian DNA is primarily catalyzed bythree active DNA methyltransferases (DNMTs). Several studieshave highlighted the indispensability of both the “maintenance”methyltransferase, DNMT-1, and the de novo methyltransferases,DNMT-3a and DNMT-3b, during embryogenesis. Targeted disrup-tion of the genes encoding each of these enzymes led to embryoniclethality and dysmorphologies [19–23]. Specific developmentalroles for these enzymes are suggested by the association of inad-equate DNMT-1-mediated maintenance of DNA methylation and

uctive Toxicology 58 (2015) 140–148 141

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Fig. 1. Photomicrograph of a representative GD 10.5 embryo. The region demar-

P. Mukhopadhyay et al. / Reprod

entromeric instability-facial anomalies (ICF) syndrome in humans26,27]. Interestingly, 5-azacytidine, a powerful inhibitor of DNA

ethylation, is capable of inducing cleft palate in developingetuses following in utero exposure [28].

Based on their ability to bind methylated DNA, the methyl-pG-binding domain (MBD) proteins operate as “interpreters” ofhe DNA methylation mark and thus, as crucial modulators ofiverse epigenetic processes [29]. MBD-1, MBD-2, MBD-4, andeCP-2, have been identified as transcriptional repressors that

ecruit Dnmts, histone deacetylases and chromatin remodelerso methylated DNA [30]. In this regard, methylation-dependentenomic targeting by MBD has been shown to regulate expres-ion of 5-hydroxymethylcytosine marked genes [31,32]. While theethality of Mbd3-null embryos suggests a critical role for this geneuring embryogenesis [33,34], the precise mechanisms by whichBD proteins govern normal embryonic development remain to

e defined [35,36].Maternal exposure to heavy metal teratogens, such as, arsenic

nd cadmium, has also been documented to trigger developmen-al dysmorphogenesis via alteration of global DNA methylation and

odulation of expression of DNMTs and MBDs. Significant down-egulation of Dnmt3a and Dnmt3b, significant decline in globalNA methylation and developmental dysmorphogenesis (e.g. ven-

ral body wall defects) were reported in embryos exposed intero to cadmium [37]. Another study documented arsenic-inducedltered expression of DNMTs and MBDs, a significant decrease in-adenosylmethionine (SAM) (the source of the “methyl” group forNA methylation), and global DNA hypomethylation—resulting ineural tube defects in exposed embryos [38].

Prenatal cigarette smoke exposure is thought to target cells ofhe developing midfacial complex to cause orofacial anomalies (e.g.,L/P). Thus, primary cultures of 1-BA (first branchial arch) cellsere utilized to determine the effects of maternal cigarette smoke

xposure on the expression of several DNMTs and MBDs. We showhat exposure to cigarette smoke extract (CSE) led to global DNAypomethylation, and reveal for the first time, significant protea-omal degradation of two DNA methyltransferases (DNMT-1 andNMT-3a), as well as two methyl CpG-binding proteins, MeCP-2nd MBD-3, in 1-BA cells. Moreover, pretreatment of these cellsith the proteasomal inhibitor, MG-132 completely reversed the

SE-induced degradation of Dnmts and Mbds.

. Materials and methods

.1. Establishment of primary cultures of murine first branchialrch-derived (1-BA) cells

ICR mice (Harlan Laboratories, Indianapolis, IN) were housednder climate-controlled conditions at a temperature of 22 ◦C withn alternating 12-h dark–light cycle and were provided access toood and water ad libitum. Mature male and female mice were

ated overnight and the presence of a vaginal plug the follow-ng morning (day 0 of gestation) was considered as evidence of

ating. Pregnant mice were euthanized on day 10.5 of gesta-ion, and embryos removed from pregnant dams. First branchialrches were microdissected (Fig. 1) from the embryos in ster-le, cold phosphate-buffered saline (PBS), minced and dissociated

ith 0.05% trypsin/0.1% EDTA in PBS for 10 min at 37 ◦C with con-tant shaking. Trypsin was inhibited by the addition of Opti-MEMedium (Life Technologies, Grand Island, NY, USA) containing 5%

etal bovine serum (FBS) (Sigma, St. Louis, MO, USA). Cells were

ells/dish in Opti-MEM containing Earle’s salts, 25 mM HEPESuffer and supplemented with 2 mM glutamine, 5% FBS, 150 �g/mLtreptomycin, and 100 U/mL penicillin (complete medium) (Life

cated by the black line was microdissected and utilized to establish primary culturesof first branchial arch-derived (1-BA) cells. BA1 = first branchial arch; BA2 = secondbranchial arch.

Technologies) and maintained at 37 ◦C in an atmosphere of 95%air/5% CO2, with medium replaced every 48 h. Each experimentwas performed using cells derived from first branchial arch tissuesdissected from a single litter of embryos.

2.2. Cigarette smoke extract

Cigarette smoke extract (CSE), purchased from Murty Pharma-ceuticals (Lexington, KY), was prepared by burning 1R3E standardcigarettes and extracting the condensate into dimethyl sulfoxide(DMSO) to generate a 40 mg/mL stock solution.

2.3. Global DNA methylation assay

First branchial arch-derived (1-BA) cells were re-seeded into60 mm tissue culture dishes (Nalge Nunc International, Rochester,NY) at an initial density of 1.5 × 104 cells/dish. Cells were grownto confluence in the growth medium (as described in Section 2.1),washed with phosphate-buffered saline (PBS) and treated witheither vehicle (PBS) or cigarette smoke extract (CSE) at a final con-centration of 80 �g/mL for 24 h. 1-BA cell cultures, similarly grown,were treated with 10 �M 5-azacytidine (Sigma, St. Louis, MO) (aDNA methylation inhibitor used as a positive control) for 24 h. Cells(both treated and control) were incubated for 24 h at 37 ◦C fol-lowed by genomic DNA extraction and assessment of Global DNAmethylation as previously described [39].

2.4. TaqMan® quantitative real-time PCR (qRT-PCR)

Total RNAs from control or CSE-treated 1-BA cells wereextracted using the RNeasy Protect Mini Kit (Qiagen) followingthe manufacturer’s recommendations. The quality and quantityof extracted total RNAs were assessed using the Agilent 2100

1 uctive Toxicology 58 (2015) 140–148

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Table 1Effect of CSE on global DNA methylation in first branchial arch-derived (1-BA) cells.

