Symposium: Omics Research in Neurodevelopment neup_1287 447..457

Bisphenol A, an endocrine-disrupting chemical,and brain development

Kyoko Itoh, Takeshi Yaoi and Shinji Fushiki

Department of Pathology & Applied Neurobiology, Graduate School of Medical Science, Kyoto Prefectural University ofMedicine, Kyoto, Japan

Bisphenol A (BPA) is an endocrine-disrupting chemical,widely used in various industries and the field of dentistry.The consequent increase in BPA exposure among humanshas led us to some concerns regarding the potential delete-rious effects on reproduction and brain development. Theemphasis of this review is on the effects of prenatal andlactational exposure to low doses of BPA on brain develop-ment in mice. We demonstrated that prenatal exposure toBPA affected fetal murine neocortical development byaccelerating neuronal differentiation/migration during theearly embryonic stage, which was associated with up- anddown-regulation of the genes critical for brain development,including the basic helix-loop-helix transcription factors. Inthe adult mice brains, both abnormal neocortical architec-ture and abnormal corticothalamic projections persisted inthe group exposed to the BPA. Functionally, BPA exposuredisturbed murine behavior, accompanied with a disruptedneurotransmitter system, including monoamines, in thepostnatal development period and in adult mice. Wealso demonstrated that epigenetic alterations in promoter-associated CpG islands might underlie some of the effectson brain development after exposure to BPA.

Key words: behavior, Bisphenol A, brain development,DNA methylation, monoamine.

INTRODUCTION

An endocrine-disrupting chemical, bisphenol A (BPA; 2,2-bis (4-hydroxy-phenyl) propane) is produced in highvolumes and is the base chemical (monomer) used to makepolycarbonate plastic food and beverage containers, the

resin lining of cans, and dental sealants.1–5 It is also found in“carbonless” paper used for receipts, as well as a widerange of other common household products, such ascompact disks and flooring.6 BPA can migrate from con-sumer goods into food, and a recent risk assessment sug-gests that canned vegetables contribute 10–40% of thedaily BPA intake, whereas canned fruits contribute 3–6%.7

The widespread contamination of BPA in the environment,and consequent BPA exposure among humans, have led usto examine the potentially deleterious effects of this com-pound. It was recently reported that BPA levels did notdecline rapidly with fasting time in urine samples obtainedduring the National Health and Nutrition ExaminationSurvey (NHANES), which suggested substantial nonfoodexposure, and accumulation in body tissues such as fat, orboth.8,9 The geometric mean urinary concentration of BPA(30.3 mg/L) among premature infants undergoing intensivetherapeutic medical interventions was reported to be oneorder of magnitude higher than that among the generalpopulation.10 Studies are urgently needed to determine theenvironmental source of BPA and the effects of exposureto BPA on premature infants, especially on reproductiveand endocrine organs, as well as brain development duringthe prenatal period.

Here we demonstrated that, in mice,prenatal exposure tolow doses of BPA affected brain development, and includedneocortical histogenesis, spatiotemporal gene expressions,the behavior and neurotransmitter systems in adult mice,and epigenetic modifications in fetal mice brains.

PRENATAL EXPOSURE TO LOW DOSESOF BPA PERTURBED MURINE

NEOCORTICAL HISTOGENESIS IN FETALAND ADULT MICE BRAINS

Morphological analyses and gene expression offetal mice brains (Figs 1,2)

We studied whether prenatal exposure to low doses of BPAaffected the morphology and expression of some genes

Correspondence: Kyoko Itoh, MD, PhD, Department of Pathology &Applied Neurobiology, Graduate School of Medical Science, KyotoPrefectural University of Medicine, Kawaramachi Hirokoji, Kajii-cho465, Kamigyo-ku, Kyoto 602-8566, Japan. Email: kxi14@koto.kpu-m.ac.jp

Received 12 November 2011; revised and accepted 1 December2011; published online 12 January 2012.

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Neuropathology 2012; 32, 447–457 doi:10.1111/j.1440-1789.2011.01287.x

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related to brain development in the murine fetal neocor-tex.11 Low doses of BPA, 20 mg/kg of body weight orvehicle, were injected subcutaneously into pregnant mice(ICR/Jcl strain) daily from embryonic day (E)0.5. In orderto evaluate cell proliferation, neuronal differentiation andmigration, 5-bromo-2-deoxyuridine (BrdU) was injectedintraperitoneally into pregnant mice employing variousregimens and the brains were processed for immunohis-tochemistry.The BrdU-labeled cells examined 1 h after theBrdU injection showed no significant differences betweenthe BPA-exposed and control groups, which indicated thatthe proliferation of precursor cells was not affected. TheBrdU-labeled cells, analyzed 2 days after the BrdU injec-tion, were decreased in the ventricular zone at E14.5 andE16.5, whereas they were increased in the cortical plate atE14.5 in BPA-exposed mice, compared with the controlgroup, which indicated that differentiation and the migra-tion of neurons were accelerated in BPA-exposed mice(Fig. 1).

