Paternal bisphenol a diet changes prefrontal cortex proteome and provokes behavioral dysfunction in male offspring

Chemosphere 184 (2017) 720e729

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Paternal bisphenol a diet changes prefrontal cortex proteome and provokes behavioral dysfunction in male offspring Guangying Luo a, b, Ruifen Wei a, c, Shaolin Wang d, **, Jundong Wang a, * a

Shanxi Key Laboratory of Environmental Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi, 030801, China School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, 270 Xueyuan Road, Wenzhou, Zhejiang, 325027, China c Eye Hospital, Wenzhou Medical University, 270 Xueyuan Road, Wenzhou, Zhejiang, 325027, China d College of Veterinary Medicine, China Agricultural University, Beijing 100193, China b

h i g h l i g h t s
We examined effects of paternal BPA diet on prefrontal cortex proteome of male offspring.
Prefrontal cortical ER stress and myelin deficiency related molecules were changed.
We observed influences of paternal BPA diet on behaviors of male offspring.
Paternal BPA exposure alters social and anxiety-like behavior in male mice.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 December 2016 Received in revised form 10 June 2017 Accepted 13 June 2017 Available online 14 June 2017

Relatively little attention has been given paternal effects on next generation. Given that Bisphenol A (BPA), a ubiquitous compound in maternal diet, may disrupt brain development and behavior, we hypothesized that paternal BPA diet (PBD) could affect offspring development. Prefrontal cortex (PFC), a vital brain region, is involved in emotion and social behavior. To test whether PBD could alter developing PFC, we carried out a proteomics approach for PFC in male juvenile offspring that responded to PBD (50 mg BPA/kg diet). We found that PBD altered the expressions of binding immunoglobulin protein (BIP), CCAAT/-enhancer-binding protein homologous protein (CHOP) and B-cell lymphoma-2 (BCL-2), which could reflect endoplasmic reticulum (ER) stress. In addition, downregulation of myelinogenesis genes and myelin basic protein (MBP) could provoke myelin deficiency. Furthermore, PBD significantly increased anxiety-like behavior and impaired social behavior in male offspring. Taken together, these results revealed the alterations of ER stress and myelin destruction related molecules induced by PBD might be a potential mechanism for the behavior deficits in their male offspring. These findings remind us of the importance of paternal effects in the further environmental exposure research. © 2017 Elsevier Ltd. All rights reserved.

Handling Editor: A. Gies Keywords: Paternal BPA diet Anxiety-like behavior Social behavior Immunoglobulin protein Myelin basic protein

Accumulating data have indicated a crucial biological role of parents in physiological development and mental health of their offspring (Lieb et al., 2000; Repetti et al., 2002). In this regard, most evidence seems to only regard maternal effects, such as fetal malnutrition-induced psychiatric and metabolic sequelae in rodents and human (Hales and Barker, 2001; Harris and Seckl, 2011; Symonds et al., 2009), maternal behavior and stress-induced epigenetic programming in rodents (Weaver et al., 2004;

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (S. Wang), [email protected] (J. Wang). http://dx.doi.org/10.1016/j.chemosphere.2017.06.050 0045-6535/© 2017 Elsevier Ltd. All rights reserved.

Gheorghe et al., 2010). Whereas, the exact mechanisms of maternal effects are difficult to define since offspring development heavily relies on the in utero and the postnatal maternal care. Increasing evidence has showed a paternal role in the early life origins of physiological disorders and mental illness (Flouri and Buchanan, 2003; Lamb, 1975; Sarkadi et al., 2008; Rodgers et al., 2013; Morgan and Bale, 2011; Dunn and Bale, 2011). Importantly, the influences of paternal effects on offspring phenotype is quite direct as fathers usually contribute nothing but sperm to offspring (Morgan and Bale, 2011; Dunn and Bale, 2011; Carone et al., 2010; Dias and Ressler, 2014). Hence, research about the paternal effects is possible and urgently needed for extending the cognition of parental role in offspring development.