Sample typea % Global DNA methylationb,c % Change (vs. control)

Control 3.33 ± 0.14 –20 �g/mL CSE 3.35 ± 0.05 –40 �g/mL CSE 3.27 ± 0.61 1.8% decrease80 �g/mL CSE 2.90 ± 0.11* 13.0% decrease10 �M azacytidine 2.65 ± 0.08** 20.4% decrease

* P < 0.01 vs. control.** P < 0.001 vs. control.a 1-BA cells were treated with vehicle (control) or CSE (80 �g/mL), or 10 �M

azacytidine for 24 h as described in Section 2.b Global DNA methylation was quantified using the Methylamp Global DNA

42 P. Mukhopadhyay et al. / Reprod

.5. Preparation of nuclear extracts and Western blotting

First branchial arch-derived (1-BA) cells were exposed to vehi-le (PBS) or cigarette smoke extract (CSE) and maintained for4 h as described under “Global DNA Methylation Assay”. Nuclear-nriched protein extracts were prepared from CSE- or vehiclePBS)-treated 1-BA cells using the NE-PER kit (Pierce, Rockford, IL)ollowing the manufacturer’s recommendations. Steady state levelsf various DNMT and MBD proteins were determined by West-rn (immuno) blotting using commercially available primary andecondary antibodies as previously described [39].

.6. Proteasome inhibitor

First branchial arch-derived (1-BA) cells, were grown to conflu-nce in 60 mm tissue culture dishes (Nalge Nunc International), andre-treated with either vehicle (DMSO) or 0.5-, 1.0- or 1.5-�M ofhe proteasome inhibitor MG-132 (Sigma) for 3 h. The inhibitor washen removed, and fresh medium containing DMSO (vehicle), orSE (various concentrations) was added, and the cells maintained

or an additional 24 h as described under “Global DNA Methyla-ion Assay”. Control experiments, in which cells were treated withnly 1.5-�M MG-132 (for 3 h) were also performed. Subsequently,uclear protein extracts were prepared and examined by westernnalysis as detailed in the previous paragraph.

.7. DNMT-1 ELISA

Nuclear extracts from 24-h vehicle- or CSE-treated (alone orfter 3-h MG-132 pre-treatment) 1-BA cells, were assayed forNMT-1 protein using the Epiquik DNMT-1 assay kit (Epigentek)s previously described [39].

.8. Densitometric analysis of Western blots

Densitometric analyses of DNMT-1, -3a, -3b, MeCP-2, MBD-2,BD-3 and �-actin protein bands were accomplished using Image

(version 1.38) software as previously described [39].

.9. Statistical analyses

Statistical analyses were performed using R version 3.1.2 (http://ww.R-project.org). Effects of CSE on (1) percent global DNAethylation, (2) normalized protein band intensities (relative to

teady state levels of proteins), and (3) normalized Ct values (�Ct)rom qRT-PCR for gene expression were all analyzed using one-wayNOVA models. For the global methylation experiments, controlamples were compared to samples treated with 20-, 40- and0 �g/mL CSE and 10 �M Azacytidine. For experiments #2 (proteinand intensity) and #3 (Ct values) control samples were comparedo samples with increasing CSE exposure (20 �g/mL, 40 �g/mL,nd 80 �g/mL CSE). Means and standard deviations from threendependent experiments, followed by differences or fold-changeselative to control samples were presented with simultaneous 95%onfidence limits. P-values of <0.05 were considered statisticallyignificant.

. Results

.1. Effect of cigarette smoke extract on 1-BA cell global DNAethylation

In a separate study (unpublished observation), we have assessedhe effect of increasing doses of CSE (20-, 40-, 80- and 100 �g/mL)n the survival and proliferation of 1-BA cells in primary culture.esults from those experiments revealed that 24-h exposure to CSE

Methylation Quantification Ultra Kit as described in Section 2.c Data represent mean ± standard deviation, obtained from three independent

experiments.

at doses higher than 80 �g/mL significantly reduces the survival aswell as proliferation of 1-BA cells. Based on these findings we firsttested the impact of the highest, tolerable dose (80 �g/mL) of CSE,on global DNA methylation as well as on the expression of variousDNMTs and methyl CpG-binding proteins, within 1-BA cells. Resultsfrom these experiments demonstrated significant reduction inglobal DNA methylation as well as significant downregulation ofgenes encoding various Dnmts and methyl CpG-binding proteins(see below). Finally, we repeated the aforementioned experimentsassessing the effect of 20- (low dose), 40- (intermediate dose), and80- (high dose) �g/mL of CSE on 1-BA cells (i.e., performed a doseresponse).

Cigarette smoke extract (80 �g/mL) significantly (>13%) dimin-ished global DNA methylation in 1-BA cells (Table 1). Whiletreatment with 20- or 40 �g/mL CSE had no appreciable impacton global DNA methylation (Table 1), exposure to 5-azacytidine – apowerful inhibitor of DNA methylation – effected a >20% reductionin global DNA methylation in 1-BA cells (Table 1).

3.2. Effect of cigarette smoke extract on expression of DNA methyltransferases and methyl CpG/CpG domain binding proteins innuclear extracts of 1-BA cells

Expression of DNMT-1, -3a, -3b, MeCP-2, MBD-2 and MBD-3proteins were assessed by immunoblotting of vehicle- or CSE-exposed 1-BA cell nuclear extracts. Two major bands in themolecular weight range of 200–190 kDa and 140–120 kDa weredetected for DNMT-1 and DNMT-3a, respectively, while a singlemajor ∼110 kDa band was detected for DNMT-3b, on immunoblotsof nuclear extracts of 1-BA cells treated for 24 h with either vehi-cle or 20- or 40 �g/mL CSE (Fig. 2a–c; upper panels). Under similarconditions, a single major 75 kDa band for MECP-2, a 49 kDa bandfor MBD-2, and two 34–30 kDa bands for MBD-3 were detected(Fig. 2d–f; upper panels). Exposure to 80 �g/mL CSE for 24 hresulted in a marked reduction in steady state levels of DNMT-1, DNMT-3A, and MeCP-2 proteins (Fig. 2a,b,d, upper panels; andTable 2). The 20- or 40 �g/mL CSE dose also caused reduction insteady state levels of these three proteins (Fig. 2a,b,d, upper pan-els; and Table 2). This dose-dependent reduction was verified viadensitometric analysis of film-detected chemiluminescent signals(Table 2). While steady state levels of the 34 kDa MBD-3 pro-tein were markedly reduced following exposure to 80 �g/mL CSE(Fig. 2f, upper panel; and Table 2), steady state levels of the 30kDa MBD-3 protein increased in a dose-related manner after 24 htreatment with CSE (Fig. 2f, upper panel; and Table 2). Notably,the CSE-induced effects were protein-specific in that steady state

