review article bisphenol a effects on mammalian...

Review ArticleBisphenol A Effects on Mammalian Oogenesis and EpigeneticIntegrity of Oocytes A Case Study Exploring Risks of EndocrineDisrupting Chemicals

Ursula Eichenlaub-Ritter1 and Francesca Pacchierotti2

1Faculty of Biology Gene TechnologyMicrobiology University of Bielefeld 33601 Bielefeld Germany2Laboratory of Toxicology Unit of Radiation Biology and Human Health ENEA CR Casaccia00123 Santa Maria di Galeria Rome Italy

Correspondence should be addressed to Ursula Eichenlaub-Ritter eiriuni-bielefeldde

Received 16 January 2015 Revised 5 May 2015 Accepted 5 May 2015

Academic Editor Dong-Wook Han

Copyright copy 2015 U Eichenlaub-Ritter and F Pacchierotti This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Bisphenol A (BPA) originally developed as a synthetic oestrogen is nowadays extensively used in the production of polymericplastics Under harsh conditions these plastics may release BPA which then can leach into the environment Detectableconcentrations of BPA have been measured in most analysed samples of human serum plasma or urine as well as in follicularfluid foetal serum and amniotic fluid Here we summarize the evidence about adverse BPA effects on the genetic and epigeneticintegrity of mammalian oocytes We conclude that increasing evidence supports the notion that low BPA concentrations adverselyaffect the epigenome of mammalian female germ cells with functional consequences on gene expression chromosome dynamicsin meiosis and oocyte development Specific time windows during which profound chromatin remodelling occurs and maternalimprints are established or protected appear particularly vulnerable to epigenetic deregulation by BPA Transgenerational effectshave been also observed in the offspring of BPA-treated rodents although the epigenetic mechanisms of inheritance still need tobe clarified The relevance of these findings for human health protection still needs to be fully assessed but they warrant furtherinvestigation in both experimental models and humans

1 Introduction

According to the ldquoDevelopmental Origin of Health and Dis-easerdquo hypothesis (DOHaD) that was proposed over a decadeago [1 2] lifestyle nutrition and exposures during pregnancycan influence the health of the offspring from birth to muchlater in life The DOHaD hypothesis proposes that faminenutritional deficits or diabetes in the mother can predisposethe offspring to diseases or reduce its fertility especially ifexposures occur during critical periods of embryogenesis orfoetal development [3ndash5] Prenatal exposure of the develop-ing germline may entail the additional risk to transmit theinduced damage to the following generation

Environmental exposures that affect metabolism or hor-monal homeostasis do not necessarily induce DNA muta-tions but may influence gene expression by disturbances in

epigenetic regulation Recently it has been shown in rodentsthat in utero undernourishment alters the germline DNAmethylome of 1198651 adult males in a locus-specific mannerAlthough altered DNA methylation did not persist in 1198652 tis-sues dysregulated expression of genes neighbouring affectedloci was observed suggesting the possibility of intergenera-tional transmission of environmentally induced disease notmediated by Mendelian inheritance [6]

According to the US Environmental Protection Agencyendocrine disrupting chemicals (EDCs) are defined asldquoexogenous agent(s) that interfere(s) in synthesis secretiontransport metabolism binding action or elimination of nat-ural blood-borne hormones that are present in the body andare responsible for homeostasis reproduction and develop-mental processesrdquo [7] As such theymay transiently alter geneexpression patterns in exposed cells organs and individuals

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 698795 11 pageshttpdxdoiorg1011552015698795

2 BioMed Research International

BPA polymer

O

O

(n) (n)

Monomer bisphenol A BPA44998400-(propane-22-diyl)diphenol

HO OH O

CH3

CH3CH3

CH3

Figure 1 BPA monomer and polymer

by interfering in hormonal homeostasis for example byacting as agonist or antagonist in hormone receptor-mediatedsignalling Moreover it has recently been shown that expo-sure to EDCs may induce transgenerational phenotype alter-ations possibly caused by differential DNA methylation ingene promoter regions termed epimutations [8ndash12]

2 Bisphenol A

Monomer bisphenol A (441015840-(propane-22-diyl)diphenol)(BPA) (Figure 1) was developed in 1891 as a synthetic oestro-gen (xenoestrogen) BPA indeed binds to oestrogen receptorsin vivo and in vitro [13] but due to its low oestrogenic activ-ity (asymp103ndash105 less than the natural steroid oestradiol) itwas replaced by diethylstylbestrol (DES) which had muchstronger oestrogenic properties DES is sadly known for theteratogenic and carcinogenic effects observed in the genitalorgans of daughters of women using DES in pregnancy toprevent spontaneous abortion [14] Recently an epigeneticinfluence of DES on the regulation of histone [15] and DNA[16] methyltransferases has been shown that could play a rolein the induction of its reproductive effects The case of DEScould be considered an alarming sentinel of the importanceof epigenetic mechanisms in EDCs adverse effects

Even though BPA was not marketed as a hormonal activesubstance in the last decades it found application as plasti-cizer in the production of polymeric plastics mainly polycar-bonate (71) and epoxy resins (29) [17] For a long timepolymeric BPA was considered harmless as it does not inter-act with steroid receptors (Figure 1) Over the last 50 yearsthe use of BPA-containing polymers in common items suchas plastic bottles toys lining of aluminium cans and pipesdental sealants and thermal receipt paper led to increasingBPA production which reached about 5 million tons in 2010[17] Unfortunately polycarbonate plastics damaged by heatUV harsh alkaline treatment or after vigorous washing wereshown to release monomeric BPA By now it is estimatedthat the worldwide release of BPA into the environment isexceeding one million poundsyear [18]

3 Environmental andHuman BPA Contamination

In the USA average BPA groundwater concentrations rangebetween 00041 and 19mgm3 and up to 20mgm3 BPAwere

measured in some areas of Great Britain [17] BPA can beefficiently biodegraded in water and soil by microorganismsand by photolysis in water at wavelengths above 290 nm [1719 20] However in spite of environmental biodegradation01ndash790 120583gkg BPA were detected in fresh weight (fw) foodandup to 086mgm3 in drinkingwater or commercial drinks[17] biomonitoring studies detected BPA in human serumplasma or urine of over 90 US and Canadian citizens[21] Daily dietary BPA intakes of about 002ndash008120583gkgdayand 022ndash033 120583gkgday have been estimated for adultsand infants respectively (for references see [17]) BPA israpidly metabolised to bisphenol A-glucuronide in liver butunconjugated BPA has been detected in serum and bloodof the general population at concentrations of 44mgL and25mgL respectively and over 50mgL BPA were measuredin workers [18 22] BPA has been also detected in follicularfluid foetal serum and amniotic fluid (average 1-2 ngmL)[23] and in umbilical cord serum of human mid gestationembryos [24]

4 BPA Effects on Mammalian Oogenesis

Although potential adverse effects of low BPA concentrationson reproduction are still a matter of debate most studiessuggest that BPA is an ovarian toxicant and reduces oocytequality in animal models and in humans [23 25] Poten-tial mechanisms of BPA action on hormonal homeostasisinclude binding to nonclassic membrane oestrogen recep-tors (mERs) binding to glucuronide receptor activation ofnuclear oestrogen-related receptor gamma (ERR120574) suppres-sion of thyroid hormone receptor transcription decrease ofcholesterol transport through the mitochondrial membraneincrease of fatty acid oxidation stimulation of prolactinrelease and impairment of aromatase expression (reviewedin [18 25ndash27])

Mammalian oocytes are amongst the most long-livedcells in the body Primordial germ cells start differentiatingalready in the early postimplantation embryo aftermigrationto the genital ridges (Figure 2) [28] Nests of primary oocytesentering meiosis I are formed in the human ovary by the3rd month of pregnancy Pairing and recombination betweenhomologous chromosomes take place in the foetal ovarybefore birth Around the 7th month of pregnancy oocytesfinally develop to the late dictyotene stage when they becomemeiotically arrested By that time the synaptonemal com-plexes have disappeared and the homologous chromosomes