The expression levels of several genes that play criticalroles during brain development were measured using totalRNA extracted from the embryonic telencephalon atvarious embryonic stages. The expression of activator typeof basic helix-loop-helix (bHLH) genes, including Math3and Ngn2, the repressor type of bHLH, Hes1, and thyroidhormone-related genes such as Thra and L1cam, was sig-nificantly up-regulated at E14.5 in the BPA-exposedgroup, which appeared to correlate with the morphologicalchanges detected at E14.5 in the BPA-exposed group(Fig. 2). In addition, L1cam mRNA was up-regulated in theBPA-exposed group. L1cam plays an important role in cellmigration and axon growth, as well as guidance, duringbrain development,12 and it was reported to be down-regulated by thyroid hormone in the cerebral cortex.13

Taken into account the report by Zoeller et al.14 that devel-opmental exposure to BPA in rats produced an endocrineprofile similar to that observed in thyroid resistance syn-drome, our L1cam data also supported the supposition that

Fig. 1 (A) Immunohistochemistry for5-bromo-2-deoxyuridine (BrdU) in thecontrol (a,b,c) and bisphenol A (BPA)-exposed (d,e,f) groups using fetal telencepha-lon 2 days after BrdU injection at E14.5 (a,d),E16.5 (b,e), and E18.5 (c,f). BrdU-labeledcells were abundant in the cortical plate inthe BPA-exposed group at embryonic day(E)14.5 (a,d). VZ, ventricular zone; IZ,intermediate zone; CP, cortical plate. (B)Migration/differentiation analysis. The rela-tive positions of BrdU-labeled cells in themurine neocortex at 2 or 3 days after BrdUinjection. The percentage of BrdU-labeledcells was significantly decreased in the ven-tricular zone at E14.5 and E16.5, whereas itwas increased in the cortical plate at E14.5, 2days after BrdU labeling in the BPA-treatedgroup, compared with the control group.Error bars show the standard errors of themeans. PPL, primordial plexiform layer.*P < 0.05, **P < 0.01. Cited from Nakamuraet al.11

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BPA might act antagonistically against thyroid hormonesduring brain development. Hes1 was identified as a puta-tive thyroid hormone-responsive gene in the fetal cortex.15

Taken together, these results indicated that prenatal expo-sure to low doses of BPA affected murine neocorticaldevelopment by accelerating neuronal differentiation/migration with aberrant gene expression, mainly throughdisrupting thyroid hormone function. It is worthy of notehere that thyroid hormone appears to control fate specifi-cation15 and migration16,17 of neurons in the cerebral cortexbut does not appear to affect their proliferation.18

Morphological analyses of adult brain (Fig. 3)

In a previous study, we investigated whether or not such aperturbed neocortical histogenesis results in changes ofcortical cytoarchitecture and neural connections in adultmice brains.19 The BrdU-positive cells labeled at E14.5were significantly increased in the Vth and VIth corticallayers of BPA-exposed mice when examined at postnatal 3weeks (P3W), whereas they were confined to the IVthlayer in the control mice. These differences disappeared atP12W.

It is recognized that neuronal positioning is a prerequi-site for proper neuronal circuit formation. The thalamo-cortical projections demonstrated by DiI-labeling wereabnormal at P3W and P12W in BPA-exposed mice (Fig. 3).These abnormal trajectories may reflect malpositioning ofneurons, although the abnormal pattern persisted evenafter malpositioned neurons disappeared. Thalamic fibersare known to enter the intermediate zone without reaching

the subplate at E14.5, and they start to invade the lowerportion of the cortical plate at E16.5.20–22 It is tempting tospeculate that BPA affects both the arrival time of fiberscoming from the thalamus and the positioning of corticalneurons, resulting in abnormal connections between thethalamus and cortex.