P. Mukhopadhyay et al. / Reproductive Toxicology 58 (2015) 140–148 143

Fig. 2. Immunoblots (upper panels) demonstrating steady-state levels of DNMT-1 (a), DNMT-3a (b), DNMT-3b (c), MeCP-2 (d), MBD-2 (e) and MBD-3 (f) proteins in nuclearextracts derived from murine first branchial arch-derived (1-BA) cells following treatment (24 h) with either 20-, 40- or 80 �g/mL Cigarette smoke extract (CSE) or vehiclecontrol (DMSO). Equal amounts of protein (20 �g) were resolved by SDS-PAGE on 12% polyacrylamide bis–tris gels, transferred to PVDF membranes, probed with specificantibodies and immunoreactive species detected by chemiluminescence, as detailed in Section 2. Molecular weights are indicated to the left of each panel. The lower panels( noblote

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a–f) depict immunoblots of the normalization loading control, �-actin. Each immuxtracts from control and CSE treated 1-BA cells.

le isoforms and/or post-transcriptionally modified forms of theroteins, as reported earlier [36]. In order to ensure equal load-

ng of proteins, and for sample normalization during densitometricnalysis, all blots were reprobed with an antibody recognizing theousekeeping protein �-actin (Fig. 2a–f; lower panels).

.3. Cigarette smoke extract differentially altered expression ofenes encoding DNMT and methyl CpG/CpG domain bindingroteins in 1-BA cells

Effect of 24 h, 20-, 40- or 80 �g/mL CSE exposure on the expres-ion of genes encoding the three DNMTs and MBDs were assessedy TaqMan qRT-PCR and cycle threshold (Ct) values for each geneere compared [40]. Results from the qRT-PCR analyses revealed

ignificant downregulation of mRNA expression of all three DNMTsnd MBDs in 1-BA cells exposed to 40- or 80 �g/mL CSE, whereas0 �g/mL CSE (or the vehicle) did not alter the expression of theseenes (Table 3). Expression of Dnmt-1, Dnmt-3a, Dnmt-3b, Mbd-2,eCP-2 and Mbd-3 was significantly decreased 1.9- and 2.2-fold,

.7- and 1.85-fold, 1.6- and 1.8-fold, 1.45- and 1.40-fold, 2.06-nd 2.2-fold and 1.4- and 1.5-fold, following exposure to 40- or0 �g/mL CSE, respectively (Table 3). Expression of the housekeep-

ng gene encoding 18S rRNA (as well as those encoding Hsp-90

is representative of no less than three independent blots from three unique sets of

3.4. Cigarette smoke extract diminishes cellular levels of DNAmethyltransferases and methyl CpG/CpG domain binding proteinsin 1-BA cells via the proteasome pathway

Reduction in protein levels after exposure to CSE (observed incase of DNMT-1, DNMT-3a, MeCP-2 and MBD-3 proteins; Fig. 3a–d)as well as appearance of lower molecular weight bands (seen inimmunoblots of DNMT-1, DNMT-3a, MeCP-2 and MBD-3; Fig. 3a–d)could be the result of CSE-induced proteomic degradation of theseproteins. This notion is reinforced by the reversal of CSE-inducedreduction in protein levels for all four proteins (Fig. 3a–d; upperpanels), and by the reduction (Fig 3b; upper panel) or elimina-tion (Fig. 3a and c; upper panels) of the lower molecular weightbands in immunoblots of DNMT1, DNMT-3a and MeCP-2 pro-teins respectively, following pretreatment with the proteasomeinhibitor, MG-132 (1.5 �M). Twenty four hour exposure of 1-BAcells to only MG-132 (1.5 �M) did not result in any degradationof the aforementioned proteins (Fig. 3a–d; upper panels). A dose-dependent (0.5-, 1.0- and 1.5 �M) effect of MG-132 in rescuing theproteasomal degradation of the two DNMTs and MBDs was alsoobserved. A representative Western blot, documenting the dose-dependent rescue of the MeCP2 protein is shown in Fig. 4a. Equalabundance of �-actin in various lanes was also confirmed (Fig. 4b).

3.5. Determination of DNMT-1 protein levels in 1-BA cells

Treatment of 1-BA cells with 80 �g/mL CSE for 24 h revealeda significant reduction in DNMT-1 protein level (*P < 0.05, one-

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Table 2Densitometric analysis of DNMT and methyl CpG/CpG domain binding protein immunoblots of first branchial arch-derived (1-BA) cell nuclear extracts.

Proteina Treatmentb Fold increase or decrease in protein level (vs. vehicle)b

DNMT-1 Vehicle (control) Ref20 �g/mL CSE +1.03 ± 0.0640 �g/mL CSE −1.35 ± 0.06*

80 �g/mL CSE −1.88 ± 0.20**

DNMT-3 Vehicle (control) Ref20 �g/mL CSE −3.76 ± 0.42*

40 �g/mL CSE −5.64 ± 1.42**

80 �g/mL CSE −32.04 ± 9.51**

DNMT-3b Vehicle (control) Ref20 �g/mL CSE +1.09 ± 0.1240 �g/mL CSE +1.17 ± 0.0880 �g/mL CSE +1.12 ± 0.08

MeCP-1 Vehicle (control) Ref20 �g/mL CSE −1.38 ± 0.0940 �g/mL CSE −1.63 ± 0.0680 �g/mL CSE −9.22 ± 2.65**

MBD-2 Vehicle (control) Ref20 �g/mL CSE +1.10 ± 0.1340 �g/mL CSE +1.13 ± 0.0680 �g/mL CSE +1.10 ± 0.05

MBD-3 Vehicle (control) Ref20 �g/mL CSE +1.09 ± 0.0540 �g/mL CSE +1.15 ± 0.0780 �g/mL CSE +5.63 ± 1.29**

