Bisphenol A exposure disrupts metabolic health across multiple generations in the mouse

Bisphenol A exposure disrupts metabolic healthacross multiple generations in the mouse

Martha Susiarjo1,2,3, Frances Xin1,2,3, Amita Bansal3,4, Martha Stefaniak1,Changhong Li5, Rebecca A. Simmons*2,3,4, and Marisa S. Bartolomei*1,2,3

1Department of Cell and Developmental Biology, University of Pennsylvania Perelman School ofMedicine, 9–123 Smilow Center for Translational Research, Philadelphia, PA 19104; 2Center ofExcellence in Environmental Toxicology, University of Pennsylvania Perelman School of Medicine, 1316Biomedical Research Bldg II/III, Philadelphia, PA 19104; 3Center for Research on Reproduction andWomen’s Health, Perelman School of Medicine, University of Pennsylvania, 1341 BRB II/III, Philadelphia,PA 19104; 4Division of Neonatology, Department of Pediatrics, Perelman School of Medicine, Universityof Pennsylvania, 1308 BRB II/III, Philadelphia, PA 19104; 5Division of Endocrinology and Metabolism, TheChildren’s Hospital of Philadelphia, 802B Abramson Research Center, Philadelphia, PA 19104

Accumulating evidence has suggested that suboptimal early life environment produces multigen-erational developmental defects. A proposed mechanism is stable inheritance of DNA methylation.Here we show that maternal bisphenol A (BPA) exposure in C57BL/6 mice produces multigenera-tional metabolic phenotypes in their offspring. Using various methods including dual energy X-rayabsorptiometry analyses, glucose tolerance tests, and perifusion islet studies, we showed thatexposure to 10 �g/kg/day and 10 mg/kg/day of BPA in pregnant F0 mice was associated with higherbody fat and perturbed glucose homeostasis in F1 and F2 male offspring, but not female offspring.To provide insight into the mechanism of the multigenerational metabolic abnormalities, weinvestigated maternal metabolic milieu and inheritance of DNA methylation across generations.We showed that maternal glucose homeostasis during pregnancy was altered in the F0 but not F1female mice. The results suggested that compromised maternal metabolic milieu may play a rolein the health of the F1 offspring, but cannot account for all of the observed multigenerationalphenotypes. We further demonstrated that the metabolic phenotypes in the F1 and F2 BPA maleoffspring were linked to fetal overexpression of the imprinted Igf2 gene and increased DNAmethylation at the Igf2 differentially methylated region 1 (DMR1). Studies in H19�3.8-/� mousemutants supported the role of fetal Igf2 overexpression in altered adult glucose homeostasis. Weconclude that early life BPA exposure at representative human exposure levels can perturb met-abolic health across multiple generations in the mouse through stable inheritance of DNA meth-ylation changes at the Igf2 locus.

Epidemiological and animal studies have demonstratedthat early life environment plays a critical role in adult

metabolic health (1). Accumulating evidence has furthersuggested that the impacts of the early life environmentcan be transmitted across multiple generations. A pro-posed mechanism is epigenetic inheritance, possiblythrough stable changes in DNA methylation (2–5). Nev-ertheless, key components of the developmental origins ofhealth and disease concept remain to be addressed, in-

cluding the identification of developmentally relevant locithat are vulnerable to environmental perturbations, andevidence that early environment can alter DNA methyl-ation profiles of these loci across multiple generations.

We previously reported that endocrine-disruptingchemical (EDC) exposure perturbs imprinted gene regu-lation (6). Imprinted genes harbor epigenetic modifica-tions, including differential DNA methylation at imprint-ing control regions (ICRs), which results in parental-allele

ISSN Print 0013-7227 ISSN Online 1945-7170Printed in U.S.A.Copyright © 2015 by the Endocrine SocietyReceived December 22, 2014. Accepted March 17, 2015.