BioMed Research International 3

MaintenanceEstablishmentErasure

Low

CpG

met

hyla

tion

Oogenesis Embryogenesis

PassiveActive

Dnmt1Dnmt3a

Dnmt3l

AID

Tet3ELP3AID

Fertilization

Dnmt1Dnmt3a

Dnmt3b

Imprinted genes

Growth Proliferation

Zfp57Kap1Stella

PGC Immature GV Mature GV MII Zygote 2-cell Blastocyst

Hig

h

Dnmt1minus

Impr

inted

gene

s(m

atern

al)

Non

impr

inte

d ge

nes

Global

meth

ylatio

n 5 998400mCrarr

5 998400hmCrarr

5 998400C

5 998400mCrarr5 998400C

Figure 2 Critical stages for epigenetic reprogramming of chromatin in mammalian female germ cells including periods of imprint erasureduring formation of primordial germ cells maternal imprint establishment during oocyte growth and imprint maintenance after fertilizationof the egg and development to the blastocyst Statuses of maternal imprints are indicated by red lines of global methylation in maternalchromatin in dotted blue lines of global paternal methylation in green dotted lines and of development and tissue-specific methylation inorange dotted lines Some enzymes that participate in demethylation (activation induced cytidine deaminase) DNAmethylation (Dnmts) ormaintenance ofmethylation (zinc finger protein 57 Zfp57 tripartite motive containing Kap1Trim28 developmental pluripotency associated3 StellaDppa3) are indicated next to the respective lines showing changes in DNA methylation adapted from [84]

remain attached by one or more meiotic exchanges and chi-asmata until much later in oogenesis when oocytes resumemeiosis in the sexually adult female in the meantime thesister chromatids of each chromosome remain tightly linkedby cohesion complexes (reviewed in [29])

Exposure of mice from midgestation until birth to dailydoses of 400 ng BPA resulted in synaptic abnormalities andincreased rates of recombination between homologous chro-mosomes in the oocytes Interestingly these effects resembledthose observed inmice homozygous for a targeted disruptionof the gene encoding for oestrogen receptor 120573 [30] Increasedrecombination was also observed in oocytes of rhesus mon-keys prenatally exposed to BPA [31] and in human oocytestreated in vitro with BPA [32] Moreover the expression ofgenes involved in recombination and DNA repair was alteredin the BPA-exposed human foetal oocytes [33] It is knownthat alterations in the number and localization of chiasmatacan adversely affect chromosome segregation and predisposethe oocytes to aneuploidy (reviewed in [29]) The studies onaltered recombination in BPA-exposed foetal oocytes there-fore suggest that the female offspring of BPA-exposed moth-ers might be at risk for meiotic chromosome nondisjunction

In addition to effects on chromosome synapsis in theoocytes BPA was shown to induce also alterations of folliclematuration Normally at birth the oocyte nests break downand primordial follicles are formed by recruitment of a single

layer of flattened granulosa cells around the dictyate-arrestedoocytes Primordial follicles will develop to the secondarytertiary and finally large antral stage only from pubertyonwards when folliculogenesis proceeds under the influenceof gonadotropic hormones (follicle stimulating hormoneFSH and luteinizing hormone LH) Mature follicles containmultilayered outer mural granulosa cells and layers of cumu-lus granulosa cells surrounding a fully grownmeiotically anddevelopmentally competent oocyte within a large fluid-filledspace the antrumThe follicle is surrounded by a basal mem-brane and by layers of luteal cells that are involved in steroido-genesis Growth of the oocyte and full development of the fol-licle is a lengthy process that takes about 120 days in humans

In rhesusmonkeys chronic exposure to BPAduring preg-nancy leading to serum concentrations of 22ndash33 ngmLcaused in the offspring a significant increase in the fre-quency of abnormal follicles containing multiple oocytes[31] Disturbances in nest breakdown and primordial follicleformation were also noted in mice upon in utero exposure[34] In vitro experiments supported the notion that BPAimpairs follicular development Germ cell nest breakdownand primordial follicle assembly were significantly reducedwhen newborn mouse ovaries were exposed to 10 or 100 120583MBPA in culture medium [35] Similarly 100120583gmL BPA(440 120583M) inhibited follicle growth and induced atresia in amouse follicle in vitro model [36] by mechanisms that were


independent of the genomic oestrogenic pathway [37] Con-tinuous 12-day exposure of mouse follicles in culture fromthe early preantral up to the large antral stage to 30 120583MBPA led to reduced granulosa cell proliferation and arrestof some oocytes at meiosis I [38] whereas lower BPA con-centrations did not significantly affect follicular developmentand hormone release Finally in vitro treatment of humanoocytes with low BPA concentrations was shown to increasethe frequency of oocyte degeneration [32]

Since the entire pool of primary oocytes that can ever beovulated is already formed in the foetal ovary disturbancesin oocyte meiosis and follicle formation recruitment andsurvival induced by any mechanism can contribute topremature ovarian depletion a clinically recognized condi-tion in women (termed ldquopremature ovarian insufficiencyrdquoPOI) In addition the fidelity of chromosome segregation atfirst and second meiosis might be compromised when theoocyte pool becomes prematurely depleted Whether BPAexposure may influence follicle pool size in humans is stillcontroversial High urinary BPA levels were associated withreduced antral follicle counts in a cohort of 209 womenundergoing infertility treatments [39] whereas no correlationwas found between serum BPA levels and antral folliclecounts in another study on a smaller cohort of 44 patients[40] Nevertheless several data suggest a negative impact ofBPA on woman fertility Urinary BPA levels were negativelycorrelated with numbers and quality of oocytes retrieved instimulated cycles for assisted reproduction [41 42] Increasedurinary [41 42] or serum [43] BPA concentrations were alsoassociated with decreased peak oestradiol levels Finally astudy on 137 patients undergoing assisted reproduction sug-gested that high urinary BPA levels might be associated withup to 50 higher chance of implantation failures in compari-son to patients with low or no evidence of BPA exposure [44]

Once follicles have developed to the large antral Graafianstage release of one (in monoovulatory species like humans)or ofmultiple (inmultiovulatory species like rodents) oocytesfrom meiotic arrest occurs under the influence of gona-dotropins and the LH surge downstream from signalling bycomplex and redundant pathways Resumption of meiosis Inormally results in gene expression changes and molecularsignalling in cumulus cells and oocyte via epidermal growthfactor-like hormones and critical changes in the concen-tration of cyclic nucleotides Fully grown developmentallycompetent oocytes then become transcriptionally quiescentand their chromatin is remodelled to surround the nucleolusin a characteristic fashion [45] Following the LH surgethe maturation promoting factorcyclin-dependent kinase 1pathway of the oocyte becomes activated chromatin becomescondensed and histones are characteristically deacetylatedand posttranslationally modified in specific ways [46] Aspindle is then formed in the ooplasm and oocytes completethe first meiosis with the reductional division of homologouschromosomes reach themetaphase II stage when they arrestagain and are finally ovulated surrounded by the expandedcumulus complex

In 2003 it was reported that low chronic BPAdosesmightinduce aneuploidy in oocytes exposed prior to resumption ofmeiosis [47] Oral treatment of mice with 20 40 or 100 ngg

bw BPA for 6ndash8 days prior to isolation and in vitromatura-tion of oocytes inducedmeiotic arrest spindle abnormalitiesand misalignment of metaphase II chromosomes Theseexperimental results supported the hypothesis that a suddenunexpected increase of aneuploid oocytes that had previouslyoccurred in the mouse colony had been caused by accidentalrelease of BPA from damaged plastic bottles and cagesAn independent study conducted under similar exposureconditions to verify these findings showed BPA inductionof subtle spindle abnormalities in metaphase II oocytes butnot aneuploidy [48] Similarly no evidence of aneuploidyinduction was obtained in metaphase II oocytes collectedfrom mice treated with a single BPA dose with 7 dailyadministrations or exposed for 7 weeks to BPA in drinkingwater [49] Differences in the animal diet were suggestedto explain these inconsistencies when it was shown that thephytoestrogen content in animal feed could influence the rateof spindle aberrations induced in metaphase II oocytes by7 daily low dose administrations of BPA [50] Other studiesshowed an influence of the diet on BPA-induced changes inDNA methylation [51] supporting an interaction betweenBPA biological activity and dietary factors