* P < 0.05 vs. control.** P < 0.01 vs. control.a Steady state levels of DNMT-1, DNMT-3a, DNMT-3b, MeCP-2, MBD-2 and MBD-3 proteins (the uppermost band) were determined by immunoblotting (Fig. 2) of nuclear

extracts of 1-BA cells treated (24 h) with either vehicle (DMSO), 20-, 40- or 80 �g/mL cigarette smoke extract (CSE).b The relative levels of DNMT-1, DNMT-3a, DNMT-3b, MeCP-2, MBD-2 and MBD-3 on immunoblots (Fig. 2) were analyzed by densitometry using the ImageJ (version 1.38)

software, as described in Section 2. Densitometric analysis of protein steady state levels was conducted on no less than three independent blots of nuclear extracts of 1-BAcells treated with either vehicle, 20-, 40- or 80 �g/mL CSE. �-Actin was used as an internal control for sample normalization. The densitometric data for each protein bandwas normalized to that of �-actin in that lane. Fold change was determined as follows: intensity of the DNMT-1 band (from 20-, 40- or 80 �g/mL CSE sample)/intensity ofthe DNMT-1 band (from vehicle-treated sample). The data are presented as the mean ± standard deviation from three independent experiments. (+) indicates higher proteinlevels in CSE treated samples in comparison to vehicle-treated samples, whereas (−) indicates lower protein levels in CSE treated samples in comparison to vehicle-treatedsamples.

Fig. 3. Immunoblots (upper panels) demonstrating steady-state levels of DNMT-1 (a), DNMT-3a (b), MeCP-2 (c), and MBD-3 (d), proteins in nuclear extracts derived frommurine first branchial arch-derived (1-BA) cells. These cells were pre-treated (3 h) with either DMSO (vehicle/control) or 1.5 �M of the proteasomal inhibitor, MG-132,followed by a 24-h treatment with either 80 �g/mL CSE or vehicle control (DMSO), as detailed in Section 2. Equal amounts of protein (20 �g) were resolved by SDS-PAGE on12% polyacrylamide bis–tris gels, transferred to PVDF membranes, probed with specific antibodies and immunoreactive species detected by chemiluminescence as detailedin Section 2. Molecular weights are indicated to the left of each panel. The lower panels (a–d) depict immunoblots of the normalization control, �-actin. Each immunoblotis representative of no less than three independent blots from three unique sets of extracts from control, inhibitor and CSE treated 1-BA cells. C: pre-treatment withDMSO + treatment with DMSO; CS: pre-treatment with DMSO + treatment with 80 �g/mL CSE; MG ± CS: pre-treatment with 1.5 �M MG-132 + treatment with 80 �g/mL CSE;MG: pre-treatment with 1.5 �M MG-132 + treatment with DMSO.

P. Mukhopadhyay et al. / Reproductive Toxicology 58 (2015) 140–148 145

Table 3Effect of cigarette smoke extract (CSE) on the expression of genes encoding various DNMT and methyl CpG/CpG domain binding proteins in first branchial arch-derived(1-BA) cells.

Gene Treatmenta �Ctb,c Fold changed (2−��Ct) (95% confidence limits)

DNMT-1 Control 8.13 ± 0.0520 �g/mL CSE 8.17 ± 0.12 −1.03 (−1.15,1.09)40 �g/mL CSE 9.05 ± 0.14 −1.9 (−2.13,−1.7)*

80 �g/mL CSE 9.27 ± 0.21 −2.21 (−2.47,−1.97)*

DNMT-3a Control 8.82 ± 0.1220 �g/mL CSE 8.89 ± 0.11 −1.05 (−1.15,1.04)40 �g/mL CSE 9.59 ± 0.11 −1.71 (−1.87,−1.56)*

80 �g/mL CSE 9.71 ± 0.12 −1.85 (−2.03,−1.69)*

DNMT-3b Control 9.53 ± 0.1220 �g/mL CSE 9.62 ± 0.06 −1.06 (−1.18,1.05)40 �g/mL CSE 10.21 ± 0.13 −1.6 (−1.77,−1.44)*

80 �g/mL CSE 10.38 ± 0.19 −1.8 (−2,−1.62)*

MeCP-2 Control 11.34 ± 0.120 �g/mL CSE 11.48 ± 0.06 −1.1 (−1.21,−1)40 �g/mL CSE 12.38 ± 0.1 −2.06 (−2.26,−1.88)*

80 �g/mL CSE 12.62 ± 0.18 −2.43 (−2.67,−2.21)*

MBD-2 Control 7.62 ± 0.0720 �g/mL CSE 7.72 ± 0.06 −1.07 (−1.18,1.03)40 �g/mL CSE 8.16 ± 0.11 −1.45 (−1.61,−1.31)*

80 �g/mL CSE 8.21 ± 0.21 −1.5 (−1.66,−1.36)*

MBD-3 Control 7.74 ± 0.0920 �g/mL CSE 7.85 ± 0.1 −1.08 (−1.21,1.04)40 �g/mL CSE 8.22 ± 0.12 −1.4 (−1.57,−1.25)*

80 �g/mL CSE 8.34 ± 0.23 −1.52 (−1.7,−1.36)*

* P < 0.001 vs. control.a cDNA samples were prepared from Control, 20-, 40- or 80 �g/mL CSE treated 1-BA cells and subjected to TaqMan® quantitative real-time PCR (QRT-PCR) for each target

gene. Analyses were performed in triplicate using data from three independent experiments.b Ct values represent the number of cycles during the exponential phase of amplification necessary to reach a predetermined threshold level of PCR product as measured

by fluorescence. The more template present at the start of a reaction, the fewer the cycles required to synthesize enough fluorescent product to be recorded as statisti-cally above background. All data were normalized to the amplification signal from the housekeeping gene, 18S rRNA. The �Ct values represent these normalized signals,�Ct = Ctsample − Ct18SrRNA. Data presented represent mean �Ct ± standard deviation for three replicates.

c Negative methodological control reactions, which lacked reverse transcriptase, did not amplify any detectable product.d Fold-change (FC) values were determined according to the relationship: FC = 2−��Ct, where ��Ct is the difference in �Ct values between CSE treated and control samples