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specific gene expression. Consistent with reports that ex-posure to the EDC bisphenol A (BPA) is associated withmodification of DNA methylation in rodents (7), weshowed that maternal BPA exposure at levels representa-tive of human exposure (10 �g/kg/d or 10 mg/kg/d) infemale (F0) mice prior to and during pregnancy alteredDNA methylation of ICRs in placentas and embryos frommidgestation mouse conceptuses (6). In utero BPA expo-sure in these offspring was associated with loss of imprint-ing (LOI) of several imprinted genes on chromosome 7 (6).Because some of aberrantly expressed imprinted genes areimplicated in growth and/or glucose homeostasis, includ-ing insulin-like growth factor 2 (Igf2) and cyclin-depen-dent kinase inhibitor 1c (Cdkn1c), we postulated thatBPA-induced altered imprinted gene expression disruptsgrowth potential and the future metabolic health of the F1offspring. Moreover, BPA-induced DNA methylationchanges at imprinted loci can be inherited stably acrossgenerations, potentially leading to multigenerationaltransmission of altered phenotypes. Furthermore, thegerm cells of the F1 progeny are developing at the timewhen in utero BPA exposure occurs, and exposure canreprogram the germ cell epigenome. Because these germcells represent the future F2 offspring, we additionallyhypothesized that health of the subsequent generationmay be affected.

In the current study, we tested the hypothesis that earlydevelopmental BPA exposure alters growth and metabolicphenotypes in the F1 and F2 offspring. Dual energy X-rayabsorptiometry (DEXA) analyses, glucose tolerance tests(GTT), and insulin secretion studies revealed that F1 andF2 adult male offspring from BPA exposure groups werefatter, more glucose intolerant, and had altered insulinsecretion relative to controls. These effects were highly sexspecific because the females were unaffected. Further anal-ysis demonstrated that F2 embryos inherited a subset ofthe imprinting features of the F1 embryos, including over-expression of the Igf2 gene and elevated DNA methylationat the Igf2 differentially methylated region 1 (DMR1). Totest whether Igf2 contributed to the perturbed glucose ho-meostasis in the F1 and F2 BPA mice, we studied the met-abolic phenotype of another mouse model, the H19�3.8-/�,which also exhibits increased Igf2 expression. Our datasuggest that Igf2 overexpression during early develop-ment is responsible, at least in part, for abnormal glucosehomeostasis later in life.

Materials and Methods

Mouse informationSix week old virgin C57BL/6 females were purchased from

Jackson Laboratory and assigned to the following diets: (a) mod-

ified AIN 93G diet (TD 95 092 with 7% corn oil substituted for7% soybean oil; Harlan Teklad) as “control”; (b) modified AIN93G diet supplemented with 50 �g/kg BPA (TD 110 337; HarlanTeklad) as “lower dose”; or (c) modified AIN 93G supplementedwith 50 mg/kg BPA (TD 06 156; Harlan Teklad) as “upperdose”. Teklad Diets (Harlan Laboratories Inc, Madison, WI)provided all ingredients except BPA (�99% in purity; Sigma-Aldrich, St. Louis, MO). Based on the average body weight (bw)of an adult mouse (25 g) and daily food consumption (5 g) weestimated exposure levels per mouse to be 10 �g/kg bw/d (lowerdose) and 10 mg/kg bw/d (upper dose). The doses were selectedbased on our previous study demonstrating effects of exposure atthese doses on DNA methylation and RNA expression at im-printed loci (6). Unconjugated BPA in serum from pregnant micemeasured in this previous study indicated levels representative ofhuman exposure (6). The lower dose represented safe humanexposure level (at or below 50 �g/kg bw/d) (8). After two weeksof treatment, females were time-mated to C57BL/6 males and theday a plug was detected was assigned as E0.5. Pregnant femaleswere designated as the “F0”. At E16.5-E17.5, a subset of thepregnant F0 mice was included in the glucose tolerance studies.The remaining F0 females were allowed to deliver and raise theiroffspring until weaning – the offspring were designated as the“F1”. At PND 21, all F1 mice were weaned on to the TD 95 092/control diet, so these mice were exposed to BPA during gestationand lactation periods only. Body weight was recorded weeklystarting at postnatal day (PND) 1 until PND 98 and at the timeDEXA was performed (between PND 98 and 117). A subset ofadult F1 females was time-mated to control C57BL/6 males andincluded in the glucose tolerance studies at E16.5-E17.5 or al-lowed to deliver offspring naturally. These offspring were des-ignated as the “F2”. For molecular analysis of Igf2, we mated F1females to Cast7 males as previously described (6). For our mu-tant mouse studies, the H19�3.8-/� deletion mice were derived aspreviously described (9, 10). To generate the H19�3.8-/� off-spring, H19�3.8 -/� heterozygous female mice were mated to wildtype male mice. All mouse work was conducted with the ap-proval of the University of Pennsylvania Institutional AnimalCare and Use Committee.