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We selected Bisphenol A (BPA), a widely investigated environmental xenobiotic, as a model paternal stressor. BPA is a ubiquitous compound because it is used extensively in dental sealants, plastic bottles and bags, medical equipment, and as paper coating in thermal printers (Corrales et al., 2015). A quite severe threat to the physiological and mental health of humans and wildlife has been correlated to developmental BPA exposure (Erler and Novak, 2010; Flint et al., 2012; Kang et al., 2006; Ja
sarevic et al., 2011; Luo et al., 2013; Cox et al., 2010; Halldin et al., 2001). It is worth noting that diet is the major route of BPA exposure (Kang et al., 2006). Effects of maternal BPA diet on offspring have been confirmed in the literatureedevelopmental exposure to BPA has been reported to disrupt the adult expression of sexually selected traits (Ja
sarevi c et al., 2011) and maternal BPA diet provokes anxiety-like behaviors, spatial learning dysfunction and impacts hippocampal CA1 neuronal morphology in rodents (Ja
sarevi c et al., 2013; Luo et al., 2014; Wang et al., 2014). Furthermore, epidemiological data from human populations linked maternal and prenatal BPA exposure to behavioral disorders of their offspring (Braun et al., 2009; Perera et al., 2012; Rochester, 2013; Sathyanarayana et al., 2011). However, at present there is scant evidence for effects of paternal BPA diet (PBD) on CNS in their offspring. It has as yet been shown that paternal BPA exposure modifies hippocampal acetylcholinesterase (AChE) activities along with spatial memory deficits in mice (Fan et al., 2013). To expand this finding, in this study, we report the effect of PBD on the prefrontal cortex (PFC) in their offspring. PFC, the most evolved brain region in CNS, is vital to executing and regulating action, thought and emotion, which are associated with emotion and social behavior (Arnsten, 2009a,b; Hains and Arnsten, 2008). Previously studies have revealed that many critical molecules and specific brain pathways, mostly located in the PFC, are involved in behavioral development (Arnsten, 2009a,b). Impaired ultrastructure and reduced myelination of prefrontal cortex has been related to many psychiatric disorders, such as schizophrenia, major depression, obsessiveecompulsive disorder, anxiety, and bipolar disorder (Bartzokis, 2005; Uranova et al., 2001; Bercury and Macklin, 2015; Lehmann et al., 2017). Endoplasmic reticulum (ER) stress is caused by the disruption to lipid synthesis and accumulation of unfolded or misfolded proteins. Decreased myelination is partly ascribed to ER stress (Bercury and Macklin, 2015). The study from Fan et al., suggested that paternal BPA exposure could suppress AChE activities in hippocampus. However, it is not clear the impact of paternal BPA exposure on the PFC development in juvenile offspring. The primary objective of this study was to examine the effect of preconception paternal exposure to BPA on the offspring. In this study, male mice exposed to a BPA diet (50 mg BPA/kg diet) or a control diet were used as sires to mate with dams, the dams and their offspring were fed a control diet. We used a proteomics approach toward understanding the primary damaging events that occur in PFC in male juvenile mice. In addition, we also determined potentially PFC-regulated behaviors among early adult and adult offspring.

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binding immunoglobulin protein (BIP), anti-CCAAT/-enhancerbinding protein homologous protein (CHOP), anti-B-cell lymphoma-2 (BCL-2), anti-myelin basic protein (MBP), anti-GAPDH antibodies and goat anti-rabbit IgG horseradish peroxidase (HRP) conjugated were obtained from Bioss (Beijing, China). Trizol Reagent, real time PCR primers and SYBR® QRT-PCR kit were obtained from the Takara Biotechnology Company (Dalian, China). All regents preparing for proteomic analysis were purchased from BioRad Laboratories.

1.2. Animals and treatments A total of sixty male and sixty female 28-day-old CD-1® (ICR) mice from Beijing Vital River Laboratories were housed individually in special pathogen-free polypropylene cages at 20 ± 2  C, on a 12:12 h light/dark cycle. Glass water bottles were needed through the whole study. Male F0 founders were assigned to a BPA (modified AIN-93G diet supplemented with 50 mg/kg diet of BPA) diet or a control (modified diet: AIN-93G diet with 7% corn oil substituted for 7% soybean oil) diet at 5 weeks of age (n ¼ 30, 30, for control and BPA diets, respectively). At 15 weeks, BPA males begin to mate with females consuming control diet. During mating, one male and one female were housed together, with free access to control diet from 0700 to 1700 h, for 7 consecutive days. Males returned to their cages overnight to their assigned diets, whereas females consumed control diet throughout mating, gestation and lactation (Fig. 1). PBD exposure did not alter body weight, sex ratios or litter sizes of F1 offspring (data not shown). To avoid possible litter effects, male mice were randomly selected from a total of 30 BPA and 30 control litters. After weaning (at PND28), 40 male offspring (n ¼ 20, 20, for control and BPA diets, 20 male/30 litters, respectively) were sacrificed using cervical dislocation and brains removed and rinsed in ice-cold saline. Based on mouse brain anatomical landmarks (Paxions and Franklin, 2004), PFC was dissected from 2-mm slices over ice for proteomics analysis, western blot and quantity realtime RT-PCR assay. Because prefrontal cortical proteome of PBD on female offspring did not show significant different proteins (data not shown), only male offspring were used in these experiments. The total number of F1 offspring analyzed was 120 males to determine whether PBD related to behaviors. The number of F1 offspring in each behavior test required 40 males (n ¼ 20, 20, for control and BPA diets, 20 male/30 litters, respectively). These mice