[37]. Statistical analysis comparing the control with the three CSE treatment groups was done using one-way ANOVA of the �Ct values. 95% confidence intervals for the FCw imits

d chang

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ere calculating by taking the appropriate transformation of the 95% confidence lown regulation of gene expression relative to control samples and a positive fold-

ay analysis of variance [ANOVA]). Pre-treatment of 1-BA cellsith 1.5 �M of the proteasomal inhibitor MG-132 resulted in total

revention of CSE-induced DNMT-1 protein degradation (*P < 0.05,ne-way ANOVA) (Fig. 5). This verified the involvement of the pro-easome pathway in CSE-induced degradation of DNMT and MBDroteins.

ig. 5. Histogram depicting DNMT-1 protein levels in murine first branchial arch-derived-h MG-132 pre-treatment)-treated 1-BA cells, were assayed for DNMT-1 protein using

tilizing protein standards of known concentrations (2, 5, 10, and 20 ng). The amount oD/standard slope) × sample dilution.

for the estimated difference in �Ct values. A negative fold-change value indicatese value indicates up regulation.

4. Discussion

Orofacial clefting (CL/P, or CPO), including clefts associated withexposure to maternal cigarette smoke, is a prevalent categoryof human congenital anomalies with significant adverse medi-cal, financial and social consequences to those affected. In depth

(1-BA) cells. Nuclear extracts from 24-h, vehicle-, or 80 �g/mL CSE (alone or afterthe Epiquik DNMT-1 ELISA assay kit (Epigentek). A standard curve was generatedf DNMT-1 protein was estimated as: DNMT protein (ng/mL) = (sample OD − blank

146 P. Mukhopadhyay et al. / Reproductive

Fig. 4. Immunoblots demonstrating steady-state levels of MeCP-2 (a), protein innuclear extracts derived from murine first branchial arch-derived (1-BA) cells.These cells were pre-treated (3 h) with either DMSO (vehicle/control) or 1-, 3-or 5 �M of the proteasomal inhibitor, MG-132, followed by a 24-h treatmentwith either 80 �g/mL CSE or vehicle control (DMSO), as detailed in Section 2.Equal amounts of protein (20 �g) were resolved by SDS-PAGE on 12% poly-acrylamide bis–tris gels, transferred to PVDF membranes, probed with specificantibodies and immunoreactive species detected by chemiluminescence as detailedin Section 2. Molecular weights are indicated to the left of each panel. Thelower panel (b) depicts one representative immunoblot of the normalizationcontrol, �-actin. Each immunoblot is representative of no less than three inde-pendent blots from three unique sets of extracts from control, inhibitor and CSEtreated 1-BA cells. C: pre-treatment with DMSO + treatment with DMSO; CS: pre-treatment with DMSO + treatment with 80 �g/mL CSE; CS ± 1: pre-treatment with0.5 �M MG-132 + treatment with 80 �g/mL CSE; CS ± 2: pre-treatment with 1.0 �MM1

kmsAsdtodauocatsDcteFtsmi

ossecvsnco

cells from other regions of the embryo that apparently develop nor-

G-132 + treatment with 80 �g/mL CSE; CS±3: pre-treatment with 1.5 �M MG-32 + treatment with 80 �g/mL CSE.

nowledge of the cellular and molecular mechanisms governingorphogenesis of the midfacial region is essential for any con-

ideration given to therapeutic intervention of this birth defect.n array of conditions such as maternal exposure to cigarettemoke, alcohol, heavy metals (e.g., arsenic and cadmium), variousrugs, microbial infection, and deficiency of essential nutrients (e.g.,race elements and folate) have been implicated in the etiologyf CL/P, or CPO [6,9,41–44]. Although, a number of studies haveocumented increased occurrence of CL/P with maternal activend passive cigarette smoking [6,9,12,13], the precise molecularnderpinnings directing in utero tobacco smoke exposure-inducedrofacial clefting are still obscure. One potential mechanism thatould account for the pathogenesis of such orofacial defects isltered expression of crucial genes due to aberrant methylation ofheir regulatory regions. Support for this notion comes from recenttudies demonstrating modulation of global and gene-specificNA methylation in offspring subsequent to maternal exposure toigarette smoke [15,17]. In a recent study, infants exposed in uteroo cigarette smoke demonstrated elevated methylation at a differ-ntially methylated region (DMR) regulating Insulin-like Growthactor 2 (IGF2) [17]. Inasmuch as IGF2 represents a gene essen-ial for normal embryonic growth and development [45,46], thisuggests that the plasticity of this DMR may represent a crucialechanism underlying in utero adjustments to environmental tox-

cants/teratogens, such as cigarette smoke [17].Global hypomethylation was also reported in cord blood DNA

f newborns of mothers who smoked [16]. This observation is con-istent with data from the present study which demonstrated aignificant global DNA hypomethylation in 1-BA cells followingxposure to CSE. Underscoring the possibility that exposure toigarette smoke contributes to adverse developmental outcomesia altered methylation, is the demonstration that in utero tobaccomoke exposure resulted in altered placental methylation of a

Toxicology 58 (2015) 140–148

DNA methylation [50] and altered placental expression of CYP1A1due to hypomethylation of CpG sites proximal to its regulatoryregion [51] have been reported. One role for CYP1A1, which encodesa member of the cytochrome P450 superfamily of enzymes, ismetabolism of polycyclic aromatic hydrocarbons found in cigarettesmoke [52]. Moreover, fetuses exhibiting polymorphic variants ofNAT1, an enzyme involved in phase II detoxification of compo-nents of cigarette smoke, possess an increased risk for orofacialclefts [53]. Collectively, these studies allow the suggestion that inutero cigarette smoke exposure can epigenetically modulate geneexpression in a manner that adversely affects the developing fetus.

In the present study, exposure to cigarette smoke extract (CSE)significantly altered global DNA methylation, and diminished bothmRNA and protein levels of DNMT-1 and DNMT-3A. DNMT-3B wasreduced only at the mRNA level with no significant change in pro-tein levels. These findings are consistent with previous studiesdemonstrating CSE-induced genomic hypomethylation and dimin-ished DNMT-1 expression in small airway- and bronchial epithelialcells [54]. Cigarette smoke extract has also been shown to trig-ger a significant decrease in DNMT-1 mRNA and protein levelsin frontal cortex and GABAergic neurons of mice injected withnicotine and to downregulate Dnmt3b mRNA in lung cancer cells[55,56]. Benzo[a]pyrene (BaP), another major toxic constituent ofcigarette smoke, downregulated both DNMT-3a and DNMT-3b inmouse embryonic fibroblast cells, and led to a significant decreasein global and gene specific DNA methylation during embryogenesis[57,58].