Igf2 mRNA expression analysisTotal RNA was extracted from E9.5-E10.5 tissues using the

Trizol reagent according to manufacturer’s guidelines and quan-tified using the Nanodrop. Using Invitrogen Super Script Tran-scriptase III and random hexamers, we subsequently generatedcDNA. Allele-specific expression analysis has been describedpreviously (6). For total expression, real time qPCR analysis wasconducted using the Applied Biosystems 7900HT Fast Real TimePCR System with the following protocol: 5 �L Master Mix, 0.12�L of reverse and forward primers, and 2.76 �L of water and 2�L cDNA (50 �g). The following primers were used:5�CGCTTCGTTTGTCTGTTCG3� (forward) and 5�GCAGCACTCTTCCACGATG3� (reverse) with product size of 94bp. Three reference genes were used in this study: Arppo, Gapdh,and Nono.

DNA methylation analysisDNA was extracted using the standard phenol chloroform

method and quantified using the Nano Drop spectrophotometer.One microgram of DNA was bisulfite treated using the EpiTect

2 BPA and multigenerational metabolic phenotypes Endocrinology


Bisulfite Kit (Qiagen) following manufacturer’s protocol. Pyro-sequencing was conducted to measure the methylation status ofthe promoter region of the Igf2 DMR1 (chr7: 149 851 180–149 851 655; NCBI37/MM9) as previously described (6).Briefly, 50 ng of bisulfite treated DNA was used in the PCR. Thefollowing primers were used: 5�- TGAGGTTAGATTAGGTT-GTAAGTT-3� (forward) and 5�- CTTCCCTACCCCTTA-AACC –3� (biotinylated reverse). For the sequencing, the fol-lowing primers were used: 5�-GGATTTTGTTAGGTAGGA-3�(CpG sites 1 and 2) and 5�-TTTTAGAGGTTTTTGGAGAA-3�(CpG sites 3 and 4).

Glucose tolerance testAt PND 98–117 (for the adult F1 and F2 offspring) or at

day16.5–17.5 of gestation (for the pregnant F0 and F1 females),mice were fasted overnight, and subsequently injected with 2g/kg body weight of glucose intraperitoneally. At 0, 15, 30, 60and 120 minutes, blood was sampled from the tail vein andanalyzed by a handheld glucometer.

Insulin measurement and islets perifusion studyFasting blood insulin levels were measured by ELISA (AL-

PCO). Isolation of pancreatic islets, perifusion, and insulin as-says were performed as previously described (11, 12). In brief,islets were isolated by collagenase digestion and cultured with 10mM glucose in RPMI 1640 medium (Sigma) for 2 days. Isletswere perifused with a Krebs-Ringer bicarbonate buffer (KRBB)containing 0.25% BSA at a flow rate of 1 mL/min. Glucoseramps were performed at increments of 0.5 mM/min. For max-imum insulin release, 30 mM KCl was used. Insulin was mea-sured by homogeneous time resolved fluorescence technology(Cisbio Kit).

DEXATo assess body composition, dual energy X-ray absorptiom-

etry (DEXA) scans were performed (GE Lunar PIXImus x-raydensitometer) on a subset of male and female adult (PND 98–117) F1 and F2 mice as previously described (13). Briefly, eachmouse was anesthetized using isoflurane. Each mouse was anes-thetized throughout the duration of the scan (�5 minutes). Bodyfat, lean mass, bone mineral content, and bone mineral density(BMD) measures were collected.

Statistical AnalysisThe significance of differences among multiple groups and

between two groups was examined using analysis of variance(ANOVA) and student t test, respectively. All values are pre-sented as means � SEM. A P value of � 0.05 was consideredsignificant. All data were analyzed using Prism data analysissoftware.