In vitro experiments in mouse oocytes showed that highconcentrations of BPA induced spindle aberrations chro-mosome congression abnormalities and meiotic arrest butnot aneuploidy [48 52] suggesting that an efficient spindleassembly checkpoint was able to prevent chromosome segre-gation errors in healthy young oocytes An inverse relation-ship between BPA concentration and percentage of oocytesthat progressed to metaphase II and a dose-dependentincrease in aberrant spindles and unaligned chromosomes atmetaphase II were also reported for human oocytes exposedin vitro to 20 200 ngmL or 20120583gmL BPA (88 880 nM88 120583M) [53]

Fertilization triggers release ofmetaphase II oocytes fromsecond meiotic arrest This entails second polar body extru-sion and completion of oocyte second meiosis during whichsister chromatids separate from each other (reviewed in [29])There is a paucity of data about BPA effects on the secondmeiotic division in oocytes Chronic exposures of mice to05mgL BPA in drinking water resulted in the prematureseparation of sister chromatids in their metaphase II oocyteswhich however had no consequence upon the fidelity ofchromosome segregation during the secondmeiotic divisionas demonstrated by the normal chromosome constitution ofzygotes under the same exposure condition [49]

5 BPA Epigenetic Effects on Female GermCells and Their Consequences

Oogenesis from primordial germ cell differentiation to ferti-lization and preimplantation embryonic development entailprofound epigenetic changes (Figure 2) After global DNAdemethylation in primordial germ cells female specificgenomic imprinting is set during oocyte development in asite-specific sequential fashion in imprinting control regions(ICRs) The whole process is completed prior to resumption


of meiosis [54ndash56] The zygote and the early preimplanta-tion embryo are further subjected to extensive chromatinremodelling and DNA methylation changes active hydrox-ymethylation and global DNA demethylation in the malechromatin and passive global demethylation in the femalechromatin (Figure 2) Most of the enzymes for these eventsare maternally provided by the oocyte before full zygoticgene activation The remodelling and epigenetic changesin male and female chromatin proceed according to ahighly regulated sex-specific program [57] which spares theremoval of DNA methylation on genomic imprints fromsperm and oocyte that are important for normal preimplan-tation development and are retained in tissues to regulatemonoallelic gene expression frompaternal ormaternal alleles[58 59] Disturbances in genomic imprinting and in DNAmethylation pattern or histone pattern and chromatin con-formation can contribute to epigenetic diseases such as theAngelman Beckwith-Wiedemann Prader-Willi and Russell-Silver syndromes [60ndash62] and predispose the offspring oreven the following generations to cancer and other diseasesrelated to epigenetic instability

The first report showing BPA epigenetic effects camefrom studies in the agouti viable yellow (119860vy) mutant mousemodel The 119860vy allele carries an Intracisternal A Particle(IAP) retrotransposon insertion upstream of the locus TheAgouti gene is usually expressed during a narrow windowin embryogenesis and codes for a signalling molecule thatcan lead to either production of black eumelanin or yellowpheomelanin from a hair-cycle-specific promoter in exon2 [63 64] In the 119860vy allele transcriptional control of theAgouti coding sequence is driven by promoter elements inthe retrotransposon containing 9CpGs whose methylationlevel can vary among individual isogenic mice [51 64 65]Hypomethylation of the 9CpGs leads to the binding of theagouti protein to the melanocortin 4 receptor in all tissuesectopic gene expression and shifting of the coat colour fromwild type pseudoagouti brown to mottled to yellow In addi-tion the overproduction of the agouti protein associated tohypomethylation causes obesity diabetes and tumorigenesisin adult mice through itsmultiple actions on gene expression

When 119886119886 female mice received a phytoestrogen-freediet doped with 50mgkg bw BPA in the weeks prior tomating with 119860vy119886 males and throughout the gestation andlactation time periods coat colour shift towards yellow andobesity were observed in the 119860vy119886 heterozygous offspringThese effects were related to decreased methylation in the9CpG sites within the 119860vy allele Dietary supplementationwith either methyl donors (folic acid betaine vitamin B12and choline) or phytoestrogen prevented hypomethylationresulting in more offspring with brown or mottled browncoat colour and normal weight [51] Although the alteredphenotype of BPA-exposedmice could be transgenerationallytransmitted the CpG methylation pattern was not inheritedin the blastocyst suggesting that other epigenetic mecha-nisms like histone-mediated chromatin alterations might beresponsible for the transgenerational effects in this model

The insulin-like growth factor II receptor (Igf2r) and thepaternally expressed gene 3 (Peg3) are first imprinted infemale mice after birth when follicles and oocytes start to

develop in the ovary Chao and coworkers exposed CD-1mice to low doses of BPA (20 or 40 120583gkg bw) either bydaily hypodermal injections frompostnatal day 7 to postnatalday 14 or by intraperitoneal injections administered eachfifth day between postnatal days 5 and 20 [66] BPA notonly dose-dependently inhibited methylation of Igf2r andPeg3 differentially methylated regions but also lowered thetranscription of DNA methyltransferase Dmnt1 Dmnt3aDmnt3b andDnmt3l genes in the oocytes Since the oestrogenreceptor (ER)may recruit coactivator complexes with histoneacetyltransferase or methyltransferase activities to activatedownstream target genes [67] Chao and coworkers examinedthe expression of ERs and found a significant increase inER120572 mRNA and protein levels at the highest BPA dose ERinhibitor ICI182780 abolished the reduction in Dnmt geneexpression in the ovary of BPA-exposed mice These obser-vations suggested that ER signalling mediated the epigeneticeffects induced by BPA in the oocytes [66]

Further evidence for BPA effects on methylation in dif-ferentially methylated regions of imprinted genes duringoogenesis and early embryogenesis came from [68] Exposureof mice to BPA (10 120583gkg or 10mgkg bw) during late stagesof meiosis and oocyte growth from 2 weeks prior to matinguntil day 95 of gestation resulted in significant alterations inthe expression of imprinted genes Peg3 Snrpn H19Igf2 andKcnq1 in embryonal and placental tissues and affected foetalplacental and postnatal development The higher BPA dosedisrupted the parental specific monoallelic expression of theSnrpn Igf2 and Kcnq1ot1 genes in a tissue-specific mannerand resulted in the biallelic expression of the paternallyexpressed Snrpn gene in the placenta suggesting that mater-nal imprinting of Snrpn was disturbed before fertilizationor that Snrpn was susceptible to loss of imprinting duringearly embryogenesis Expression of the normally repressedmaternal allele of Kcnq1ot1 in the placentas ranged from 123to 723 of total expression whereas no differences werefound between control and BPA-treatedmice on theKcnq1ot1maternal allele expression in the embryo [68] suggestingloss of methylation control in a tissue-specific fashion Thelow BPA dose did not significantly affect imprinted geneexpression except for Snrpn andKcnq1ot1 loci in the placentaTo determine whether the altered expression pattern ofimprinted genes was linked to abnormal DNA methylationDNA methylation was analysed from the placentas andembryos ofmice exposed to the high BPA dose during oocytegrowth and early embryogenesis A small but significant (119875 lt005 through ANOVA) reduction in the mean methylationlevel of the Snrpn imprinting control region was detectedby pyrosequencing furthermore the decrease of methylationwas attributed to the normally hypermethylated maternalallele by bisulfite mutagenesis sequencing which allowsassaying allele-specific methylation levels Pyrosequencinganalysis of the H19Igf2 imprinting control region in BPA-exposed embryos revealed also a slightly but significantlyreduced averagemethylation of the 6 analysed CpG sites (119875 lt0001) Analysis of globalDNAmethylation by LuminometricMethylation Assay (LUMA) in control and BPA-exposedsamples found a significant difference in the placentas butnot in 95-day embryos after exposure to the high dose only