We also demonstrate that cigarette smoke extract (CSE) signifi-cantly diminished both mRNA and protein levels of the two methylCpG-binding proteins, MeCP-2, and MBD-3, in 1-BA cells. As withDNMT-3b, CSE caused a decrease in MBD-2 mRNA levels withoutany measurable change in protein levels. Currently, almost nothingis known regarding the effect(s) of cigarette smoke exposure, on theexpression of the methyl CpG-binding proteins—key regulators ofthe epigenome as well as the transcriptome. Our results representthe first evidence of the impact(s) of CSE exposure on the expressionof MeCP-2, MBD-2 and MBD-3 within an embryonic cell type. Col-lectively, these data support the premise that CSE, via modulationof expression of the DNMTs and the MBDs, can modulate cellularDNA methylation and gene expression. The data provide supportfor the possibility that the teratogenic effect(s) of components ofcigarette smoke may be epigenetically mediated.

We have further shown that CSE-induced diminution ofDNMT-1, DNMT-3a, MeCP-2 and Mbd-3 was due to extensiveproteasome-mediated nuclear degradation. Pre-treatment of 1-BAcells with the proteasome inhibitor MG-132 inhibited CSE-induceddegradation of each of the DNMT and MBD proteins. This supportsour hypothesis that CSE-induced alteration of the DNA methylationmachinery (DNA methyltransferases and methyl CpG binding pro-teins) in cells of the developing first branchial arch is mediated inpart by the ubiquitin/proteasomal degradation pathway and in partby the alteration in the transcription/translation machinery. Fur-ther support for this hypothesis comes from numerous additionalstudies demonstrating proteasome-mediated protein degradationby components of cigarette smoke, and resultant adverse effects oncrucial cellular processes [18,59–61]. Our data support the conclu-sion that proteasomal degradation of crucial effectors/modulatorsof DNA methylation (such as DNMT-1, -3a, MeCP-2, and MBD-3)represents a potential epigenetic mechanism by which exposureto cigarette smoke may result in an orofacial cleft.

Taken in isolation, results of the current study indeed do notdemonstrate that 1-BA cells are any more, or less, sensitive than

mally. However, these studies are based on data from our laboratorythat (1) in utero CSE exposure in mice results in an increased fre-quency of orofacial clefting and dysmorphology of tissues derived

uctive

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P. Mukhopadhyay et al. / Reprod

rom 1-BA cells used in the present study, (2) Whole genomeequencing of CSE-exposed 1-BA tissue demonstrates detectablelterations in DNA methylation, and (3) Prenatal exposure to CSEesulted in hypomethylation of the Peg3 gene in cells of the 1-BA in

ice. These data, which support rationale for our design and con-lusions, are unpublished observations from our laboratory and areeing incorporated into manuscripts in preparation.

onflict of interest

The authors declare that there are no conflicts of interest.

cknowledgements

The authors thank Dr. Guy Brock for help with bioinformatics.his research was supported in part by NIH grant P20 RR017702rom the COBRE Program of the NIGMS and NIH grants AA13205,D053509 and DE018215.

eferences

[1] L. Stuppia, M. Capogreco, G. Marzo, D. La Rovere, I. Antonucci, V. Gatta, et al.,Genetics of syndromic and nonsyndromic cleft lip and palate, J. Craniofac.Surg. 22 (2011) 1722–1726.

[2] F. Rahimov, A. Jugessur, J.C. Murray, Genetics of nonsyndromic orofacial clefts,Cleft Palate Craniofac. J. 49 (2012) 73–91.

[3] C.W. Senders, E.C. Peterson, A.G. Hendrickx, M.A. Cukierski, Development ofthe upper lip, Arch. Facial Plast. Surg. 5 (2003) 16–25.

[4] J.O. Bush, R. Jiang, Palatogenesis: morphogenetic and molecular mechanismsof secondary palate development, Development 139 (2012) 231–243.

[5] E.P. Buchanan, A.S. Xue, L.H. Hollier Jr., Craniofacial syndromes, Plast.Reconstr. Surg. 134 (2014) 128e–153e.

[6] J. Grewal, S.L. Carmichael, C. Ma, E.J. Lammer, G.M. Shaw, Maternalpericonceptional smoking and alcohol consumption and risk for selectcongenital anomalies, Birth Defects Res. A Clin. Mol. Teratol. 82 (2008)519–526.

[7] M.A. Honein, S.A. Rasmussen, J. Reefhuis, P.A. Romitti, E.J. Lammer, L. Sun,et al., Maternal smoking and environmental tobacco smoke exposure and therisk of orofacial clefts, Epidemiology 18 (2007) 226–233.

[8] D. Ramirez, E.J. Lammer, D.M. Iovannisci, C. Laurent, R.H. Finnell, G.M. Shaw,Maternal smoking during early pregnancy, GSTP1 and EPHX1 variants, andrisk of isolated orofacial clefts, Cleft Palate Craniofac. J. 44 (2007) 366–373.

[9] G.M. Shaw, S.L. Carmichael, S.E. Vollset, W. Yang, R.H. Finnell, H. Blom, et al.,Mid-pregnancy cotinine and risks of orofacial clefts and neural tube defects, J.Pediatr. 154 (2009) 17–19.

10] M. Shi, K. Christensen, C.R. Weinberg, P. Romitti, L. Bathum, A. Lozada, et al.,Orofacial cleft risk is increased with maternal smoking and specificdetoxification-gene variants, Am. J. Hum. Genet. 80 (2007) 76–90.

11] Z.L. Jia, B. Shi, C.H. Chen, J.Y. Shi, J. Wu, X. Xu, Maternal malnutrition,environmental exposure during pregnancy and the risk of non-syndromicorofacial clefts, Oral Dis. 17 (2011) 584–589.

12] Z. Li, J. Liu, R. Ye, L. Zhang, X. Zheng, A. Ren, Maternal passive smoking and riskof cleft lip with or without cleft palate, Epidemiology 21 (2010) 240–242.