Results

BPA-exposed F1 male offspring have lower birthweights, and develop obesity in adulthood

To assess whether BPA exposure in utero produced amultigenerational metabolic phenotype, we had to estab-lish whether the F1 offspring in our experimental model

exhibited impaired glucose homeostasis. We first deter-mined whether BPA exposure during gestation and lacta-tion (see Materials and Methods) affected postnatalgrowth of C57BL/6 offspring. At PND 1, F1 lower doseBPA male offspring had significantly reduced body weightrelative to controls (P .01; Figure 1A). The body weightof F1 male offspring from this group remained lower thancontrols at PND 14 and 21 (P .008 and P .02, re-spectively; Figure 1A). In contrast, the body weight of F1male offspring from upper dose BPA exposure groups didnot differ from controls at these time-points (Figure 1A).Notably, F1 lower dose BPA males exhibited acceleratedweight gain postweaning, and by PND 28, weights weresimilar in all exposure groups. Between PND 98–117, F1lower dose BPA adult mice had significantly increasedbody weight relative to controls (30.3 � 1.2 g and 25.9 �1.0 g, respectively; P .002). The upper dose BPA malesshowed a trend towards increased body weight comparedwith controls (27.3 � 0.6; P .07). We did not observeany difference in food intake of F1 male mice from thedifferent exposure groups (Supplemental Figure 1). Con-sistent with their body weight profiles, F1 male offspringfrom lower and upper dose BPA exposure groups had sig-nificantly higher body fat content between PND 98–117relative to control males (Figure 1B; P � .05). Interest-ingly, upper dose BPA mice had significantly reducedBMD and bone mineral content (Supplemental Figure 2,A-B). We did not detect significant differences in bodyweight, body fat content, and bone mineral content anddensity in the F1 females at all time-points analyzed (Sup-plemental Figure 2, A-C, and 3A).

BPA-exposed F1 male, but not female, offspringhave impaired glucose homeostasis

Epidemiological, clinical, and experimental animalstudies have demonstrated that perinatal growth restric-tion followed by postnatal accelerated growth is linked tothe development of adult obesity and glucose intolerance(14). To determine whether increased body fat content inBPA-exposed F1 male offspring was associated with per-turbed glucose homeostasis, we performed intraperitonealGTTs and measured fasting insulin levels. Fasting glucoseconcentrations in F1 upper dose and lower dose BPA maleoffspring were not significantly different relative to con-trol mice (data not shown). In contrast, there was a trendin increased insulin levels in the F1 upper dose mice(1.29 � 0.82 ng/mL; n 5; P .08) and significantlyelevated levels in the lower dose BPA males (0.44 � 0.15ng/mL; P .04; n 4) relative to controls (0.24 � 0.08ng/mL; n 6). On a GTT, glucose area under the curve(AUC) was greater in F1 upper dose BPA male offspringcompared to control male offspring (Figure 1C; P .04),

doi: 10.1210/en.2014-2027 endo.endojournals.org 3


consistent with the phenotype of glucose intolerance. F1lower dose BPA male offspring showed a trend towards anincrease in glucose AUC compared with control males(Figure 1C; P .06). In contrast to the males, F1 upperdose and lower dose female offspring had similar glucoseAUC as control offspring (Supplemental Figure 3B), sug-gesting that BPA exposure-induced glucose intolerancewas highly sex-specific.

BPA exposure alters glucose-stimulated insulinsecretion

Impaired glucose tolerance is characterized by reducedinsulin secretion and/or peripheral insulin resistance. Toinvestigate whether impaired glucose tolerance in BPA ex-

posed F1 male offspring is associated with insulin secre-tory defects, we measured insulin secretion in response toa glucose gradient and potassium chloride (KCl) in iso-lated islets (15, 16). F1 upper dose BPA males exhibitedsignificantly elevated basal rates of insulin release relativeto controls (Figure 1D; P � .01), consistent with insulinresistance. In response to an increasing glucose gradient,we did not detect a significant difference in insulin releasebetween F1 control and upper dose BPA males (Figure 1E).Furthermore, KCl induced insulin release was not differ-ent in islets of upper dose BPA male mice relative to con-trols, consistent with an intact insulin secretory apparatus.These findings suggest that glucose intolerance in F1 upperdose BPA mice is not linked to impaired insulin secretion,

but rather due to insulin resistance.In contrast, lower dose BPA maleshad similar basal rates of insulin re-lease (Figure 1D), but significantlyreduced maximal glucose stimulatedinsulin release compared to controls(Figure 1E; P � .01).

