Does the prenatal bisphenol A exposure alter DNA methylation levels in the mouse hippocampus?: An analysis using a high-sensitivity methylome technique

Does the prenatal bisphenol A exposure alter DNA methylation levels in the mouse hippocampus?: An... Background: There is still considerable debate about the effects of exposure to bisphenol A (BPA) an endocrine disrupter at low doses. Recently, many studies using animal models have shown that prenatal BPA exposure induces behavioral and neuronal disorders due to epigenetic changes in the brain. However, striking evidence of epigenomic changes has to be shown. Methods: To investigate whether low-dose BPA exposure in the fetal stage can alter CpG methylation levels in the central nervous system, the hippocampus of the inbred C57BL/6 J mouse as the target tissue was collected to detect alterations in CpG methylation levels using a highly sensitive method of genome-wide DNA methylation analysis, methylated site display–amplified fragment length polymorphism (MSD-AFLP). Results: BPA showed the sex-hormone like effects on male reproductive organs. Although we examined the methylation levels of 43,840 CpG sites in the control and BPA (200 μg/kg/day)-treated group (6 mice per group), we found no statistically significant changes in methylation levels in any CpG sites. Conclusions: At least under the experimental condition in this study, it is considered that the effect of low-dose BPA exposure during the fetal stage on hippocampal DNA methylation levels is extremely small. Keywords: Bisphenol A, Hippocampus, DNA methylation, Epigenetics Background study [6]. Subsequently, in experimental animal stu- Bisphenol A (BPA), an endocrine disruptor, has still been the dies, exposureofpregnantanimals to BPA ata low focusofattentionofscientistsinthe fieldofenvironmental doses (2–20 μg/kg) altered the development of male health since the last century. Even today, many researchers reproductive organs such as the prostate [7–9]. It was in field of hygiene and environmental health science are also reported that the sperm count decreased in adult investigating exposure level in human population [1, 2], and male rats even at a low dose of BPA exposure reporting studies focusing on mechanistic aspects [3–5]. An (20 μg/kg/day) [9]. This dose was comparable to the initial study found that BPA monomer released from a poly- maximum exposure levels (375–857 ng/kg/day) for in- carbonate dish has an estrogenic activity in a cell culture fants [10]. Subsequently, similar experiments on a low-dose of BPA exposure were performed by re- search groups associated with some industrial com- panies [11–13]. However, they failed to reproduce the * Correspondence: ohsako@m.u-tokyo.ac.jp above described results. Furthermore, the rat two- Toshiki Aiba and Seiichiroh Ohsako contributed equally to this work. 1 generation and three-generation reproductive toxicity Laboratory of Environmental Health Science, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, studies carried out by independent groups did not 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654, Japan detect changes in the reproductive organs by BPA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Aiba et al. Genes and Environment (2018) 40:12 Page 2 of 8 diet throughout the experiments [14, 15]. These in- Methods consistent conclusions brought the story of BPA into Reagents complex. The reagents and materials used in this study were as After a while, with the focus of studies shifting away follows. BPA and corn oil were purchased from Wako from reproductive toxicity, a number of studies started to Pure Chemical Industries (Osaka, Japan). T4 DNA ligase show that low-dose BPA exposure in the perinatal period and restriction enzymes Hpa II, Msp I, Sbf I were from affected behavioral and brain development [16–18]. In New England Biolabs (MA, USA). AllPrep DNA/RNA particular, the effects of low-dose BPA exposure on the mini kit was from Qiagen (Hilden, Germany). Oligonu- hippocampus were observed in many studies. BPA was cleotides were from Operon (Alameda, Calif., USA) and found to decreased hippocampal spine number as induced streptavidin-coated magnetic beads (Dynabeads M-280 by estrogen in the African green monkey (Chlorocebus Streptavidin) were from Dynal (Oslo, Norway). TITAN- aethiops sabaeus)[19], inhibit acetylcholine production in IUM Taq DNA polymerase was from