13] T. Wu, M.D. Fallin, M. Shi, I. Ruczinski, K.Y. Liang, J.B. Hetmanski, et al.,Evidence of gene-environment interaction for the RUNX2 gene andenvironmental tobacco smoke in controlling the risk of cleft lip with/withoutcleft palate, Birth Defects Res. A Clin. Mol. Teratol. 94 (2012) 76–83.

14] D.A. Brandini, M.A. Sala, R.A. Lopes, M. Semprini, M.G. Contrera, Effects ofcigarette smoke on the Meckel’s cartilage of rat fetus: morphologic,morphometric and stereologic study, Braz. Dent. J. 16 (2005) 62–66.

15] C.V. Breton, H.M. Byun, M. Wenten, F. Pan, A. Yang, F.D. Gilliland, Prenataltobacco smoke exposure affects global and gene-specific DNA methylation,Am. J. Respir. Crit. Care Med. 180 (2009) 462–467.

16] R. Guerrero-Preston, L.R. Goldman, P. Brebi-Mieville, C. Ili-Gangas, C. Lebron,F.R. Witter, et al., Global DNA hypomethylation is associated with in uteroexposure to cotinine and perfluorinated alkyl compounds, Epigenetics 5(2010) 539–546.

17] S.K. Murphy, A. Adigun, Z. Huang, F. Overcash, F. Wang, R.L. Jirtle, et al.,Gender-specific methylation differences in relation to prenatal exposure tocigarette smoke, Gene 494 (2012) 36–43.

18] D. Adenuga, H. Yao, T.H. March, J. Seagrave, I. Rahman, Histone deacetylase 2is phosphorylated, ubiquitinated, and degraded by cigarette smoke, Am. J.Respir. Cell Mol. Biol. 40 (2009) 464–473.

19] C.Y. Howell, T.H. Bestor, F. Ding, K.E. Latham, C. Mertineit, J.M. Trasler, et al.,

20] M. Kaneda, M. Okano, K. Hata, T. Sado, N. Tsujimoto, E. Li, et al., Essential rolefor de novo DNA methyltransferase Dnmt3a in paternal and maternalimprinting, Nature 429 (2004) 900–903.

Toxicology 58 (2015) 140–148 147

21] E. Li, T.H. Bestor, R. Jaenisch, Targeted mutation of the DNA methyltransferasegene results in embryonic lethality, Cell 69 (1992) 915–926.

22] S. McGraw, C.C. Oakes, J. Martel, M.C. Cirio, P. de Zeeuw, W. Mak, et al., Loss ofDNMT1o disrupts imprinted X chromosome inactivation and accentuatesplacental defects in females, PLoS Genet. 9 (2013) e1003873.

23] M. Okano, D.W. Bell, D.A. Haber, E. Li, DNA methyltransferases Dnmt3a andDnmt3b are essential for de novo methylation and mammalian development,Cell 99 (1999) 247–257.

24] L.J. Yin, Y. Zhang, P.P. Lv, W.H. He, Y.T. Wu, A.X. Liu, et al., Insufficientmaintenance DNA methylation is associated with abnormal embryonicdevelopment, BMC Med. 10 (2012) 26.

25] N. Hu, P. Strobl-Mazzulla, T. Sauka-Spengler, M.E. Bronner, DNAmethyltransferase3A as a molecular switch mediating the neuraltube-to-neural crest fate transition, Genes Dev. 26 (2012) 2380–2385.

26] R.S. Hansen, C. Wijmenga, P. Luo, A.M. Stanek, T.K. Canfield, C.M. Weemaes,et al., The DNMT3B DNA methyltransferase gene is mutated in the ICFimmunodeficiency syndrome, Proc. Natl. Acad. Sci. U. S. A. 96 (1999)14412–14417.

27] K. Martins-Taylor, D.I. Schroeder, J.M. LaSalle, M. Lalande, R.H. Xu, Role ofDNMT3B in the regulation of early neural and neural crest specifiers,Epigenetics 7 (2012) 71–82.

28] T. Hatta, A. Matsumoto, K. Moriyama, H. Otani, Opposite effects of thematernal immune system activated by interleukin-1beta vs. PSK and OK432on 5-azacytidine-induced birth defects, Congenit. Anom. 43 (2003) 46–56.

29] B. Hendrich, A. Bird, Identification and characterization of a family ofmammalian methyl-CpG binding proteins, Mol. Cell. Biol. 18 (1998)6538–6547.

30] A. Miremadi, M.Z. Oestergaard, P.D. Pharoah, C. Caldas, Cancer genetics ofepigenetic genes, Hum. Mol. Genet. 16 (Spec no. 1) (2007) R28–R49.

31] T. Baubec, R. Ivanek, F. Lienert, D. Schubeler, Methylation-dependent and-independent genomic targeting principles of the MBD protein family, Cell153 (2013) 480–492.

32] O. Yildirim, R. Li, J.H. Hung, P.B. Chen, X. Dong, L.S. Ee, et al., Mbd3/NURDcomplex regulates expression of 5-hydroxymethylcytosine marked genes inembryonic stem cells, Cell 147 (2011) 1498–1510.

33] J. Guy, B. Hendrich, M. Holmes, J.E. Martin, A. Bird, A mouse Mecp2-nullmutation causes neurological symptoms that mimic Rett syndrome, Nat.Genet. 27 (2001) 322–326.

34] K. Kaji, J. Nichols, B. Hendrich, Mbd3, a component of the NuRD co-repressorcomplex, is required for development of pluripotent cells, Development 134(2007) 1123–1132.

35] O. Bogdanovic, G.J. Veenstra, DNA methylation and methyl-CpG bindingproteins: developmental requirements and function, Chromosoma 118(2009) 549–565.

36] P.A. Defossez, I. Stancheva, Biological functions of methyl-CpG-bindingproteins, Prog. Mol. Biol. Transl. Sci. 101 (2011) 377–398.

37] T. Doi, P. Puri, A. McCann, J. Bannigan, J. Thompson, Epigenetic effect ofcadmium on global de novo DNA hypomethylation in the cadmium-inducedventral body wall defect (VBWD) in the chick model, Toxicol. Sci. 120 (2011)475–480.

38] Z.J. Han, G. Song, Y. Cui, H.F. Xia, X. Ma, Oxidative stress is implicated inarsenic-induced neural tube defects in chick embryos, Int. J. Dev. Neurosci. 29(2011) 673–680.