Effects of BPA-induced genomicimprinting defects are partiallytransmitted to the F2 offspring

Following the establishment thatthe F1 offspring had altered bodycomposition, glucose tolerance andinsulin secretion, we subsequentlystudied the phenotypes of the nextgeneration. An important questionwith in utero environmental expo-sures is whether offspring of the F1exposed mice (ie, the F2 mice) alsoexhibit altered phenotypes in the ab-sence of further exposure. In the cur-rent model, the F2 offspring were ex-posed to BPA as developing germcells. Previously, we demonstratedthat upper dose BPA exposure re-sulted in biallelic expression of theimprinted Igf2 gene and increasedtotal mRNA expression in the F1 em-bryos (6). To determine whethergenomic imprinting defects were ob-served in the F2 offspring, a subset ofC57BL/6 F1 females were time-mated to unexposed Cast7 males,and embryos were collected at E9.5–10.5. We assessed allele-specific andtotal mRNA expression of the Igf2gene. In contrast to the F1 embryos,

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Figure 1. Metabolic profiles of F1 control and BPA male offspring: A. F1 Weekly body weightmeasurement in the F1 male offspring from control, lower dose, and upper dose BPA exposuregroups (*P � .05); B and C. At PND 98–117, F1 adult male offspring from BPA exposure groupsare fatter and more glucose intolerant relative to controls; D and E. Perifusion islet studiesshowing basal and glucose-stimulated insulin secretion.



we did not observe biallelic expression of the Igf2 gene inthe F2 embryos (data not shown). F2 upper dose BPAembryos, however, significantly overexpressed total Igf2mRNA relative to controls (Figure 2A).

Our previous study revealed that Igf2 overexpression inthe F1 upper dose BPA embryos was linked to increasedaverage DNA methylation at 4 CpG sites of the DMR1 (6),specifically at CpG sites 1, 2, and 4 (Supplemental Figure4). In contrast to the F1 embryos, we did not detect sig-nificant changes in the average DNA methylation levels atthese 4 CpG sites of the DMR1 in the F2 embryos (data notshown). When individual CpG sites were further ana-lyzed, the F2 upper dose BPA embryos showed elevatedDNA methylation at CpG site 2 relative to controls (Figure2B).

The metabolic phenotypes of the F1 offspringwere transmitted to the next generation

Because the F2 offspring shared the molecular pheno-types of the previous generation, we asked whether the F2offspring would exhibit a similar metabolic phenotype. Todetermine if the F2 offspring also exhibited an altered met-abolic profile, a subset of F1 females was mated to unex-posed C57BL/6 males, and adult F2 offspring were exam-ined for body weight, body fat, glucose tolerance, andinsulin secretion. The body weights of lower and upperdose male offspring were statistically not different thancontrols at PND1 (1.43 � 0.05 g, 1.59 � 0.04 g, and1.49 � 0.05 g, respectively; P � .05), or at adulthood(28.0 � 0.8 g, 28.8 � 0.9 g, and 27.8 � 1.1 g, respectively;P � .05). Similar to the F1 offspring (Figure 1B), however,F2 male offspring from upper dose BPA group were sig-nificantly fatter than controls at PND 98–117 (Figure 3A;

P � .05). Despite a higher body fat content in upper doseBPA F2 mice, fasting glucose (data not shown) and insulinconcentrations (0.33 � 0.06 ng/mL and 0.35 � 0.03mg/mL in control and upper dose BPA F2 males; P � .05)did not differ between groups. Glucose AUC, however,was significantly greater in F2 upper dose BPA males com-pared with control males (Figure 3B; P � .01). In contrast,the F2 lower dose BPA males showed similar glucose AUCrelative to controls. These results suggest that similar tothe F1 offspring, F2 upper dose BPA males also exhibitglucose intolerance.