(119875 lt 005) Exposure of females to the high BPA doseonly from day 55 to day 125 of gestation that is outsideof the critical windows of DNA methylation acquisition inthe oocytes and epigenetic reprogramming in embryos didnot significantly affect expression of imprinted genes Asmight be expected from the important role of imprintedgenes in placental development aberrant imprinting inducedby BPA exposure was associated with abnormal placentalphenotypes [68] In conclusion this study revealed that expo-sure to environmentally relevant doses of BPA during crit-ical windows of oocyte development and growth and earlyembryogenesis can perturb expression and methylation ofimprinted genes with the most significant effects observedin the placenta It remains to be established whether loss ofimprinting per se andor disturbance of imprint maintenancewere due to direct effects on the early embryoplacenta orwere preprogrammed in the oocyte prior to conception

Other studies although not specifically focused on oo-cytes support the hypothesis that BPA exposure may affectmethylation of cytosines in DNA of imprinted and nonim-printed genes outside and within coding regions A genome-wide analysis showed that perinatal exposure to 50 120583gkgor 50mgkg BPA in diet induced nonmonotonic dose-dependent alterations of DNA methylation patterns in liverAltered methylation was predominantly found within CpGisland shores and overall several hundred novel BPA-sen-sitive methylation sites were identified involving pathways inmetabolism and stimulus response [69]

In another study it was shown that exposure of pregnantmice throughout gestation to low doses of BPA (20 120583gkgbw) altered the epigenome in the forebrain of the offspringinducing hypomethylation at NotI locus and deregulation ofgene expression [70] Transgenerational changes in behaviourwere also noted in mice upon gestational BPA exposures [71]

An epigenetic impact of BPA was demonstrated also onmale germ cells Male offspring of rats perinatally exposed toBPA had reduced sperm counts and other changes in phe-notypes not only in the first but also in the 1198653 generation[9 10] Induction of sperm epimutations and male-mediatedtransgenerational inheritance of obesity and reproductivedisturbances were also shown after BPA exposure of rats[12 72] When female mice were exposed during gestationand lactation to low BPA doses (40 120583gkg bw) deregulatedglucose homeostasis in the 1198652 generation was observeddecreased global methylation and differential methylation ofa specific CpG site in the glucokinase promoter in the 1198651sperm suggested that the 1198652 phenotype could be caused byepigenetic alterations induced in the male paternal germlineby BPA prenatal exposure [73]

Finally exposure to BPA appears to affect DNA methy-lation also in humans a study in human foetuses found anorgan-specific association between changes of global DNAmethylation and BPA exposure [74] a cross-sectional studyof epigenomic alterations in prepubescent girls from Egyptrevealed that increasing urinary BPA levels were associatedwith changes in methylation in particular reduced methyla-tion in genes involved in immune function metabolism andon the X chromosome [75]

6 From Epigenetic Alterations toChromosome Segregation Errors in OocytesExposed to BPA

An impact of BPA on the oocyte epigenome was confirmedby in vitro experiments using the same preantral follicle cul-ture model in which nonlinear negative effects had beenshown on spindle integrity chromosome congression andmeiotic progression [38] Follicles were chronically exposedin vitro to 3 or 300 nM BPA for 12 days during which theymatured under the influence of follicle stimulating hormoneup to the large antral stage when stimulation of ovulationby recombinant hCG and recombinant EGF caused resump-tion of oocyte maturation and development of oocytes tometaphase II at day 13 of culture [38 76] Follicle survival anddevelopment oocyte growth and maturation rates and chro-mosome alignment on the metaphase II plate were comparedbetween controls and BPA-exposed groups Concomitantlypossible BPA-induced epigenetic alterations at the level ofDNA methylation and posttranslational histone modifica-tions were analysed in single oocytes The specific cultureconditions were shown not to affect the physiological DNAmethylation pattern of maternally imprinted genes in theoocytes [77] Thus the methylation patterns of differentiallymethylated regions in the maternally imprinted Snrpn Igf2rMest genes and in the paternally imprinted H19 gene wereanalysed using limiting dilution bisulfite pyrosequencing[78] A cut-off of at least 50 abnormally methylated CpGsites was established to define epimutations

Changes of posttranslational histone modifications hadbeen previously imputed to BPA [79] and alterations ofH3K9trimethylation in pericentromeric heterochromatin had beenassociated in cultured oocytes to meiotic arrest unalignedchromosomes and spindle defects [80] a phenotype similarto that observed after treatmentwith a lowBPAconcentration[38] Furthermore biallelically different histone posttransla-tional epigenetic marks are functionally relevant for a correctexpression of imprinted genes as supported by the evidencethat Beckwith-Wiedemann syndrome patients exhibit bial-lelic instead ofmonoallelic gene expression and similarmarksfor trimethylated histone H3 lysine 9 (H3K9me3) [81] Basedon these notions relative histone H3K9 trimethylation andH4K12 acetylation were assessed by quantitative confocalmicroscopy of control or BPA-exposed mouse metaphase IIoocytes from preantral follicle cultures

Only the low BPA concentration (3 nM) caused a slightbut significant acceleration of follicular growth Overall 75of all analysed maternally imprinted alleles were abnormallydemethylated in the group exposed to 3 nM BPA a percent-age significantly higher (119875 lt 005) compared to the controland 300 nM BPA group [76]The specific rates of abnormallydemethylated alleles were 167 in Mest 74 in Igf2r and48 in Snrpn alleles No BPA effect was detected on thepaternally imprinted H19 allele Single changes in cytosinemethylation presumably not relevant for gene expressionwere not significantly affected by BPA exposures The obser-vations suggest that low chronic BPA exposure during oocytegrowth can either adversely influence maternal imprinting


DAPI

H3K9me3

DAPI

H3K9me3

In vitro controlIn vitro controlDAPI

H4K12ac

DAPI

H4K12ac

DAPI CREST DAPI CREST

5120583m5120583m

2120583m

(a) (b)

(e)

(f)

(c) (d)

(a998400) (b998400) (c998400) (d998400)

In vitro control

Dnmt

G9a

ATRX

HDAC

Auro

ra k

inas

e

P

Ac

M Methylation

DNAMM M

MM M

CGCGCG

H3S10

PAc

PhosphorylationAcetylation

Histone

H3K

9

Regu

latio

n at

m

atur

atio

n

Epig

enet

ic

cont

rol

3nM BPA 3nM BPA

3nM BPA

iKD128

plusmn03120583

m

iKD122

plusmn03120583

m

Figure 3 Changes in histone posttranslational modifications in low dose BPA-exposedmetaphase II mouse oocytes (a-b1015840) Images of histoneH3K9 trimethylation in control and BPA-exposed oocytes some unaligned chromosomes (b) and reduced trimethylated pericentromericheterochromatin (b1015840) in the BPA group (c-d1015840) Unchanged pattern of histone H4K12 acetylation (e) Decreased distance between centromeresof sister chromatids in metaphase II chromosomes of BPA-exposed oocytes (blue) shown in fixed oocytes that were stained by CRESTautoantibodies for centromeres (red) (f) Model indicating relevance of H3K9 trimethylation for recruitment of Dnmts (right side) andother factors like ATRX (ATP-dependent helicase that belongs to SWISNF family of chromatin remodelling factors) and Aurora kinase thatmight play a role in centromere regulation microtubule attachment and chromosome alignment through phosphorylation of different targetproteins and histone H3S10 (left side) For further explanation see text and [76]

per se or affect imprint stability The particular sensitivity ofthe maternal Mest allele to epimutations by low BPA con-centrations may relate to influences of BPA on bidirectionalsignalling between the oocyte and its surrounding cumulusgranulosa cells by gap junctional communication as altered

methylation of Mest was also detected in mouse oocytes ofconnexin 37 deficient transgenic mice [82]