1. Materials and methods 1.1. Chemicals Bisphenol A (BPA, >99% pure) was purchased from SigmaAldrich (USA); Modified AIN-93G diet (a low phytoestrogen diet with 7% corn oil substituted for 7% soybean oil) and modified AIN93G diet supplemented with 50 mg/kg diet of BPA was purchased from HFK Bioscience CO., LTD (Beijing, China). RIPA lysis buffer, ECL (Enhanced Chemiluminescence) Plus reagent and the BCA Protein Assay kit were purchased from Beyotime (Shanghai, China); Anti-

Fig. 1. Paternal BPA diet (PBD) paradigm. Male CD-1 mice were exposed to a BPA diet for 10w before mating. After mating, dams consumed a control diet. The prefrontal cortexes were collected for molecules assay at PND28, and behaviors were tested at PND30-70.

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were used to conduct behavioral assay from 30d to 70d. The behavioral tests were performed between 0900 and 1600 h with constant luminance of 50 lux. In total, 60 male and 60 female founders, as well as 160 male F1 offspring were used in this study. All experiments were approved by the Institutional Animal Care and Use Committee of China and carried out in accordance with the approved guidelines. In this study, our BPA diet preparation and dose (50 mg BPA/kg diet) selection entirely based on Sieli et al., their results indicated that the presence of food might contribute to increase internal exposure to bioactive BPA, and thus diet exposure was presumably the more relevant manner for modeling the nature route of human contract to BPA. Indeed, some studies have reported that the estimated daily treatment of 50 mg BPA/kg diet in dams was 10 mg/kg body weight (bw) (Susiarjo et al., 2013; Weinhouse et al., 2014), which was considered to be far below the diet-administered maximum nontoxic dose (200 mg/kg body weight per day) for rodents, within the presumptive no observed effect level for mice (Takahashi and Oishi, 2003). Furthermore, such an exposure manner (50 mg BPA/kg diet) has been used in many studies on the effects of BPA in rodents (Ja
sarevi c et al., 2011, 2013; Rosenfeld et al., 2013; Anderson et al., 2013; Kim et al., 2014). In all, 50 mg BPA/kg diet (about 10 mg/kg BW/d), likely to provide circulating serum concentrations close to those observed BPA for humans (Sieli et al., 2011), is still a matter of concern. 1.3. Sample preparation and proteomic analysis Frozen PFC was handled with a lysis buffer including 7 M urea, 4% (w/v) CHAPS, 2 M thiourea, Bio-Lyte 3/10 and 1% (w/v) protease inhibitor cocktail. Total protein concentration was measured using modified Bradford protein assay. Two-dimensional electrophoresis analyses of prefrontal cortical proteins were performed essentially as previously described (Eccleston et al., 2011). During rehydration of ReadyStrip IPG strips (pH 4e7; 17 cm) overnight (~16 h), prefrontal cortical protein (405 mg) was loaded to ReadyStrip IPG strips. The following morning, isoelectric focusing (IEF) was conducted using the following conditions: 250 V for 30min, ramp to 1000v for 1 h, ramp to 10 000 V for 5 h, and then hold at 10000 V for 6 h, ramp down to 500 V forever. For SDS-PAGE, IPG strips were placed horizontally on top of a 12% separation gel and sealed using overlay agarose solution, and gels were run at 16 mA for 0.5 h, and then run at 24 mA for 5.5 h. After electrophoresis, gels were stained with Bio-Rad's BioSafe Coomassie Stain and imaged using Bio-Rad's GS-800. Using PDQuest 2D analysis software (Bio-Rad), gels were analyzed for protein abundance essentially as previously described (Eccleston et al., 2011). Gels for the control and BPA treatments were manually verified to match individual proteins identified. The protein abundance of all gels was compared. Based on the overall best spot quality, the master gel was automatically chosen by PDQuest 2D analysis software. Proteins of each gel were automatically matched with the corresponding proteins in the master gel. Incorrectly matching proteins were manually calibrated in the master gel. Different spots were excised from gels and performed as previously described during protein identification. Using 4800 Plus MALDI TOF/TOFTM Analyzer, samples were analyzed and the obtained peptide masses were submitted to the Mascot 2.2 database (www.matrixscience.com) for protein identification. The biological functions of these proteins were annotated using the Universal Protein Resource website (www.uniprot.org). 1.4. Total protein extraction and western blotting analysis Total protein from the PFC was extracted using RIPA lysis buffer