We then focused on the development of representativethalamocortical connections, that is, cortical barrel forma-tion. Specifically, we analyzed whether BPA exposureaffected the formation of the cortical barrel, the barreloidof the thalamus and the barrelette of the brainstem, interms of the histology and the expression of genes involvedin the barrel development.23 The barrel, barreloid and bar-relette of the adult mice were examined by cytochrome Coxidase (COX) staining. It turned out that there were nosignificant differences in the total and septal areas andthe patterning of the posterior medial barrel subfield(PMBSF), barreloid and barrelette, between the BPA-exposed and control groups in the adult mice. When weexamined the development at postnatal day 1 (PD1), PD4and PD8, we demonstrated that the cortical barrel vaguelyappeared at PD4 and completely formed at PD8 in bothgroups. In comparison, the expression levels of S100b,Slc6a4, Htr1b, Maoa, Scla3, and Gap43 in the cortex, thala-mus and pons at PD1, PD4 and at PD8 were spatiotempo-rally altered depending on the sex and the treatment.Theseresults suggest that the trigeminal projection and the tha-lamic relay to the cortical barrel were spared after prenataland lactational exposure to low doses of BPA, althoughprenatal exposure to BPA was shown to disrupt the corti-cothalamic projection.

Fig. 2 The temporal expression levels ofMash1, Math3, Ngn2, Hes1, Hes5, L1cam,Thra and Thrb, in the fetal forebrain. TotalRNA was extracted from telencephalon andthe expression levels of the target transcriptsand glyceraldehyde-3-phosphate dehydroge-nase (Gapdh) were measured by real timeRT-PCR. The expression level of the targettranscripts in each sample was normalizedwith that of Gapdh. *P < 0.05, **P < 0.01:each level in BPA-exposed mice was com-pared with that in control mice at the sameage. Cited from Nakamura et al.11

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PRENATAL AND EARLY POSTNATAL BPAALTERED THE MOLECULAR PROFILES

OF THE SUBPLATE NEURONS, WITHSPECIAL REFERENCES TO

CORTICOGENESIS

We then analyzed the effects of prenatal exposure to BPAon the functional state of the subplate neurons, becausethey are supposed to play an important role in corticalhistogenesis and the formation of thalamocortical tract.24,25

By using laser capture microdissection, we were selectivelyable to dissect the subplate neuron-enriched area fromfetal mice brains, followed by extraction of RNA and theanalysis of gene profiles with a cDNA microarray. Microar-ray analyses revealed that 4896 probes with uniquesequences out of a total of 45 101 probes showed up- ordown-regulation in the BPA-exposed group, compared tothe control group. After confirmation of the gene expres-sion levels by real time RT-PCR, we found 36 genesshowing more than two-fold up- or down-regulation in the

BPA-exposed group. It turned out that those genes/geneproducts were closely related with each other in terms ofthe molecular characteristics, including promotor binding,protein-modification or direct regulation (manuscript inpreparation). We plan to elucidate the functional role ofthose gene/gene products that cause the outcome of BPAexposure observed in fetal as well as adult mice brains.

PRENATAL AND EARLY POSTNATALBPA EXPOSURE ALTERED THENEUROTRANSMITTER SYSTEM,

RESULTING IN BEHAVIORALCHANGES IN MICE

Behavioral analyses (Fig. 4)

We performed behavioral analyses in order to investigatewhether prenatal and lactational BPA exposure affectedbehavior in adult mice.26 Pregnant mice were injected sub-

Fig. 3 DiI-labeling in the somatosensorycortex at postnatal week 3 (P3W) (A, C)and P12W (B, D) in control mice (A, B) andbisphenol A (BPA)-exposed mice (C, D).Retrogradely-labeled cortical neurons werebroadly distributed at P3W and P12W andshowed no convergence in the IVth layerat P12W in BPA-exposed mice. Scalebar = 100 mm (A–D). I, II/III, IV,V,VI denotethe layer of neocortex. Cited from Nakamuraet al.19

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cutaneously with 20 mg/kg of BPA daily from E0 until P3W.Behavioral tests, including an open-field test, an elevatedplus maze, and a Morris water maze test, were conducted atP3W and P10–15W. The total distance in the elevated plusmaze test at P3W and in the open-field test at P10W wassignificantly decreased in the BPA-exposed group, com-pared with the control group (Fig. 4). No significant differ-ences in other batteries were observed between the sex orbetween the groups. As shown by the Morris water mazetest, mice in both of the groups showed a decrease in thetrial duration (escape latency) with days, and no significantdifference was shown between the groups over the 5 daysof training. These results suggested that the motor activitywas suppressed in the BPA-exposed group, althoughthe motivation to explore, anxiety, and spatial learningmemory seemed to be unaffected in the protocol that weemployed. In previous studies, perinatal BPA-exposurechanged the motivation to explore, anxiety levels,27–31 andspatial learning/memory.29–31 Although the mechanismsof the behavioral changes in BPA-exposed mice remainunknown, it is tempting to speculate that low doses of BPA

exposure might underlie the recent increase in the numberof children with neurobehavioral disorders.