Takara Bio mice [20], and reduce the spine density in the mouse CA1 (Kusatsu, Japan) and LightCycler® 480 SYBR Green I region [21], suggesting that perinatal BPA exposure may Master was from Roche (Diagnostics GmbH, Mannheim, cause developmental disturbance. Germany). POP-7™ Polymer, GeneScan™ 500 LIZ® Size Such effects of BPA can be regarded as the Developmental Standard and BigDye® Terminator v3.1 Cycle Sequencing Origins of Health and Disease (DOHaD), that is fetal/neo- Kit were from ThermoFisher Scientific Inc., (San Diego, natal environmental factors are the causes of diseases after CA, USA). maturation [22–24]. It is hypothesized that exposure to en- vironmental factors changes epigenomic states, such as Animals and treatments DNA methylation levels, which remain after birth Pregnant C57BL/6 J mice were purchased from CLEA [25, 26]. Furthermore, many challenging studies re- Japan (Tokyo, Japan). The mice were bred at temperature ported that gestational exposure to environmental of 23 °C, in 12/12 h light/dark cycle (animal facility, 08: 00– chemicals, such as vinclozolin and arsenite, causes in 20: 00). Laboratory rodent feed (Lab MR stock, Nosan, epigenetic transgenerational changes in rodents [27– Yokohama, Japan) and distilled water were provided ad libi- 29]. Now the chemical-induced epigenomic alteration tum. BPA in vehicle (corn oil) or vehicle only was orally ad- is extremely controversial issue not only in DOHaD ministered for 12 consecutive days at a dose of 200 μg/kg/ studies but also in general biology concerning evolution day from the 6th to 17th days of gestation (GD); (Fig. 1). [30]. In our series of environmental epigenetics studies, Pup mice were culled to 5 males and 3 females on postnatal we found that a single and low dose of 2,3,7,8-tetrachloro- day 9 (PND 9) and maintained under the same conditions. dibenzo-p-dioxin induced hypomethylation of CpGs in Body weight and anogenital distance (AGD) were measured Cyp1a1 gene promoter region in mouse liver [31]. This from PND 0 to PND 49. Male (PND 84) and female (PND hypomethylation was induced by adulthood exposure, but 87) mice were killed by cervical dislocation. Body weight also by in utero exposure [32]. Thus, we convinced that a and weights of some tissues [liver, heart, kidney, urogenital low level of environmental pollutants causes the alterna- complex (UGC), testes, and uterus] were measured. The tion of DNA methylation status. hippocampus was dissected on ice, stored at − 80 °C, and Indeed, several animal studies have shown that used for DNA methylation analysis. The animal experiment prenatal exposure to BPA causes epigenetic changes protocol was approved by the Animal Care and Use Com- [33–38]. However, there are only a few studies using mittee of the University of Tokyo (I-P11–015). a sufficient number of inbred animals for comparison and/or by genome-wide DNA methylation analysis. MSD-AFLP Recently, we have developed a technique called meth- MSD-AFLP analysis was performed in accordance with ylated site display-amplified fragment length poly- our previous report [39]. Briefly, genomic DNA was iso- morphism (MSD-AFLP) analysis, which is a sensitive lated from the male pup hippocampus using the AllPrep and affordable method of CpG methylation profile DNA/RNA Mini Kit. Genomic DNA (100 ng) was analysis [39]. MSD-AFLP analysis is cost-effective be- digested with the primary restriction enzymes Sbf I and cause multiple samples can be analyzed at the same Msp I, and then ligated using adaptor A (Upper, b-TCC time. MSD-AFLP analysis is also a very sensitive tech- GAC TGG TAT CAA CGC AGA GTA CTA GAG TTG nique because it can detect slight changes in DNA CA; Lower, p-ACT CTA GTA CTC TGC GTT GAT methylation levels. Therefore, in this study, a low-dose ACC AGT CGG A; here, b indicates 5′-biotiylation and BPA (200 μg/kg/day) was administered to pregnant p, 5′-phospholation). The ligated DNA fragment was B57BL/6 J mice and the changes of DNA methylation digested with methylation-sensitive Hpa II. After the level in the hippocampus of offspring were detected by ligation reaction of adaptor B (Upper, AAT GGC TAC MSD-AFLP analysis. ACG AAC TCG GTT CAT GAC C; Lower, p-CGT Aiba et al. Genes and Environment (2018) 40:12 Page 3 of 8 Daily dose of BPA (0 or 200 µg/kg/day) PND 84 (male) GD6 GD17 PND 9 PND 49 PND 87 (female) Gestation Birth Culling Dissection (Cont, dam=5; BPA, dam=6) Fig. 