according to the manufacturer's instructions. SDS-PAGE gels were cast with a 10%, 12% or 15% acrylamide resolving layer and 5% stacking layer to separate total protein. Western blot analysis was performed using an appropriated dilution of primary antibodies (anti-BIP, 1:200; anti-CHOP, 1:250; anti-BCL-2, 1:200; anti-MBP, 1:200; anti-GAPDH, 1:1000; Beijing Bioss, China) and secondary antibodies (goat anti-rabbit IgG horseradish peroxidase conjugated; Beijing Bioss, China). The immunoreactive bands were detected using the ECL methods. Equal protein loading was guaranteed by probing the blots with anti-GAPDH. Densitometry of the western blot protein bands was analyzed using AlphaView FluorChemQ. GAPDH was used as an endogenous control to reflect the relative abundance of the target protein. 1.5. Total RNA extraction and real-time qRT-PCR analysis Total RNA was isolated from the PFC using Trizol reagent according to the manufacturer's instructions. Specific primers of Bip, Chop, Bcl-2, myelin oligodendrocyte glycoprotein (Mog), myelinassociated glycoprotein (Mag), myelin basic protein (Mbp), myelinassociated oligodendrocytic basic protein (Mobp), and proteolipid protein (Plp) and 18S rRNA were designed with Primer3.0 plus (see Supplemental Material, Table S1). Real time qRT-PCR was conducted using the Mx3000 P™ qRT-PCR system (Stratagene, USA) and SYBR® QRT-PCR kit. 18S rRNA was used as an endogenous control to reflect the relative abundance of the target gene. Data were analyzed and were expressed as relative gene expression using the 2-DDCt method. Real time qRT-PCR was conducted in triplicate for each sample. 1.6. Open field test (OFT) As previously described (Salas et al., 2003), the mice activity in the center and in the periphery of a novel open field (40  40  40 cm) was quantified to measure exploratory activity and anxiety. At PND30, each mouse was placed in the novel open area, and behaviors were recorded for 5 min using a color video camera (SSC-G213, SONY). The open field was cleaned with water and 70% ethanol between each trial. The total distance moved, duration and frequency to enter the center of a novel open arena were analyzed by SMART V 3.0 tracking system (Panlab, Spain). 1.7. Elevated zero maze (EZM) As previously described (Kulkarni et al., 2008), the mice locomotor activity on the open arms versus the closed maze arms of elevated zero maze (EZM) was quantified to measure anxiety. The EZM consisted of a 6 cm wide ring with an outer diameter of 40 cm containing four equal quadrants of alternating closed or open sections. At PND60, each mouse was placed in the closed arm of EZM, and behaviors were recorded for 5 min using a color video camera (SSC-G213, SONY). The maze was cleaned with water and 70% ethanol between each trial. The latency and frequency of entry into each of 2 open and 2 closed arms were quantified by SMART V 3.0 tracking system (Panlab, Spain). 1.8. Social behavior As previously described (Won et al., 2012), social behavior was tested by a 3-chamber apparatus, which consisted of one rectangular box (40  60  20 cm). This box was divided into 3 equal parts using the two dividing walls. Each wall has a tractable doorways accessing to the side chambers. Each side of chambers has a closed cage for mice. At PND70, sociability and preference for social novelty of mice were performed. Before starting test, each test mouse

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was permitted to habituate for 10min to this 3-chamber apparatus. For sociability test, the mouse was placed in the middle part and a same gender stranger 1 (S1) mouse was randomly placed in a closed cage. Behaviors were recorded for 10 min using a color video camera (SSC-G213, SONY). For preference for social novelty, a same gender stranger 2 (S2) mouse was placed in other empty closed cage, and behaviors were again recorded for 10 min using a color video camera (SSC-G213, SONY). The chamber was cleaned with water and 70% ethanol between each trial. The time of spent exploration toward the “Stranger 1” versus the “Empty” or

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“Stranger1” versus “Stranger 2” were quantified by SMART V 3.0 tracking system (Panlab, Spain). The preference index in exploration with the stranger [1] or [2] was calculated.