After exposure to 3 nM BPA congression failures andloosely aligned chromosomes at the metaphase II plate wereobserved (Figures 3(a) and 3(b)) Concomitantly the relative


H3K9me3 fluorescence was significantly lower compared tothe control group (119875 lt 0001) (Figures 3(a1015840) and 3(b1015840))whereas there was no difference in the intensity of fluo-rescence associated with H4K12 acetylation (Figures 3(c1015840)and 3(d1015840)) Interestingly under the same treatment con-ditions the average interkinetochore distance was slightlybut significantly reduced from 128 plusmn 03 120583m to 122 plusmn03 120583m (Figure 3(e)) H3K9 is acetylated in immature Ger-minal Vesicle pig oocytes but becomes deacetylated andtrimethylated during meiosis I and at metaphase II [83]Trimethylated H3K9 is also a hallmark of heterochromatinof mature metaphase II oocytes in the mouse and onlygradually disappears from chromatin after fertilization toincrease again later in preimplantation development [57]One can speculate that the changes in histone methylationof metaphase II chromosomes induced by low chronic BPAtreatment may influence recruitment of DNA methyltrans-ferases and chromatin remodelling proteins like ATRX orAurora kinase that are critical for heterochromatin forma-tion at centromeres (schematically depicted in Figure 3(f))thereby affecting microtubule attachment and chromosomesegregation [76] In other words the changes in histone post-translational modification and DNA methylation detectedin oocytes after BPA exposure could represent the ldquomissinglinkrdquo explaining the effects of low BPA concentrations onchromosome meiotic segregation

7 Conclusions

By now there is increasing evidence that low BPA concentra-tions adversely affect the epigenome of mammalian femalegerm cells with functional consequences on gene expressionand oocyte development and quality There are specific timewindows during which profound chromatin remodellingoccurs and maternal imprints are established or protectedthat appear particularly vulnerable to epigenetic deregulationby BPA these correspond to primordial germ cell formationand oocyte meiotic prophase in the foetus oocyte matura-tion after puberty and early preimplantation developmentafter fertilization Transgenerational effects have also beenobserved in the offspring of BPA-treated rodents althoughthe epigenetic mechanisms of inheritance still need to beclarified Thus BPA exposure might have long-lasting con-sequences on the female reproductive health ranging fromreduced fertility to offspring defects Finally the studies onhistone posttranslational modifications suggest that BPA canalso predispose the oocytes to altered chromosome behaviourin meiosis particularly at low concentrations The relevanceof these findings for human health protection still needs to befully assessed but they warrant further investigation in bothexperimental models and humans

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors are grateful to TomTrapphoff for help with prep-aration of the figures The studies were supported by theGerman Research Foundation (FOR 1041)

References

[1] D J P Barker J G Eriksson T Forsen and C Osmond ldquoFetalorigins of adult disease strength of effects and biological basisrdquoInternational Journal of Epidemiology vol 31 no 6 pp 1235ndash1239 2002

[2] P Bateson D Barker T Clutton-Brock et al ldquoDevelopmentalplasticity andhumanhealthrdquoNature vol 430 no 6998 pp 419ndash421 2004

[3] P D Gluckman and M A Hanson ldquoDevelopmental and epi-genetic pathways to obesity an evolutionary-developmentalperspectiverdquo International Journal of Obesity vol 32 supple-ment 7 pp S62ndashS71 2008

[4] T J Roseboom and E D Watson ldquoThe next generation ofdisease risk are the effects of prenatal nutrition transmittedacross generations Evidence from animal and human studiesrdquoPlacenta vol 33 no 2 pp e40ndashe44 2012

[5] P Iozzo M Holmes M V Schmidt et al ldquoDevelopmentalORIgins of healthy and unhealthy AgeiNg the role of maternalobesitymdashintroduction to DORIANrdquo Obesity Facts vol 7 no 2pp 130ndash151 2014

[6] E J Radford M Ito H Shi et al ldquoIn utero undernourishmentperturbs the adult sperm methylome and intergenerationalmetabolismrdquo Science vol 345 no 6198 Article ID 12559032014

[7] E Diamanti-Kandarakis J-P Bourguignon L C Giudice etal ldquoEndocrine-disrupting chemicals an Endocrine Society sci-entific statementrdquoEndocrine Reviews vol 30 no 4 pp 293ndash3422009

[8] A Baccarelli and V Bollati ldquoEpigenetics and environmentalchemicalsrdquoCurrent Opinion in Pediatrics vol 21 no 2 pp 243ndash251 2009

[9] S Salian T Doshi and G Vanage ldquoPerinatal exposure of rats toBisphenol A affects the fertility of male offspringrdquo Life Sciencesvol 85 no 21-22 pp 742ndash752 2009

[10] S Salian T Doshi and G Vanage ldquoImpairment in proteinexpression profile of testicular steroid receptor coregulators inmale rat offspring perinatally exposed to Bisphenol Ardquo LifeSciences vol 85 no 1-2 pp 11ndash18 2009

[11] V Bollati andA Baccarelli ldquoEnvironmental epigeneticsrdquoHered-ity vol 105 no 1 pp 105ndash112 2010

[12] M Manikkam R Tracey C Guerrero-Bosagna and M KSkinner ldquoPlastics derived endocrine disruptors (BPA DEHPand DBP) induce epigenetic transgenerational inheritance ofobesity reproductive disease and sperm epimutationsrdquo PLoSONE vol 8 no 1 Article ID e55387 2013

[13] J B Matthews K Twomey and T R Zacharewski ldquoIn vitro andin vivo interactions of bisphenol A and its metabolite bisphenolA glucuronide with estrogen receptors 120572 and 120573rdquo ChemicalResearch in Toxicology vol 14 no 2 pp 149ndash157 2001

[14] S H Swan ldquoIntrauterine exposure to diethylstilbestrol long-term effects in humansrdquo Acta Pathologica Microbiologica etImmunologica Scandinavica vol 108 no 12 pp 793ndash804 2000

[15] T G Bredfeldt K L Greathouse S H Safe M-C Hung M TBedford and C L Walker ldquoXenoestrogen-induced regulation


of EZH2 and histone methylation via estrogen receptor signal-ing to PI3KAKTrdquo Molecular Endocrinology vol 24 no 5 pp993ndash1006 2010

[16] K Sato H Fukata Y Kogo J Ohgane K Shiota and C MorildquoNeonatal exposure to diethylstilbestrol alters expression ofDNA methyltransferases and methylation of genomic DNA inthe mouse uterusrdquo Endocrine Journal vol 56 no 1 pp 131ndash1392009

[17] A Careghini A F Mastorgio S Saponaro and E SezennaldquoBisphenol A nonylphenols benzophenones and benzotria-zoles in soils groundwater surface water sediments and fooda reviewrdquo Environmental Science and Pollution Research vol 22no 8 pp 5711ndash5741 2015

[18] GMileva S L Baker A T Konkle andC Bielajew ldquoBisphenol-A epigenetic reprogramming and effects on reproduction andbehaviourrdquo International Journal of Environmental Research andPublic Health vol 11 no 7 pp 7537ndash7561 2014

[19] Y Yang Z Wang T He Y Dai and S Xie ldquoSediment bacte-rial communities associated with anaerobic biodegradation ofbisphenol ArdquoMicrobial Ecology 2014

[20] JMichałowicz ldquoBisphenol amdashsources toxicity and biotransfor-mationrdquo Environmental Toxicology and Pharmacology vol 37no 2 pp 738ndash758 2014

[21] A M Calafat Z Kuklenyik J A Reidy S P Caudill J Ekongand L L Needham ldquoUrinary concentrations of bisphenol A and4-Nonylphenol in a human reference populationrdquo Environmen-tal Health Perspectives vol 113 no 4 pp 391ndash395 2005