39] P. Mukhopadhyay, S. Singh, R.M. Greene, M.M. Pisano, Molecularfingerprinting of BMP2- and BMP4-treated embryonic maxillarymesenchymal cells, Orthod. Craniofac. Res. 9 (2006) 93–110.

40] U.E. Gibson, C.A. Heid, P.M. Williams, A novel method for real timequantitative RT-PCR, Genome Res. 6 (1996) 995–1001.

41] H.A. Goenjian, E.S. Chiu, M.E. Alexander, H. St Hilaire, M. Moses, Incidence ofcleft pathology in Greater New Orleans before and after Hurricane Katrina,Cleft Palate Craniofac. J. 48 (2011) 757–761.

42] N. Kaminen-Ahola, A. Ahola, M. Maga, K.A. Mallitt, P. Fahey, T.C. Cox, et al.,Maternal ethanol consumption alters the epigenotype and the phenotype ofoffspring in a mouse model, PLoS Genet. 6 (2010) e1000811.

43] S.G. Obican, R.H. Finnell, J.L. Mills, G.M. Shaw, A.R. Scialli, Folic acid in earlypregnancy: a public health success story, FASEB J. 24 (2010) 4167–4174.

44] J. Thompson, J. Bannigan, Cadmium: toxic effects on the reproductive systemand the embryo, Reprod. Toxicol. 25 (2008) 304–315.

45] L.N. Kent, S. Ohboshi, M.J. Soares, Akt1 and insulin-like growth factor 2 (Igf2)regulate placentation and fetal/postnatal development, Int. J. Dev. Biol. 56(2012) 255–261.

46] J. St-Pierre, M.F. Hivert, P. Perron, P. Poirier, S.P. Guay, D. Brisson, et al., IGF2DNA methylation is a modulator of newborn’s fetal growth and development,Epigenetics 7 (2012) 1125–1132.

47] J.Z. Maccani, D.C. Koestler, E.A. Houseman, C.J. Marsit, K.T. Kelsey, PlacentalDNA methylation alterations associated with maternal tobacco smoking atthe RUNX3 gene are also associated with gestational age, Epigenomics 5(2013) 619–630.

48] D. Levanon, O. Brenner, V. Negreanu, D. Bettoun, E. Woolf, R. Eilam, et al.,Spatial and temporal expression pattern of Runx3 (Aml2) and Runx1 (Aml1)indicates non-redundant functions during mouse embryogenesis, Mech. Dev.

49] H. Yamamoto, K. Ito, M. Kawai, Y. Murakami, K. Bessho, Y. Ito, Runx3expression during mouse tongue and palate development, Anat. Rec. ADiscov. Mol. Cell. Evol. Biol. 288 (2006) 695–699.

1 uctive

[proteasome inhibitor MG132 attenuate cigarette smoke induced catabolismin C2 myotubes, Adv. Exp. Med. Biol. 788 (2013) 25–33.

[61] S.H. van Rijt, I.E. Keller, G. John, K. Kohse, A.O. Yildirim, O. Eickelberg, et al.,Acute cigarette smoke exposure impairs proteasome function in the lung, Am.J. Physiol. Lung Cell. Mol. Physiol. 303 (2012) L814–L823.

48 P. Mukhopadhyay et al. / Reprod

50] M. Suter, J. Ma, A. Harris, L. Patterson, K.A. Brown, C. Shope, et al., Maternaltobacco use modestly alters correlated epigenome-wide placental DNAmethylation and gene expression, Epigenetics 6 (2011) 1284–1294.

51] M. Suter, A. Abramovici, L. Showalter, M. Hu, C.D. Shope, M. Varner, et al., Inutero tobacco exposure epigenetically modifies placental CYP1A1 expression,Metabolism 59 (2010) 1481–1490.

52] A.A. Walsh, G.D. Szklarz, E.E. Scott, Human cytochrome P450 1A1 structureand utility in understanding drug and xenobiotic metabolism, J. Biol. Chem.288 (2013) 12932–12943.

53] E.J. Lammer, G.M. Shaw, D.M. Iovannisci, J. Van Waes, R.H. Finnell, Maternalsmoking and the risk of orofacial clefts: susceptibility with NAT1 and NAT2polymorphisms, Epidemiology 15 (2004) 150–156.

54] F. Liu, J.K. Killian, M. Yang, R.L. Walker, J.A. Hong, M. Zhang, et al., Epigenomicalterations and gene expression profiles in respiratory epithelia exposed tocigarette smoke condensate, Oncogene 29 (2010) 3650–3664.

55] H. Liu, Y. Zhou, S.E. Boggs, S.A. Belinsky, J. Liu, Cigarette smoke inducesdemethylation of prometastatic oncogene synuclein-gamma in lung cancer

56] R. Satta, E. Maloku, A. Zhubi, F. Pibiri, M. Hajos, E. Costa, et al., Nicotinedecreases DNA methyltransferase 1 expression and glutamic aciddecarboxylase 67 promoter methylation in GABAergic interneurons, Proc.Natl. Acad. Sci. U. S. A. 105 (2008) 16356–16361.

Toxicology 58 (2015) 140–148

57] X. Fang, C. Thornton, B.E. Scheffler, K.L. Willett, Benzo[a]pyrene decreasesglobal and gene specific DNA methylation during zebrafish development,Environ. Toxicol. Pharmacol. 36 (2013) 40–50.

58] C.L. Yauk, A. Polyzos, A. Rowan-Carroll, I. Kortubash, A. Williams, O.Kovalchuk, Tandem repeat mutation, global DNA methylation, and regulationof DNA methyltransferases in cultured mouse embryonic fibroblast cellschronically exposed to chemicals with different modes of action, Environ.Mol. Mutagen. 49 (2008) 26–35.

59] S.Y. Kim, J.H. Lee, J.W. Huh, J.Y. Ro, Y.M. Oh, S.D. Lee, et al., Cigarette smokeinduces Akt protein degradation by the ubiquitin-proteasome system, J. Biol.Chem. 286 (2011) 31932–31943.

60] O. Rom, S. Kaisari, D. Aizenbud, A.Z. Reznick, Essential amino acid leucine and

Cigarette smoke induces proteasomal-mediated degradation of DNA methyltransferases and methyl...

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