1.9. Statistical analysis Behavior data were analyzed by trained experimenters blind to the treated group. Statistical analysis was conducted using GraphPad Prism5 software (GraphPad Software Inc., San Diego, USA). All data were analyzed using two-tailed t-test to determine statistical

Table 1 Protein alterations of PFC in response to paternal BPA diet exposure.

Spot

Accession

Protein

No.

No.

ID

1

gi|2598562

Bip/GRP78

2

gi|6754254

HSP90-α

MW(KDa)/pI

Peptide

Protein

Protein

Count

Score/CI%

level (fold)

Main function

72546.6/5.1

24

697/100

3.43436

ER stress

85133.9/4.93

28

950/100

2.84267

Cell cycle regula on; Signal transduc on

3

gi|6755000

Pdcd6

21910.8/5.16

10

386/100

2.62980

4

gi|14290464

Anxa7

50162.4/5.91

18

475/100

-2.54084

5

gi|13435885

Atp6v1b1

44877.6/5.19

8

310/100

-2.33443

6

gi|148665899

ATP synthase

14195.4/9.35

2

108/100

2.31757

7

gi|148707168

NADH dehydrogenase

50179.2/5.94

17

383/100

2.24987

92717.4/4.74

30

825/100

2.19343

Angiogenesis; Apoptosis

Membrane fusion; exocytosis

ATP synthesis

Produce ATP

ATP synthesis

Fe-S protein 2

8

gi|14714615

HSP 90-β

Cell cycle regula on; Signal transduc on

9

gi|148667766

NADH dehydrogenase

74783.1/5.57

24

750/100

2.12723

ATP synthesis

Fe-S protein 1

10

gi|148670722

mCG7617

14574.1/5.38

4

163/100

2.11428

Cell cycle

11

gi|6671702

T-complex protein 1

60042.1/5.72

16

321/100

2.11326

Molecular chaperone

12

gi|31982300

Hemoglobin subunit

15795.2/7.14

8

246/100

2.07685

68566.7/5.62

26

1080/10

-2.06417

Oxygen transport

beta-1

13

gi|1184659

Vacuolar adenosine

Enzyme ac vity regula on

0 triphosphatase subunit A

14

gi|225543196

NDRG2

39597.5/5.4

13

392/100

2.04039

Cell differen a on

15

gi|124430543

Sorcin isoform 2

20567.8/5.11

9

423/100

2.02785

Contributes to calcium homeostasis

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significance to assess the difference between control group and BPA group. Values in graphs are expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

2. Results 2.1. Alterations of prefrontal cortical proteome reveal ER stress Our first priority was to perform prefrontal cortical proteome of juvenile male mice that were paternally exposed to BPA. Representative two-dimensional electrophoresis gels from a control and BPA groups are shown in Fig. S1. We typically detected ~1000 protein spots in each 2DE map using the Bio-Rad's PDQuest analysis software. 15 significantly altered proteins with a ratio fold change >2 or <0.5 were found by densitometry analyses in response to PBD. The location of these different proteins is displayed in a 2D gel (see Supplemental Material, Fig. S2). In addition, a list of these 15 different proteins Accession No., IDs, MW(K Da)/pI, Peptide Count, Protein Score/CI%, Protein level (fold) and Biological function is provided in Table 1. Of the 15 altered proteins in male offspring from PBD exposed, 12 were increased (circled in blue) and 3 were decreased (circled in red) in PFC (see Supplemental Material, Fig. S2). As shown in Table 1, the major functions of these proteins were cell cycle control, ATP synthesis, calcium homeostasis regulation, enzyme activity and ER stress response. Most interestingly, the highest up-regulated protein (~3.4 fold; in a red rectangle of Table 1) BIP, is one of the crucial markers for endoplasmic reticulum (ER) stress.