[22] J R Rochester ldquoBisphenol A and human health a review of theliteraturerdquo Reproductive Toxicology vol 42 pp 132ndash155 2013

[23] J Peretz L VroomanWA Ricke et al ldquoBisphenolA and repro-ductive health update of experimental and human evidence2007ndash2013rdquo Environmental Health Perspectives vol 122 no 8pp 775ndash786 2014

[24] R R Gerona T JWoodruff C A Dickenson et al ldquoBisphenol-A (BPA) BPA glucuronide and BPA sulfate in midgestationumbilical cord serum in a northern and central California pop-ulationrdquo Environmental Science and Technology vol 47 no 21pp 12477ndash12485 2013

[25] R Machtinger and R Orvieto ldquoBisphenol A oocyte matura-tion implantation and IVF outcome review of animal andhuman datardquo Reproductive BioMedicine Online vol 29 no 4pp 404ndash410 2014

[26] J Mathieu-Denoncourt S J Wallace S R de Solla andV S Langlois ldquoPlasticizer endocrine disruption highlight-ing developmental and reproductive effects in mammals andnon-mammalian aquatic speciesrdquo General and ComparativeEndocrinology 2014

[27] D E Cantonwine R Hauser and J D Meeker ldquoBisphenol Aand human reproductive healthrdquo Expert Review of Obstetricsand Gynecology vol 8 no 4 pp 329ndash335 2013

[28] P Hajkova S Erhardt N Lane et al ldquoEpigenetic reprogram-ming in mouse primordial germ cellsrdquoMechanisms of Develop-ment vol 117 no 1-2 pp 15ndash23 2002

[29] U Eichenlaub-Ritter ldquoMeiosis how to get a good start in liferdquoin Textbook of Human Reproductive Genetics K Sermon and SViville Eds pp 33ndash51 Cambridge University Press 2014

[30] M Susiarjo T J Hassold E Freeman and P A Hunt ldquoBisphe-nol A exposure in utero disrupts early oogenesis in the mouserdquoPLoS genetics vol 3 no 1 article e5 2007

[31] P A Hunt C LawsonM Gieske et al ldquoBisphenol A alters earlyoogenesis and follicle formation in the fetal ovary of the rhesus

monkeyrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 109 no 43 pp 17525ndash17530 2012

[32] M A Brieno-Enrıquez P Robles N Camats-Tarruella et alldquoHumanmeiotic progression and recombination are affected byBisphenol A exposure during in vitro human oocyte develop-mentrdquoHuman Reproduction vol 26 no 10 pp 2807ndash2818 2011

[33] M A Brieno-Enrıquez R Reig-Viader L Cabero et al ldquoGeneexpression is altered after bisphenol A exposure in human fetaloocytes in vitrordquoMolecular Human Reproduction vol 18 no 4Article ID gar074 pp 171ndash183 2012

[34] W Wang K S Hafner and J A Flaws ldquoIn utero bisphenolA exposure disrupts germ cell nest breakdown and reducesfertility with age in the mouserdquo Toxicology and Applied Phar-macology vol 276 no 2 pp 157ndash164 2014

[35] T Zhang L Li X-S Qin et al ldquoDi-(2-ethylhexyl) phthalateand bisphenol A exposure impairs mouse primordial follicleassembly in vitrordquo Environmental and Molecular Mutagenesisvol 55 no 4 pp 343ndash353 2014

[36] J Peretz R K Gupta J Singh I Hernandez-Ochoa and JA Flaws ldquoBisphenol A impairs follicle growth inhibits ster-oidogenesis and downregulates rate-limiting enzymes in theestradiol biosynthesis pathwayrdquo Toxicological Sciences vol 119no 1 pp 209ndash217 2011

[37] J Peretz Z R Craig and J A Flaws ldquoBisphenol a inhibitsfollicle growth and induces atresia in cultured mouse antral fol-licles independently of the genomic estrogenic pathwayrdquo Biol-ogy of Reproduction vol 87 no 3 article 63 Article ID Article63 2012

[38] S Lenie R Cortvrindt U Eichenlaub-Ritter and J SmitzldquoContinuous exposure to bisphenol A during in vitro follic-ular development induces meiotic abnormalitiesrdquo MutationResearchmdashGenetic Toxicology and Environmental Mutagenesisvol 651 no 1-2 pp 71ndash81 2008

[39] I Souter K W Smith I Dimitriadis et al ldquoThe association ofbisphenol-A urinary concentrations with antral follicle countsand other measures of ovarian reserve in women undergoinginfertility treatmentsrdquoReproductive Toxicology vol 42 pp 224ndash231 2013

[40] M S BloomDKim F S vomSaal et al ldquoBisphenol A exposurereduces the estradiol response to gonadotropin stimulationduring in vitro fertilizationrdquo Fertility and Sterility vol 96 no3 pp 672e2ndash677e2 2011

[41] E Mok-Lin S Ehrlich P L Williams et al ldquoUrinary bisphenolA concentrations and ovarian response among women under-going IVFrdquo International Journal of Andrology vol 33 no 2 pp385ndash393 2010

[42] S Ehrlich P L Williams S A Missmer et al ldquoUrinary bisphe-nol A concentrations and early reproductive health outcomesamong women undergoing IVFrdquo Human Reproduction vol 27no 12 pp 3583ndash3592 2012

[43] V Y Fujimoto D Kim F S vom Saal J D Lamb J A Taylorand M S Bloom ldquoSerum unconjugated bisphenol A concen-trations in women may adversely influence oocyte qualityduring in vitro fertilizationrdquo Fertility and Sterility vol 95 no5 pp 1816ndash1819 2011

[44] S Ehrlich P L Williams S A Missmer et al ldquoUrinarybisphenol A concentrations and implantation failure amongwomen undergoing in vitro fertilizationrdquo Environmental HealthPerspectives vol 120 no 7 pp 978ndash983 2012

[45] M Zuccotti V Merico S Cecconi C A Redi and S GaragnaldquoWhat does it take to make a developmentally competent


mammalian eggrdquo Human Reproduction Update vol 17 no 4pp 525ndash540 2011

[46] S-I Kageyama H Liu N Kaneko M Ooga M Nagata andF Aoki ldquoAlterations in epigenetic modifications during oocytegrowth in micerdquo Reproduction vol 133 no 1 pp 85ndash94 2007

[47] P A Hunt K E Koehler M Susiarjo et al ldquoBisphenol Aexposure causes meiotic aneuploidy in the female mouserdquoCurrent Biology vol 13 no 7 pp 546ndash553 2003

[48] U Eichenlaub-Ritter E Vogt S Cukurcam F Sun F Pac-chierotti and J Parry ldquoExposure of mouse oocytes to bisphenolA causes meiotic arrest but not aneuploidyrdquoMutation Researchvol 651 no 1-2 pp 82ndash92 2008

[49] F Pacchierotti R Ranaldi U Eichenlaub-Ritter S Attia andI-D Adler ldquoEvaluation of aneugenic effects of bisphenol A insomatic and germ cells of the mouserdquo Mutation Research vol651 no 1-2 pp 64ndash70 2008

[50] AMuhlhauser M Susiarjo C Rubio et al ldquoBisphenol A effectson the growing mouse oocyte are influenced by dietrdquo Biology ofReproduction vol 80 no 5 pp 1066ndash1071 2009

[51] D C Dolinoy D Huang and R L Jirtle ldquoMaternal nutri-ent supplementation counteracts bisphenol A-induced DNAhypomethylation in early developmentrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 104 no 32 pp 13056ndash13061 2007

[52] A Can O Semiz and O Cinar ldquoBisphenol-A induces cell cycledelay and alters centrosome and spindle microtubular organ-ization in oocytes duringmeiosisrdquoMolecular Human Reproduc-tion vol 11 no 6 pp 389ndash396 2005

[53] R Machtinger C M H Combelles S A Missmer et alldquoBisphenol-A and human oocyte maturation in vitrordquo HumanReproduction vol 28 no 10 pp 2735ndash2745 2013