To determine whether glucose intolerance in the F2BPA offspring was due to impaired insulin secretion as weobserved in the F1 offspring, we conducted perifusionstudies to measure basal and glucose-stimulated insulinrelease. We did not detect significant differences in basalinsulin secretion between groups (Figure 3C). F2 upperdose males also showed similar glucose stimulated andKCI induced insulin secretion as controls (Figure 3D).These results were consistent with our observation that theF1 upper dose males showed normal insulin secretion (Fig-ure 1E). In contrast, islets from lower dose male had sig-nificantly reduced glucose stimulated insulin secretioncompared with controls (Figure 3D; P � .01). Further-more, in the presence of KCl, similar to islets from the F1lower dose BPA males (Figure 3D), islets from the F2 lowerdose males showed a trend towards reduced insulin releaserelative to controls. These data show that the F2 maleoffspring inherit the metabolic phenotypes of the previousgeneration.

Maternal effects contributed to the perturbedmetabolic health in F1 but not F2 offspring

A previous study revealed thatBPA exposure induces gestationalglucose intolerance in mice (17). Ad-ditionally, elevated fetal Igf2 expres-sion increases maternal glucose con-centrations during pregnancyleading to gestational diabetes (18).These studies suggest that BPA ex-posure and embryonic Igf2 overex-pression in our mouse model maycause gestational glucose intoleranceduring pregnancy. Because gesta-tional glucose intolerance is a riskfactor for obesity and altered glucosehomeostasis in the offspring, it ispossible that our F1 and F2 male off-spring become fatter relative to con-trols and develop glucose intolerancedue to compromised maternal met-

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Figure 2. E10.5 F2 embryos have elevated total Igf2 mRNA expression and increased DNAmethylation at CpG site 2 of the DMR1: A. Total mRNA expression of Igf2 in whole embryosfrom control, lower dose, and upper dose BPA exposure groups relative to the reference genes.All values were normalized to controls; B. DNA methylation percentages at each CpG sites of theIgf2 DMR1. Increased DNA methylation was detected at CpG site 2 in F2 upper dose BPAembryos.



abolic health. To test this possibility, we performed GTTsin F0 and F1 pregnant mice. Pregnant F0 mice from boththe lower and upper dose exposure groups were glucoseintolerant when tested between gestation days 16.5 and17.5 (Figure 4A; P � .05 and P � .01, respectively). Incontrast to BPA-exposed F0 pregnant females, F1 femalesfrom upper dose BPA exposed and control groups showedno significant difference in glucose tolerance during preg-nancy (Figure 4B). We did not observe a significant dif-ference in maternal food intake in F0 and F1 pregnant miceamong the different exposure groups (Supplemental Fig-ure 5, A-B). These results showed that although maternalgestational glucose intolerance could explain the abnor-mal glucose homeostasis in the BPA-exposed F1 male off-spring, it did not contribute to the metabolic phenotypesof the F2 male offspring.

Overexpression of Igf2 was associated withaltered glucose homeostasis

In addition to its well-characterized role in promotingfetal growth (19), increasing evidence has suggested thatIgf2 influences adult energy metabolism (20–23). To de-termine if fetal Igf2 expression contributed to the meta-bolic phenotypes in our F1 and F2 BPA offspring, we askedwhether the metabolic profiles of other mouse models thatdemonstrate elevated Igf2 expression would be altered ina similar manner to BPA-exposed mice. A well-studiedmouse model of Igf2 overexpression is the H19�3.8 model,which contains an engineered 3.8 kb deletion of the H19/Igf2 locus including the ICR (9). The ICR is unmethylatedon the maternal allele and serves as an insulator thatblocks access of Igf2 to enhancers that are shared betweenH19 and Igf2. Mice that inherit this mutation maternally(ie, H19�3.8-/�) exhibit 2.0–2.5 fold higher levels of Igf2 at

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Figure 3. Metabolic profiles of F2 adult male offspring from control and BPA exposure groups: A and B. At PND 98–117, F2 upper dose BPAmales are fatter and more glucose intolerant relative to controls; C and D. Perifusion islet studies showing normal basal insulin secretion, butreduced glucose-stimulated insulin secretion in F2 lower dose males.



E9.5 (10). If Igf2 overexpression contributes to the met-abolic profile of our BPA-exposed mice, we would expectthat the H19�3.8-/� mice exhibit glucose intolerance. Con-sistent with this prediction, GTTs performed betweenPND 98–117 revealed that H19�3.8-/�male mice weremore glucose intolerant relative to wild type (Figure 4C;P .05). DEXA scan revealed that these mice showed atrend in increased body weight and body fat content rel-ative to wild type mice (data not shown). Interestingly,H19�3.8-/� females did not develop glucose intolerance(data not shown). These results demonstrate that Igf2overexpression contributes, but is not exclusively linked,to abnormal glucose homeostasis in mouse.