2.2. ER stress is validated in PFC Our next goal was to investigate ER stress of PFC in males in response to PBD and determine the severity of ER stress. Hence, we used Western blot to validate the BIP expression in PFC of the control and BPA, and found BIP levels in the PFC were markedly increased (p ¼ 0.0248) in the BPA group (Fig. 2A and B). In order to confirm ER stress, we further used Western blot to validate the CHOP expression in PFC of the control and BPA, and found CHOP levels in the PFC were markedly increased (p ¼ 0.0347) in the BPA group (Fig. 2D and E). Subsequently, sustained ER stress was also established by BCL-2 expression, as shown in Fig. 2G and H, the BCL-2 level in the PFC was significantly decreased (p ¼ 0.0010) in the BPA group as compared to the control group. In addition, as shown in Fig. 2C, F and 2I, the Bip (p ¼ 0.0389) and Chop (p ¼ 0.0185) mRNA expression levels in the PFC were significantly increased in the BPA group as compared to the control group, but Bcl-2 mRNA expression level had no significant difference (p > 0.05). 2.3. Myelination related molecules are changed in PFC of BPA mice Our next goal was to investigate whether PBD could affect myelination related molecules. Myelin gene transcripts were sharply decreased in the PFC, including Mog (p ¼ 0.0011), Mag (p ¼ 0.0048), Mbp (p ¼ 0.0013), Mobp (p ¼ 0.0263), and Plp (p ¼ 0.0240) (Fig. 3A). In addition, MBP, an important component of the myelin sheath, was also decreased (p ¼ 0.0456) in PFC (Fig. 3B and C).

Fig. 2. Paternal BPA diet (PBD) provokes the changes of endoplasmic reticulum (ER) stress-associated molecules in the prefrontal cortex (PFC) of male offspring. A, D and G. Representative Western immunoblot images showing levels of BIP, CHOP and BCL-2 and GAPDH in mice PFC. B, E and H. Quantitative analysis of Western blots and histogram showing the expression of BIP, CHOP and BCL-2. C, F and I. Quantitative analysis of real time qRT-PCR showing the expression of Bip, Chop and Bcl-2. Data are means ± SEM (n ¼ 6 for each group); unpaired t-test; *P < 0.05; **P < 0.01.

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Fig. 3. Paternal BPA diet (PBD) decreased myelin-associated molecules in the prefrontal cortex (PFC) of male offspring. A. Quantitative analysis of real time qRT-PCR showing the expression of Mbp, Mobp, Mog, Plp and Mag. B. Representative Western immunoblot images showing levels of MBP (molecular weight: 17e22 kDa) and GAPDH (molecular weight: 36 kDa) in mice PFC. C. Quantitative analysis of Western blots and histogram showing the expression of MBP. Data are means ± SEM (n ¼ 6 for each group); unpaired t-test; *P < 0.05; **P < 0.01.

2.4. BPA mice exhibit behavioral alterations The offspring of BPA fed fathers was referred to as “BPA mice”. In the open field test (OFT), the proportion of distance traveled of BPA mice in the center was significantly decreased (p ¼ 0.0151; Fig. 4B), and they were less active as measured in total distance traveled in the open field (p ¼ 0.0013; Fig. 4C), the percent of time in the center from BPA mice was significantly decreased (p ¼ 0.0014; Fig. 4D), and they had less entries into the center of the open field (p ¼ 0.0023; Fig. 4E). In the elevated zero maze (EZM) assay, BPA mice spent less time in the open arm (p ¼ 0.0125; Fig. 4G), and had less entries in the open arm (p ¼ 0.0045; Fig. 4H). In social interaction test, BPA mice show reduced exploration of the “Stranger 1” over the “Empty” relative to control mice (Fig. 5B), and BPA mice preferred to explore “Stranger 1” over “Empty” relative less than did control mice (p ¼ 0.0028; Fig. 5C). In preference for social novelty test, BPA mice show comparable preference on the “Stranger 2” over the “Stranger 1” relative to control mice (Fig. 5E). BPA mice preferred to explore Stranger 2 over Stranger 1, while the preference index of BPA mice similar to the preference index of control mice. There was no difference in the preference index in exploration with the stranger between BPA mice and control mice (p ¼ 0.2018; Fig. 5F).