1 Experimental design of prenatal BPA exposure to mice. Vehicle (corn oil) or BPA was orally administered to female C57BL/6 J mice during gestation at a dose of 200 μg/kg/day daily by gavage from GD 6 to GD 17. Pups were eliminated to be of the same number per mother on PND 9. Body weight and AGD were measured from PND 0 to PND 49. Male (PND 84) and female (PND 87) mice were sacrificed by cervical dislocation GTC ATG AAC CGA GTT CGT GTA GCC ATT), pre- p-value less than 0.05 was considered statistically PCR was performed using adapter-specific primers (For- significant. The significance of differences in methyla- ward, AAT GGC TAC ACG AAC TCG GTT CAT GAC tion level between groups was analyzed by adjusted ACG G; Reverse, TCC GAC TGG TAT CAA CGC p-value (q-value, false discovery rate (FDR)) calculated AGA) to amplify only the genomic DNA fragments that with Benjamini–Hochberg (BH) correction after Student’s have methylated CpG at the end. The pre-PCR amplicon t-test using R statistical software. Statistical probabilities of (MSD library) was subjected to selective PCR using 256 FDRless than0.05were considered significant. Hierarchical primer sets (Forward, f-AAT GGC TAC ACG AAC clustering analysis (HCA) and principal component analysis TCG GTT CAT GAC AII INN; Reverse, AGA GTA (PCA) of methylation patterns were performed using CTA GAG TTG CAG GNN; here, f means 5′-6-carbo- Euclidean distance, and the unweighted pair grouping xyfluorescein (6-FAM) conjugation; I, inosine). The method using arithmetic mean (UPGMA) was carried out. product of selective PCR was electrophoresed using a The Kyoto Encyclopedia Genes and Genome (KEGG) en- capillary sequencer to obtain an AFLP chart. richment analysis was performed using Gene Set Enrichment Analysis (GSEA) software. GFDB analysis The genomic position was predicted from the AFLP Results peak chart using the Genome DNA Fragment Database Reproductive outcome (GFDB) system. GFDB can simulate the MSD-AFLP Prenatal BPA exposure did not significantly affect litter size procedure of genomic DNA cleavage with any restriction or sex ratio compared with controls (Additional file 1:Table enzyme or any selective PCR [39]. S1). In both sexes, the BPA-exposed group showed no effect on body weight or AGD from birth to PND 49 (Additional MSRE–PCR file 1:FigureS1).However,BPA exposure significantly Methylation-sensitive restriction-enzyme-dependent PCR decreased the UGC weight of male on PND 84 (Table 1). (MSRE-PCR) analysis of two CpGs was carried out as de- There were no significant differences in other tissues. scribed in our previous study {Amenya, 2016 #432}. The following primers were used in this study: Chr 4: 35339023 (Forward, TCA CTC TTC ACC TGC AGG Table 1 Effects of prenatal BPA exposure on tissue weight of AAG; Reverse, GCT GTC ACT CTG TGC TCT TCT) mice at dissection and Chr X: 74707008 (Forward, CTG CTT TGC TGC Control BPA TCA GAG TTT; Reverse, CCC GAG TCC TGA GAT Male (n = 24) Female (n = 14) Male (n = 19) Female (n = 12) TAA AGG). Purified genomic DNA (100 ng) was divided Liver 4.340 ± 0.063 5.703 ± 0.284 4.231 ± 0.066 5.183 ± 0.196 into two portions. One portion was digested with Hpa II and the other was not digested. Both were subjected to UGC 1.030 ± 0.015 N/A 0.941 ± 0.026* N/A quantitative PCR analysis using Light Cycler 480 and the Testis 0.764 ± 0.012 N/A 0.743 ± 0.014 N/A methylation level of CpG was calculated as the ratio of Uterus N/A 0.686 ± 0.036 N/A 0.567 ± 0.083 target copy number from nondigested DNA to that from Heart 0.576 ± 0.014 0.514 ± 0.011 0.573 ± 0.013 0.501 ± 0.012 Hpa II-digested DNA. kidney 1.138 ± 0.017 1.158 ± 0.019 1.138 ± 0.014 1.146 ± 0.017 , Tissue weight data was expressed as percentage per body weight Statistical analysis N/A, not available The significance of differences in the tissue weight and Data were expressed as means ± SE. Statistically significant difference MSRE-PCR measurement analyzed by Student’s t-test. between means from control group was analyzed by t-test (*: p < 0.05) Aiba et al. Genes and Environment (2018) 40:12 Page 4 of 8 Overall effect of BPA exposure on CpG methylation shown in MSD-AFLP analysis were detected, neither of the We investigated the effects of prenatal BPA exposure on two CpGs showed significant difference in the MSRE-PCR the CpG methylation profile of the hippocampus in male analysis (Additional file 1:FigureS3). pups by MSD–AFLP analysis (Additional file 1:FigureS2). A total of 43,840 CpG data were obtained by using 256 se- The KEGG enrichment analysis of CpG methylation level lective primer sets. The mean methylation levels were 59.5 The enrichment analysis of CpG methylation level on ± 0.1% in the control group and 59.0 ± 0.1% in the BPA- the intragenic region and promoter region (5 kb exposed group (p = 0.22); (Fig. 2). There was no significant upstream from TSS) was performed to examine if statis- difference between control and BPA samples in the HCA tical difference is detected in KEGG pathway (Additional (Fig. 3). Moreover, PCA showed no clusters and large vari- file 1: Table S2). The hypermethylated CpGs in BPA ex- ances of both control and BPA groups (Fig. 4). These obser- posure were tended to highly enrich in the pathway of vations suggest that prenatal BPA exposure hardly affects N-Glycan Biosynthesis (NES = 1.600; FDR = 0.77). On DNA methylation in the mouse hippocampus. the other hands, the hypomethylated CpGs in BPA exposure were tended to highly enrich in the pathway of Statistical analysis for possibility of BPA-induced change Histidine Metabolism (NES = 1.602; FDR = 0.917). How- in CpG methylation ever, the minimum FDR was 0.776, no pathway with sig- We analyzed the significance of differences in methylation nificantly different methylation level was detected level between groups by q-value calculated with BH correc- according to the criteria FDR < 0.05. tion after Student’s t-test using all CpGs data determined by MSD-AFLP to explore possibility of BPA-induced Discussion changes. However, we detected no CpGs showing less than In this study we investigated whether prenatal low-dose 0.05 of q-value of FDR. Figure 5 is a volcanic plot of q-value BPA exposure causes epigenomic changes in the cerebral of FDR and fold of changes by BPA exposure. We found hippocampus. Our study showed that the weight of the only three CpGs shows the lowest q-value of FDR (q = 0.24, male UGC consisting of three tissues, i.e., the prostate, −log(q-value) = 0.620) out of 43,840 CpGs. The genomic seminal vesicle, and urethra, was significantly reduced. positions of three CpGs predicted by using the GFDB sys- Although this observation is in contrast to a previous re- tem were shown in Table 2. Using the genomic DNA sam- port on prostate development [8], our experiment also ples, in order to examine if fold of changes detected by showed sex-hormone-like activity in pups with maternal MSD-AFLP are significant, we performed MSRE-PCR ana- BPA exposure. Therefore, we focused on the hippocam- lysis. Since another Hpa II site is located on immediately pus, which is the part of the central nervous system that nearby the CpG (Chr 5: 137753995), it was impossible to is thought to be most strongly affected by BPA, as design primers that are amplifiable in MSRE-qPCR. There- shown by recent studies, and performed MSD–AFLP fore, we examined the remaining only two CpGs (Chr 4: analysis which is a new method that can detect genome- 35339023 and Chr X: 74707008). Although similar trends wide CpG methylation level changes with a difference in of difference between control and BPA in both CpGs the methylation level of less than 5% and a variation rate of the methylation level of less than 10% in an inter- tissue comparison experiment [39]. However, in this study, no statistically significant differences in methylation level between the control group and the BPA-exposed BPA (59.0 ± ± 0.1%) group were detected in all CpGs (43,840) analyzed in the Control (59.5 ± ± 0.1%) hippocampus. The effects of BPA exposure on CpG methylation level in genomic DNA in the brain and gonads have been re- Control ported. BPA at a low dose (20 μg/kg) was administered to BPA pregnant ICR/Jcl mice and the methylation level changes in the forebrain of the fetus was analyzed by the genome- wide restriction landmark genomic scanning (RLGS) method [37], which showed that the methylation levels of 48 CpGs were altered. A low dose BPA (10 μg/kg) 0 20 40 60 80 100 injection to pups of Sprague-Dawley (SD) rat has also been Methylation level (%) reported to lead to the hypomethylation of the phospho- Fig. 2 Histogram of methylation levels of all CpGs detected by diesterase type 4 gene in the prostate and increase suscepti- MSD-AFLP analysis. Blue and red lines indicate the mean methylation bility to 7,12-dimethylbenz[a]anthracene (DMBA)-induced