Quantitative analyses of monoamines inthe brain (Figs 5,6)

The mice employed in this study were processed after fin-ishing the behavioral tests mentioned above. The freshbrains were dissected into six regions for biochemicalassays at P3W and P10–15W. The concentration of theneurotransmitters was determined by high-performanceliquid chromatography. The levels of dopamine (DA)and its metabolite (DOPAC) significantly increased inthe caudate/putamen (Fig. 5) and dorsal raphe nucleus(Fig. 6), whereas serotonin (5HT) and its metabolite(5HIAA) increased in the caudate/putamen (Fig. 5),dorsal raphe nucleus (Fig. 6), thalamus and substantianigra in the BPA-exposed group at both P3W and/orP14–15W. These results suggested that prenatal and lac-tational BPA-exposure might perturb the monoaminesystem, and also that the effects lasted even in adult

Fig. 4 The total distance in the elevatedplus maze test at postnatal week 3 (P3W) andin the open-field test at P10W was signifi-cantly decreased in the bisphenol A (BPA)-exposed group, compared with the controlgroup. *P < 0.05 (2-way analysis of variance).

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mice.32 It could be argued that the alterations in monoam-ine concentrations in the brain may reflect the alterationsin response to behavioral tasks.

It was previously reported that BPA exposure affecteddopaminergic systems both in vivo and in vitro. Tyrosinehydroxylase-immunoreactive neurons in the substantianigra were significantly decreased in female offspring fol-lowing maternal as well as early postnatal exposure toBPA.33 Perinatal exposure to BPA disrupted DA receptorexpression and/or function.34–38 It may thus be concludedfrom these observations that BPA disrupts the monoam-ine concentration in various brain areas through differentmechanisms, either separately or in combination, includ-ing the ER-mediated mechanism, the interactions of DAreceptor and DA release, and the reduction of monoam-ine neurons. The mechanism by which BPA affects thedevelopment of serotonergic systems has not been thor-oughly investigated. Kawai et al. reported that 5HT-immunoreactive neurons and the immunoreactivityof the serotonin transporter (SERT) in the dorsal raphenucleus showed a tendency to increase in mice exposedto BPA compared to control mice at P9W and P13W;39

however, the underlying mechanisms remain to beclarified.

GENOME-WIDE ANALYSIS OFEPIGENOMIC ALTERATIONS IN FETAL

MOUSE FOREBRAIN AFTER EXPOSURETO LOW DOSES OF BPA40 (FIGS 7–9)

During vertebrate development, it has been postulatedthat dynamic changes in DNA methylation status are ableto regulate tissue- and cell type-specific gene expres-sion.41,42 Methylation of the cytosine residue at the CpG

dinucleotide sites is one of the targets of epigenetic modi-fications. Hypermethylation in promoter-associated CpGisland (CGI) often suppresses gene expression via chroma-tin remodeling.43 The restriction landmark genomic scan-ning (RLGS) method made it possible to demonstratemethylation patterns of NotI loci,44 and most of NotI sitesin the genome should correspond to gene loci, especiallyCGIs. An important role of the epigenetic states hasrecently been demonstrated in neural progenitor cellsduring neurogenesis.45 We therefore hypothesized that pre-natal exposure to BPA may affect the epigenome in devel-oping mouse forebrain as well, and the exposure may thuschange the transcription level of the genes.

In order to address this issue we attempted to studygenome-wide alterations by RLGS with the landmarkrestriction enzyme NotI. The genomic DNAs were pre-pared from the forebrain of BPA-exposed mice andvehicle-exposed control mice, at three developmentalstages (E12.5, E14.5, E16.5). We surveyed the methylationstatus of 2500 NotI loci (spots) that were distinguishablefrom each other. The signal intensity of a total of 55 spots(2.2%) changed between the control group and the BPA-exposed group. Out of these 55 spots, 19 spots (0.8%), 13spots (0.5%) and seven (0.3%) were induced specificallyby BPA exposures at E12.5, E14.5 and E16.5, respectively(Fig. 7). The comparison between the profiles of thecontrol group at different developmental stages revealedthat a total of 75 spots were developmental stage-dependent changed spots. Thirty-seven spots out of the 55(67%) induced by the exposure to BPA were coincidentwith developmental stage-dependent changed spots.Cloning of a part of the target loci in each group at E12.5was undertaken and it turned out that 13 spots were asso-ciated with gene areas located on different chromosomes.