3. Discussion This study provided evidence that PBD could alter ER stress related molecules in the PFC of male juvenile offspring, which also might affect myelination related molecules in the PFC. Importantly, our findings indicated that alterations of ER stress and myelination related molecules in the PFC induced by PBD could be a potential mechanism for behavioral changes. In this study, our proteomics analysis clearly identified a set of proteins with a ratio fold change >2 or <0.5 (see Supplemental Material, Fig. S2). Specially, we found BIP, the highest up-regulated protein (~3.4 fold) relative to the offspring of males fed a control diet. BIP plays an important role in protein folding and quality control of misfolded proteins in ER (Kroeger et al., 2012). Once the ER stress is provoked, cells could rapidly elevate the BIP protein level. Thus, increased BIP protein level is a subtle marker and central regulator of ER stress (Lee, 2005). Also the BIP western blot

confirmed the proteomics results. As we know, persistent ER stress may trigger apoptosis, which involves the levels of CHOP and BCL-2 (Szegezdi et al., 2006). Furthermore, we firstly used western blot to determine another subtle marker of ER stress: CHOP is also a key element of the switch from pro-survival to pro-death signaling (Kroeger et al., 2012; Szegezdi et al., 2006). We found PBD significantly elevated CHOP protein level in PFC of male offspring. Subsequently, we additionally used Western blot to confirm the decreasing of BCL-2, an anti-apoptosis factor and important regulator of apoptotic cell death in the PFC of male offspring (Szegezdi et al., 2006). Notably, we found that increased Bip mRNA and Chop mRNA expression of PFC was consistent with BIP protein and CHOP protein abundance in this model. Here, we ascertained that PBD could cause sustained ER stress by disrupting ER stress-related gene regulation, thereby possibly triggering cell apoptosis. One of the most common biological effects of sustained ER stress in brain is myelination or maintains disorder, thus myelination was a logical endpoint to evaluate in the PBD offspring (Naidoo, 2011; Raghubir et al., 2011; Lin and Popko, 2009). Myelin, a multilayered and dielectric membrane, is formed by wrapping around the axon of a neuron. Moreover, myelin has vital role in maintaining brain function since it can regulate neurotransmission and sustain neuronal survival (Cao et al., 2013a,b). Liu et al., found that ultrastructural changes of myelin were induced in the PFC of socially isolated mice. To date, many studies have linked impaired myelin with neuropsychiatric disorder, including schizophrenia, major depression, obsessiveecompulsive disorder, anxiety, and bipolar disorder (Bartzokis, 2005). Brain myelin has attracted more attention, as myelination is vulnerable in the brain development and myelin give a direct insight into molecular mechanisms for various psychiatric disorders (Bartzokis, 2005). Under healthy conditions, the integrity of myelin morphology in brain depends on major myelin molecules, such as Mog, Mag, Mbp, Mobp, Plp mRNA (Bartzokis, 2005). However, decreasing myelination related molecules altered myelin structure and oligodendrocyte function in PFC have been detected in human postmortem with psychiatric disorders (Bercury and Macklin, 2015). Impairments of myelination could directly to psychiatric disorders through energy restrictions during highly-demanding behavioral tasks and/or impaired action potential fidelity (Bercury and Macklin, 2015). Our results showed that the expression of major

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Fig. 4. Early adult BPA mice show increased anxiety-like response in open field test and elevated zero maze. A demonstrated an illustrative example of a control and a BPA mouse's travel pathway on the Open Field Test: the center zone (green). The percent of locomotion distance of BPA mice in the center was significantly decreased (B), and they were less active as measured in total distance traveled in the open field (C), BPA mice spent less time in the center of the open field (D), and they had less entries into the center of the open field (E). F demonstrated an illustrative example of a control and a BPA mouse's travel pathway on the EZM: open arms (red) and closed arms (green). BPA mice spent less time in the open arms (G), had less entries in the open arm (H). Two tailed t-test; n ¼ 15e20; *P < 0.05, **P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

myelin molecules were significantly decreased in the PFC of male offspring. Given that PFC dysfunction has been observed in many psychiatric disorders (Gamo and Arnsten, 2011), alterations of ER stress and myelination related molecules in PFC may shed light on the link between PBD and neurobehavioral problems in the offspring. Hence, our next goal was to investigate whether PBD affects behavioral development of their early adult and adult offspring. We first examined whether BPA mice displayed abnormal associative anxiety, exploration activity, and social behaviors. In the OFT, we found that BPA mice did not prefer to explore the “center zone”. Moreover, BPA mice also did not prefer to explore the “open arm” in the EZM. These results collectively suggest that BPA mice increase anxiety-like behavior. It should be noted that anxiety-like behavior might contribute to the impaired social behavior in BPA mice (Bielsky et al., 2004). We also examined whether BPA mice displayed abnormal social behavior. In social interaction test, BPA mice showed reduced sociability relative to control mice. While we found that BPA mice displayed the normal levels of social novelty recognition. These results suggest that BPA mice have partially