[54] D Lucifero M R W Mann M S Bartolomei and J MTrasler ldquoGene-specific timing and epigeneticmemory in oocyteimprintingrdquo Human Molecular Genetics vol 13 no 8 pp 839ndash849 2004

[55] H Kobayashi T Sakurai M Imai et al ldquoContribution of intra-genic DNAmethylation inmouse gametic DNAmethylomes toestablishOocyte-specific heritablemarksrdquo PLoSGenetics vol 8no 1 Article ID e1002440 2012

[56] K Shirane H Toh H Kobayashi et al ldquoMouse oocyte methy-lomes at base resolution reveal genome-wide accumulation ofnon-CpG methylation and role of DNA methyltransferasesrdquoPLoS Genetics vol 9 no 4 Article ID e1003439 2013

[57] A Burton and M Torres-Padilla ldquoChromatin dynamics in theregulation of cell fate allocation during early embryogenesisrdquoNature Reviews Molecular Cell Biology vol 15 no 11 pp 723ndash735 2014

[58] D Bourcrsquohis and C Proudhon ldquoSexual dimorphism in parentalimprint ontogeny and contribution to embryonic developmentrdquoMolecular and Cellular Endocrinology vol 282 no 1-2 pp 87ndash94 2008

[59] C W Hanna and G Kelsey ldquoThe specification of imprints inmammalsrdquo Heredity vol 113 no 2 pp 176ndash183 2014

[60] H Judson B E Hayward E Sheridan and D T Bonthron ldquoAglobal disorder of imprinting in the human female germ linerdquoNature vol 416 no 6880 pp 539ndash542 2002

[61] B Horsthemke ldquoMechanisms of imprint dysregulationrdquo TheAmerican Journal of Medical Genetics Part C Seminars inMedical Genetics vol 154 no 3 pp 321ndash328 2010

[62] S-I Tomizawa and H Sasaki ldquoGenomic imprinting and itsrelevance to congenital disease infertility molar pregnancy and

induced pluripotent stem cellrdquo Journal of Human Genetics vol57 no 2 pp 84ndash91 2012

[63] D M J Duhl H Vrieling K A Miller G L Wolff and G SBarsh ldquoNeomorphic agouti mutations in obese yellow micerdquoNature Genetics vol 8 no 1 pp 59ndash65 1994

[64] R AWaterland and R L Jirtle ldquoTransposable elements targetsfor early nutritional effects on epigenetic gene regulationrdquoMolecular and Cellular Biology vol 23 no 15 pp 5293ndash53002003

[65] R L Jirtle ldquoThe Agouti mouse a biosensor for environmentalepigenomics studies investigating the developmental origins ofhealth and diseaserdquoEpigenomics vol 6 no 5 pp 447ndash450 2014

[66] H-H Chao X-F Zhang B Chen et al ldquoBisphenol A exposuremodifies methylation of imprinted genes in mouse oocytes viathe estrogen receptor signaling pathwayrdquo Histochemistry andCell Biology vol 137 no 2 pp 249ndash259 2012

[67] J M Hall and D P McDonnell ldquoCoregulators in nuclear estro-gen receptor action from concept to therapeutic targetingrdquoMolecular Interventions vol 5 no 6 pp 343ndash357 2005

[68] M Susiarjo I Sasson C Mesaros and M S BartolomeildquoBisphenol A exposure disrupts genomic imprinting in themouserdquo PLoS Genetics vol 9 no 4 Article ID e1003401 2013

[69] J H Kim M A Sartor L S Rozek et al ldquoPerinatal bisphenolA exposure promotes dose-dependent alterations of the mousemethylomerdquo BMC Genomics vol 15 no 1 article 30 2014

[70] T Yaoi K Itoh K Nakamura H Ogi Y Fujiwara and SFushiki ldquoGenome-wide analysis of epigenomic alterations infetal mouse forebrain after exposure to low doses of bisphenolArdquo Biochemical and Biophysical Research Communications vol376 no 3 pp 563ndash567 2008

[71] J T Wolstenholme M Edwards S R J Shetty et al ldquoGes-tational exposure to bisphenol a produces transgenerationalchanges in behaviors and gene expressionrdquo Endocrinology vol153 no 8 pp 3828ndash3838 2012

[72] T Doshi C DrsquoSouza and G Vanage ldquoAberrant DNA methyla-tion at Igf2-H19 imprinting control region in spermatozoa uponneonatal exposure to bisphenol A and its association with postimplantation lossrdquoMolecular Biology Reports vol 40 no 8 pp4747ndash4757 2013

[73] G Li H Chang W Xia Z Mao Y Li and S Xu ldquoF0 maternalBPA exposure induced glucose intolerance of F2 generationthrough DNA methylation change in Gckrdquo Toxicology Lettersvol 228 no 3 pp 192ndash199 2014

[74] M S Nahar C Liao K Kannan C Harris and D C DolinoyldquoIn utero bisphenol A concentration metabolism and globalDNAmethylation across matched placenta kidney and liver inthe human fetusrdquo Chemosphere vol 124 pp 54ndash60 2015

[75] J H Kim L S Rozek A S Soliman et al ldquoBisphenol A-associated epigenomic changes in prepubescent girls a cross-sectional study in Gharbiah Egyptrdquo Environmental Health vol12 no 1 article 33 2013

[76] T Trapphoff M Heiligentag N El Hajj T Haaf and UEichenlaub-Ritter ldquoChronic exposure to a low concentration ofbisphenol A during follicle culture affects the epigenetic statusof germinal vesicles and metaphase II oocytesrdquo Fertility andSterility vol 100 no 6 pp 1758e1ndash1767e1 2013

[77] E Anckaert T Adriaenssens S Romero S Dremier and JSmitz ldquoUnaltered imprinting establishment of key imprintedgenes in mouse oocytes after in vitro follicle culture undervariable follicle-stimulating hormone exposurerdquo InternationalJournal of Developmental Biology vol 53 no 4 pp 541ndash5482009


[78] N El Hajj T Trapphoff M Linke et al ldquoLimiting dilutionbisulfite (pyro)sequencing reveals parent-specific methylationpatterns in single early mouse embryos and bovine oocytesrdquoEpigenetics vol 6 no 10 pp 1176ndash1188 2011

[79] L F Doherty J G Bromer Y Zhou T S Aldad andH S TaylorldquoIn utero exposure to diethylstilbestrol (DES) or bisphenol-A(BPA) increases EZH2 expression in the mammary gland anepigenetic mechanism linking endocrine disruptors to breastcancerrdquo Hormones and Cancer vol 1 no 3 pp 146ndash155 2010

[80] E Vogt A Kipp and U Eichenlaub-Ritter ldquoAurora kinase Bepigenetic state of centromeric heterochromatin and chiasmaresolution in oocytesrdquo Reproductive Biomedicine Online vol 19no 3 pp 352ndash368 2009

[81] R Nativio A Sparago Y Ito R Weksberg A Riccio andA Murrell ldquoDisruption of genomic neighbourhood at theimprinted IGF2-H19 locus in Beckwith-Wiedemann syndromeand Silver-Russell syndromerdquo Human Molecular Genetics vol20 no 7 Article ID ddr018 pp 1363ndash1374 2011

[82] MM Denomme C RWhite C Gillio-Meina et al ldquoCompro-mised fertility disrupts Peg1 but not Snrpn and Peg3 imprintedmethylation acquisition in mouse oocytesrdquo Frontiers in Genet-ics vol 3 article 129 Article ID Article 129 2012

[83] T Endo K Naito F Aoki S Kume and H Tojo ldquoChangesin histone modifications during in vitro maturation of porcineoocytesrdquoMolecular Reproduction and Development vol 71 no1 pp 123ndash128 2005

[84] S A Smallwood and G Kelsey ldquoDe novo DNA methylation agerm cell perspectiverdquo Trends in Genetics vol 28 no 1 pp 33ndash42 2012