Fig. 6 The levels of dopamine (DA), seroto-nin (5HT) and its metabolite (5HIAA) sig-nificantly increased in the dorsal raphenucleus. **P < 0.01, ***P < 0.001.

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We tested the methylation status of these 13 NotI lociusing methylation-sensitive quantitative PCR, and con-firmed that changes in the methylation status of theseNotI sites represent intensity changes in each correspond-ing spot. Moreover, the 12 out of 13 NotI sites werelocated within the CGI adjacent to the 50 transcriptionalend of a functional gene defined as the promoter-associated CGI (Fig. 8). These results suggest that BPAis able to alter the genome-wide methylation status ofCGIs that are associated with gene areas, and that suchchanges include not only hypermethylation, but alsohypomethylation.

We examined whether or not the mRNA expressionlevels of the genes identified in the changed spots wereaffected using quantitative RT-PCR. We selected twogenes corresponding to spots, spot 12B6; Vps52, andspot 12B9; LOC72325, respectively (Fig. 9). Both spotsshowed similar behavior; at E12.5 in the control group,the intensity of both spots was less than that of a spotfrom a haploid genomic fragment; however, at E12.5in the BPA group, their intensity was similar to a spotfrom a haploid genomic fragment. At E14.5 in bothgroups, the signal intensity was equal to the one of thesurrounding spots from diploid genomic fragments. Asshown in Figure 9, the mRNA expression of Vps52and LOC72325 was significantly up-regulated 2-fold byBPA exposure at E12.5. However, at E14.5 both wereup-regulated in BPA-independent and developmentalstage-dependent fashion. Taken together, these resultssuggest that the epigenetic alterations in promoter-associated CGIs after exposure to BPA may underliesome of the effects on brain development, although nodirect association between epigenetic alterations andmorphological changes in the developing brain has yetbeen clarified.40

CONCLUDING REMARKS

Our studies using mice disclosed that prenatal andlactational exposure to low doses of BPA affected fetalcortical histogenesis, followed by abnormal corticalcytoarchitecture in adult mice, as well as aberrantthalamocortical tract with preserved cortical barrelformation, hypoactive behavior, derangement in themonoamine system and epigenomic alterations. Despiteprevious intensive studies on the effects of BPA on thedeveloping brain, the mechanisms by which BPA affectsbrain development still remain to be clarified. We areinterested in studying how epigenetic alterations inpromoter-associated CGIs after exposure to BPA maycontribute to the phenotypic features observed in ourmouse model. An omics study may potentially offer a newperspective, not only for elucidating the molecularmechanisms of BPA exposure in the developing brain,but also for the discovery of biomarkers that might beapplicable for surveillance in humans.

ACKNOWLEDGMENTS

The authors are grateful to many graduate students andresearchers for their great contributions to this study. Thisstudy was supported in part by a Grant-in-Aid from theMinistry of Education, Culture, Sports, Science and Tech-nology, Japan (15390334, 20310036; SF).

Control BPA

Fig. 7 Restriction landmark genomic scanning with the land-mark restriction enzyme NotI. The signal intensity of a total of 55spots (2.2%) changed between the control group and the bisphe-nol A (BPA)-exposed group. Out of the 55 spots, 19 spots (0.8%),13 spots (0.5%) and 7(0.3%) were induced specifically byBPA exposures at embryonic day (E)12.5, E14.5 and E16.5,respectively.

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Fig. 8 Confirmation of methylation status and cloning of the target loci at embryonic day (E)12.5. We determined the base sequences ofsix hypomethylated (1–6) and six hypermethylated (7–12) spots.The 4-kb sequence around each NotI site was retrieved from the GenBankdatabase. By using methylation-sensitive quantitative PCR, the methylation status (hypomethylation or hypermethylation) of the 12 NotIloci represented the intensity changes of each corresponding spot. It turned out that all 12 spots were associated with gene areas locatedon different chromosomes and each of the 12 NotI sites located within the CGI adjacent to the 50 transcriptional end of a functional gene.Cited from Yaoi et al.40

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Fig. 9 The mRNA expression of Vps52 andLOC72325 coincided with the methylationstates of NotI site associated with a CGI. Thespot for each gene is indicated by an arrow ina magnified image of a restriction landmarkgenomic scanning profile. Relative levelsof mRNA expression normalized to GapdhmRNA expression level were comparedusing one-way analysis of variance (P < 0.01),followed by Scheffe’s multiple comparisontest. In the graph, significant differences incomparison with the control group at embry-onic day (E)12.5 alone are indicated by**(P < 0.05). Cited from Yaoi et al.40

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