impaired social behavior. These aberrant behaviors are reminiscent of negative symptoms of mental patients who exhibit anxiety and unsocialness (Blanchard and Cohen, 2006). Incomplete myelination may, at least in part, underlie behavioral changes observed in this study, as myelin has been associated with social and anxiety-like behaviors (Liu et al., 2012). Especially, impaired myelination in PFC has been reported in a wide range of mental illness, including depression, autism, schizophrenia and anxiety (Liu et al., 2012). Moreover, altered myelination related molecules have been shown to be stable and partially clarify the long-term consequences of PBD on behavioral development in male early adult and adult offspring (Liu et al., 2012, 2016). Mechanismly, myelination is in need of the generation of a vast amount of lipid-rich membrane and proteins, which produces a heavy load on the secretory pathway of myelinating glia and leaves them vulnerable to ER stress (Clayton and Popko, 2016). ER is a centric subcellular compartment for protein modifies and folding. Massive secretory proteins and lipids, including major myelin proteins and lipids, were synthesized and processed in ER lumen. ER stress is a transient or sustained imbalance between the unfolded protein load and the capacity to

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Fig. 5. Adult BPA mice show reduced social interaction in three chambers social behavior test. A and D demonstrated an illustrative example of a control and a BPA mouse's travel pathway on the Three Chambers Social Behavior Assay Box. B. BPA mice show reduced exploration of the Stranger1 over the Empty relative to control mice. C. BPA mice preferred to explore Stranger 1 over Empty relative less than did control mice. E. BPA mice show comparable preference on the Stranger 2 over the Stranger 1 relative to control mice. F. BPA mice preferred to explore Stranger 2 over Stranger 1, similar to control mice. Two tailed t-test; n ¼ 15e20; *P < 0.05, **P < 0.01, ***P < 0.001.

process that load (Clayton and Popko, 2016; Lin et al., 2005). Decreasing myelination related molecules partially were associated with sustained ER stress, which could be attributed to insufficient myelin proteins and lipids (D'Antonio et al., 2013). There is strong evidence that ER stress play a role in a number of disorders of myelin (Clayton and Popko, 2016). However, myelination dysfunction may not be the only immediate explanation for the alteration in PBD-induced social behavior. According, in the present study, paternal BPA exposure modified ER stress- and myelination-associated molecules in PFC, and behavioral changes in male early adult and adult offspring, which presumably arise as result of abnormal epigenome induced by PBD in male germline. The mechanisms for PBD induced abnormal behavior of male offspring may be partially attributed to epigenetics effect (Miao et al., 2014). For instance, environmental exposures have been displayed to result in DNA methylation, miRNA expression and histone acetylation in germline, and subsequently genome-wide transcriptome alterations in multiple tissues, such as brain, testis and prostate of offspring (Curley et al., 2011). Both epigenome modifications and transcriptome lie behind psychiatric disorders, reproduction and prostate cancer that might be inherited  et al., 2015). transgenerationally when affecting germline (Lombo Previous study has found that BPA exposure decreased levels sperm of DNA methylation (Miao et al., 2014), and generated b-cell dysfunction and generational transmission of glucose intolerance  et al., in the offspring by male germ line (Mao et al., 2015). Lombo observed that lower spermatozoal insulin receptor b mRNAs after PBD caused cardiac malformations and continued to the F2

generation, clearly indicating an inheritance through paternal sperm. More importantly, we have found that pubertal exposure to Bisphenol A increased anxiety-like behavior in adult F0 male mice (Luo et al., 2013), which hinted that a link between BPA male exposure and behavioral dysfunction in forthcoming generations. Further studies are therefore warranted to understand the epigenetics effect of PBD on the subsequent behavioral changes of male offspring. This is the first demonstration that PBD changes PFC associated behaviors in their male offspring. The potential mechanisms seem to include alterations of ER stress and myelination related molecules. These findings extend the knowledge of the effects of parental BPA exposure on their offspring. Funding This work was supported by the China National Natural Science Foundation (31172376 and 31372497). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.chemosphere.2017.06.050. References Anderson, O.S., Peterson, K.E., Sanchez, B.N., Zhang, Z., Mancuso, P., Dolinoy, D.C., 2013. Perinatal bisphenol A exposure promotes hyperactivity, lean body composition, and hormonal responses across the murine life course. FASEB J. 27

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