Discussion

Increasing evidence has suggested that the early life envi-ronment can significantly impact the future health of anindividual and his/her offspring. In this study, we foundthat early life exposure to the EDC BPA in the mouse was

associated with sex-specific abnormal metabolic health inmultiple generations (Supplemental Table 1). Both F1 andF2 lower and upper dose male offspring became fatterrelative to controls, and in the upper dose groups, themales developed glucose intolerance during adulthood.Our perifusion islet studies revealed that in F1 and F2lower dose BPA male mice, glucose stimulated insulin se-cretion was impaired. Interestingly, we did not observeperturbed insulin release with upper dose BPA exposure.Both F1 and F2 upper dose males exhibited normal glu-cose stimulated insulin secretion, suggesting that the glu-cose intolerance that these mice exhibit may be due toinsulin resistance rather than defects in insulin secretionper se. An increase in basal insulin release in islets of F1upper dose male mice was further suggestive of an insulinresistant phenotype. Insulin biosynthesis and release arepartly regulated by estrogen receptors (ERs). Previousstudies have demonstrated that subcutaneous injection of100 �g/kg/d BPA alters pancreatic � cell function and in-creases insulin release in OF1 male mice, and that the ef-

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Figure 4. Glucose AUC measurement in pregnant F0 and F1 mice, and in the H19�3.8-/� male mice: Pregnant F0 females (A), but not F1 (B), havesignificantly increased glucose concentration when tested between E16.5–17.5; and C. H19�3.8-/� male mice have significantly higher glucoseconcentration compared to wild type mice.



fects are dependent on an ER� signaling pathway (24). Itis possible that BPA has a nonmonotonic dose response on� cell function and exposure to 10 �g/kg/d, but not 10mg/kg/d, inhibits an ER� signaling pathway, leading todistinct dose-response effects on insulin release in ourstudy. Furthermore, we cannot rule out the possibility thatBPA exposure also alters insulin signaling pathways at keytarget tissues such as liver, skeletal muscle, or adipose tis-sue (25) in a dose-dependent manner leading to insulinresistance.

Previous studies have reported similar effects of mater-nal BPA exposure on glucose homeostasis of the F1 off-spring in mouse strains other than the C57BL/6 strain usedhere (17, 26, 27), however, to our knowledge, this is thefirst study to demonstrate that the metabolic phenotypecan be transmitted to the F2 offspring in the mouse. Li etal (2014) recently reported glucose intolerance and insulinresistance in the F2 offspring in the rat following maternalF0 exposure (28). The species, route of administration,and doses used in our study were different than Li et al(2014). Importantly, in addition to these differences, Li etal (2014) had generated their F2 mice through paternaltransmission (ie, by mating F1 male offspring), which con-trasts the maternal transmission (ie, by mating F1 femaleoffspring) used in our study. Use of maternal transmission,as opposed to paternal transmission males with demon-strated metabolic problems, limits the contribution of thepaternal metabolic milieu to the health of the F2 offspring.

There are at least two possible mechanisms that medi-ate theobservedmultigenerationalphenotypic inheritancein the current study: altered maternal metabolic milieuthat affected the health of the F1 and F2 offspring and/orepigenetic inheritance. To gain insights into the contribu-tion of maternal effects on the metabolic phenotype in theF1 and F2 offspring, we investigated gestational glucosehomeostasis in the F0 and F1 females during pregnancy.Consistent with a previous report (17), we observed thatBPA exposure induced gestational glucose intolerance inpregnant F0 mice (Figure 4A). Importantly, we report forthe first time that F1 female mice from upper dose BPAexposure group did not develop glucose intolerance dur-ing pregnancy (Figure 4B). This latter observationstrongly suggests that in our environmental exposuremouse model, the metabolic phenotype of the F2 offspringwas not caused by altered maternal metabolic milieu, butindicates an epigenetic process.

Consequently, we addressed whether the observedmultigenerational metabolic phenotype in this BPA expo-sure mouse model was associated with inheritance of al-tered DNA methylation. In the F1 embryos, BPA exposuresignificantly increased DNA methylation at the Igf2DMR1 and elevated total Igf2 mRNA expression (6).