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


BPA polymer

O

O

(n) (n)

Monomer bisphenol A BPA44998400-(propane-22-diyl)diphenol

HO OH O

CH3

CH3CH3

CH3

Figure 1 BPA monomer and polymer

by interfering in hormonal homeostasis for example byacting as agonist or antagonist in hormone receptor-mediatedsignalling Moreover it has recently been shown that expo-sure to EDCs may induce transgenerational phenotype alter-ations possibly caused by differential DNA methylation ingene promoter regions termed epimutations [8ndash12]

2 Bisphenol A

Monomer bisphenol A (441015840-(propane-22-diyl)diphenol)(BPA) (Figure 1) was developed in 1891 as a synthetic oestro-gen (xenoestrogen) BPA indeed binds to oestrogen receptorsin vivo and in vitro [13] but due to its low oestrogenic activ-ity (asymp103ndash105 less than the natural steroid oestradiol) itwas replaced by diethylstylbestrol (DES) which had muchstronger oestrogenic properties DES is sadly known for theteratogenic and carcinogenic effects observed in the genitalorgans of daughters of women using DES in pregnancy toprevent spontaneous abortion [14] Recently an epigeneticinfluence of DES on the regulation of histone [15] and DNA[16] methyltransferases has been shown that could play a rolein the induction of its reproductive effects The case of DEScould be considered an alarming sentinel of the importanceof epigenetic mechanisms in EDCs adverse effects

Even though BPA was not marketed as a hormonal activesubstance in the last decades it found application as plasti-cizer in the production of polymeric plastics mainly polycar-bonate (71) and epoxy resins (29) [17] For a long timepolymeric BPA was considered harmless as it does not inter-act with steroid receptors (Figure 1) Over the last 50 yearsthe use of BPA-containing polymers in common items suchas plastic bottles toys lining of aluminium cans and pipesdental sealants and thermal receipt paper led to increasingBPA production which reached about 5 million tons in 2010[17] Unfortunately polycarbonate plastics damaged by heatUV harsh alkaline treatment or after vigorous washing wereshown to release monomeric BPA By now it is estimatedthat the worldwide release of BPA into the environment isexceeding one million poundsyear [18]

3 Environmental andHuman BPA Contamination

In the USA average BPA groundwater concentrations rangebetween 00041 and 19mgm3 and up to 20mgm3 BPAwere

measured in some areas of Great Britain [17] BPA can beefficiently biodegraded in water and soil by microorganismsand by photolysis in water at wavelengths above 290 nm [1719 20] However in spite of environmental biodegradation01ndash790 120583gkg BPA were detected in fresh weight (fw) foodandup to 086mgm3 in drinkingwater or commercial drinks[17] biomonitoring studies detected BPA in human serumplasma or urine of over 90 US and Canadian citizens[21] Daily dietary BPA intakes of about 002ndash008120583gkgdayand 022ndash033 120583gkgday have been estimated for adultsand infants respectively (for references see [17]) BPA israpidly metabolised to bisphenol A-glucuronide in liver butunconjugated BPA has been detected in serum and bloodof the general population at concentrations of 44mgL and25mgL respectively and over 50mgL BPA were measuredin workers [18 22] BPA has been also detected in follicularfluid foetal serum and amniotic fluid (average 1-2 ngmL)[23] and in umbilical cord serum of human mid gestationembryos [24]

4 BPA Effects on Mammalian Oogenesis

Although potential adverse effects of low BPA concentrationson reproduction are still a matter of debate most studiessuggest that BPA is an ovarian toxicant and reduces oocytequality in animal models and in humans [23 25] Poten-tial mechanisms of BPA action on hormonal homeostasisinclude binding to nonclassic membrane oestrogen recep-tors (mERs) binding to glucuronide receptor activation ofnuclear oestrogen-related receptor gamma (ERR120574) suppres-sion of thyroid hormone receptor transcription decrease ofcholesterol transport through the mitochondrial membraneincrease of fatty acid oxidation stimulation of prolactinrelease and impairment of aromatase expression (reviewedin [18 25ndash27])

Mammalian oocytes are amongst the most long-livedcells in the body Primordial germ cells start differentiatingalready in the early postimplantation embryo aftermigrationto the genital ridges (Figure 2) [28] Nests of primary oocytesentering meiosis I are formed in the human ovary by the3rd month of pregnancy Pairing and recombination betweenhomologous chromosomes take place in the foetal ovarybefore birth Around the 7th month of pregnancy oocytesfinally develop to the late dictyotene stage when they becomemeiotically arrested By that time the synaptonemal com-plexes have disappeared and the homologous chromosomes



Low

CpG

met

hyla

tion


PassiveActive

Dnmt1Dnmt3a

Dnmt3l

AID

Tet3ELP3AID

Fertilization

Dnmt1Dnmt3a

Dnmt3b

Imprinted genes


Zfp57Kap1Stella


Hig

h

Dnmt1minus

Impr

inted

gene

s(m

atern

al)

Non

impr

inte

d ge

nes

Global

meth

ylatio

n 5 998400mCrarr

5 998400hmCrarr

5 998400C

5 998400mCrarr5 998400C



































DAPI

H3K9me3

DAPI

H3K9me3


H4K12ac

DAPI

H4K12ac


5120583m5120583m

2120583m

(a) (b)

(e)

(f)

(c) (d)

(a998400) (b998400) (c998400) (d998400)

In vitro control

Dnmt

G9a

ATRX

HDAC

Auro

ra k

inas

e

P

Ac

M Methylation

DNAMM M

MM M

CGCGCG

H3S10

PAc


Histone

H3K

9

Regu

latio

n at

m

atur

atio

n

Epig

enet

ic

cont

rol

3nM BPA 3nM BPA

3nM BPA

iKD128

plusmn03120583

m

iKD122

plusmn03120583

m







7 Conclusions




Acknowledgments


References
































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



Low

CpG

met

hyla

tion


PassiveActive

Dnmt1Dnmt3a

Dnmt3l

AID

Tet3ELP3AID

Fertilization

Dnmt1Dnmt3a

Dnmt3b

Imprinted genes


Zfp57Kap1Stella


Hig

h

Dnmt1minus

Impr

inted

gene

s(m

atern

al)

Non

impr

inte

d ge

nes

Global

meth

ylatio

n 5 998400mCrarr

5 998400hmCrarr

5 998400C

5 998400mCrarr5 998400C



































DAPI

H3K9me3

DAPI

H3K9me3


H4K12ac

DAPI

H4K12ac


5120583m5120583m

2120583m

(a) (b)

(e)

(f)

(c) (d)

(a998400) (b998400) (c998400) (d998400)

In vitro control

Dnmt

G9a

ATRX

HDAC

Auro

ra k

inas

e

P

Ac

M Methylation

DNAMM M

MM M

CGCGCG

H3S10

PAc


Histone

H3K

9

Regu

latio

n at

m

atur

atio

n

Epig

enet

ic

cont

rol

3nM BPA 3nM BPA

3nM BPA

iKD128

plusmn03120583

m

iKD122

plusmn03120583

m







7 Conclusions




Acknowledgments


References
































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





























DAPI

H3K9me3

DAPI

H3K9me3


H4K12ac

DAPI

H4K12ac


5120583m5120583m

2120583m

(a) (b)

(e)

(f)

(c) (d)

(a998400) (b998400) (c998400) (d998400)

In vitro control

Dnmt

G9a

ATRX

HDAC

Auro

ra k

inas

e

P

Ac

M Methylation

DNAMM M

MM M

CGCGCG

H3S10

PAc


Histone

H3K

9

Regu

latio

n at

m

atur

atio

n

Epig

enet

ic

cont

rol

3nM BPA 3nM BPA

3nM BPA

iKD128

plusmn03120583

m

iKD122

plusmn03120583

m







7 Conclusions




Acknowledgments


References
































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



7 Conclusions




Acknowledgments


References
































































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

















































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

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