Here, we demonstrated that the F2 embryos shared thesemolecular phenotypes (Figure 2, A-B, Supplemental Table1). We further asked whether Igf2 misregulation duringembryonic development was linked to abnormal growthprofile and metabolic health later in life. The Igf2 gene ishighly expressed during fetal development and the IGF2peptide hormone is primarily involved in prenatal growthregulation. Overproduction of IGF2 during developmentleads to somatic overgrowth (29); mice lacking the Igf2gene due to engineered mutation are 60% smaller thanwild type (19). Additional studies have shown that IGF2or Igf2 expression influences adult energy metabolism(20–23). In this current study, we compared the metabolicphenotype of adult F2 offspring from BPA exposure groupto H19�3.8-/� mice, which also overexpressed Igf2 duringembryonic development. We demonstrated, for the firsttime, that Igf2 overexpression in the H19�3.8 mousemodel was associated with glucose intolerance in the adultmale offspring (Figure 4C), consistent with a role for Igf2in adult metabolism. Igf2 misregulation, however, is notexclusively responsible for the development of the pheno-type. H19�3.8-/� females also overexpress Igf2 but do notdevelop glucose intolerance during adulthood. Our ob-servations suggest that Igf2 misregulation is not the solecontributor to the observed phenotypes, but most likelyrepresents a downstream effect contributing to altered me-tabolism, and that there are unknown, critical upstreamevents that control the final health outcomes.

Sex-specific differences in developmental outcomes in-duced by BPA exposure have been reported in other stud-ies (30). BPA is an estrogen mimic that has the ability tobind to estrogen receptors, although relative to endoge-nous estrogens, it has weaker binding affinity and potency.Human and animal studies have suggested that estrogenmay provide protective effects to females, which may limitdownstream metabolic effects (31). Our current study in-dicates that there are distinct downstream developmentalsusceptibilities between sexes towards metabolic abnor-malities in response to early life environmental exposure.Additionally, IGF signaling is controlled by growth hor-mone (GH) produced by the pituitary. Because GH pul-satility is highly sex-specific, factors that influence IGFsignaling may produce different outcomes between malesand females, providing another sex-specific mechanism torespond to early life environmental perturbations.

In conclusion, our study demonstrates that early lifeexposure to environmental EDCs may disrupt the meta-bolic health of the developing fetus as well as its offspring.The exact mechanisms of this phenotype transmission areunclear but our study suggests a role for Igf2, possiblythrough epigenetic misregulation. Limitations of the cur-rent study include the unknown role of the ERs in medi-



ating BPA-induced multigenerational metabolic effectsand the uncertainty as to whether BPA-induced epigeneticperturbation is linked to its estrogenicity. Although liter-ature reporting unfavorable health effects of BPA is grow-ing, BPA is considered a weak estrogen with poor affinityto the ERs. Furthermore, a recent study has questionedwhether BPA levels reported in many studies are sufficientto activate the ERs (32). We demonstrated that serum BPAlevel in the pregnant mice in our mouse model was withinrepresentative levels of human exposure (6), however,whether this exposure level would produce an adversehealth effects in humans is an ongoing research topic. Inthe future, a more global approach to analyze effects ofexposure on genome-wide DNA methylation patterns aswell as to study effects in the F3 offspring would providemore comprehensive mechanistic insights into the multi-generational inheritance of health outcomes in this modelsystem.

Acknowledgments

The authors would like to acknowledge The Penn Diabetes En-docrine Research Center Grant (P30DK19525) and the servicesof the Mouse Phenotyping, Physiology, and Metabolism Core.

Address all correspondence and requests for reprints to:[email protected] [email protected]. Request for reprints to:Marisa Bartolomei, PhD, Department of Cell and Developmen-tal Biology, 3400 Civic Center Boulevard, 9–123 Smilow Centerfor Translational Research, Philadelphia, PA 19 104.

Disclosure Summary: The authors have nothing to declare.This work is supported by K99ES022244 (MS),

T32ES019851 (FX), 1RO1DK098517 (CL), Mar of Dimes(MSB), and NIEHS ES023284 and ES013508 (MSB, RAS).

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Bisphenol A exposure disrupts metabolic health across multiple generations in the mouse

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