levels in the control and BPA-exposed groups prostate carcinogenesis [35]. On the other hand, in utero Number of CpG-site Aiba et al. Genes and Environment (2018) 40:12 Page 5 of 8 Control BPA Fig. 3 Hierarchical clustering analysis (HCA) of the effects of BPA. HCA of normalized methylation patterns of each sample using Euclidean distance and weighted pair grouping method using arithmetic mean (UPGMA) and lactational exposure of BPA (0.05, 7.5, 30 and 120 mg/ excretion rate (ADME) vary depending on the rat strain. kg/day, by oral) to the mother rats of inbred Fisher 344 strain Therefore, differences in ADME values among strains may did not result in the DMBA-induced carcinogenesis of the be reflected in blood BPA level and may have affected the ex- prostate [40]. The urinary excretion rate of BPA were 21% perimental results. Additionally, there is an argument that and 70%, and the fecal excretion rates of BPA were 42% and closed colonies such as ICR mice and SD rats show genetic 50% in SD and Fisher 344 rats, respectively [41]. Further- diversity within colonies, resulting in false positives data that more, the absorption, distribution, metabolic rate, and occasionally occur due to genetic diversity that may arise even by simply dividing pups into two groups [42]. Thus, it is preferable to use inbred strain animals to exclude genetic variation among individuals in the analysis of epigenomic changes. As an example, when using inbred Avy mice, it has been reported that prenatal BPA exposure (50-mg BPA/kg diet) decreased the CpG methylation levels of the transposon sequence in Agouti by about 10% [33]. Agouti determines body hair color and the inserted transposon methylation negatively regulates gene expression. Therefore, they con- cluded that the yellow hair of Avy mice changed into brown due to this hypomethylation. However, they did not study whether there was any alteration in other genes. Control Using inbred BALB/c mice, there was a report that BPA prenatal exposure of BPA (200 μg/kg/day, by oral) hyper- methylated the transcriptional regulatory region of the hippocampal neurotrophic factor (BDNF) of the males and consequently decreased the mRNA expression level [36]. Additionally, they also analyzed using free DNA in Fig. 4 Principal component analysis (PCA) using MSD-AFLP data. blood and interestingly showed that brain BDNF methyla- Control, red circle (n = 6); BPA, blue circle (n = 6). PCA showed large tion levels correlate with those of blood DNA [36]. The variances of both control and BPA groups with no clusters levels of 17β-estradiol and testosterone and the ratio of Aiba et al. Genes and Environment (2018) 40:12 Page 6 of 8 1.4 q = 0.05 1.2 1.0 Chr 4: 35339023 Chr 5: 137753995 Chr X: 74707008 0.8 0.6 0.4 q = 0.5 0.2 -0.5 -0.3 -0.1 0.1 0.3 0.5 Log (fold of change) Fig. 5 Effects of BPA on CpG methylation profile in hippocampus. Volcanic plot showing the difference in methylation level between control group and BPA-exposed group. The logarithmic value of the fold of change (BPA/control) is presented in the x-axis, and the logarithmic value of the q-value is presented in the y-axis. Three CpGs which show less than 0.24 of q-value are indicated by arrows these two sex hormones were different between mouse environmental chemicals. If further analysis is performed strains [43]. The difference in the endocrine system be- with a wider coverage, it may be possible to detect signifi- tween C57BL/6 J and CBA/Lac mice appears to affect the cant BPA-specific methylation level alterations even in the activity of endocrine disruptors, such as BPA. In addition, C57BL/6 J hippocampus. although 17β-estradiol had been used to positive control in the studies of effect for prenatal BPA exposure of SD rat offspring on increase DMBA-induced prostate carcino- Conclusions genesis [35], neonatal exposure of 17β-estradiol had no ef- In this study, we investigated whether BPA alters CpG fects on mutagenicity of DMBA in reproductive tissues of methylation levels in the mouse hippocampus. Despite adult Big Blue transgenic mice [44]. Thus, the difference the use of MSD–AFLP analysis, which is a high- in genetic background may change the effects of BPA. Al- precision and highly sensitive analytical method, no though we analyzed 43,840 CpGs in this study, which are changes in methylation levels as the effect of low-dose approximately 0.3% out of the total 20,000,000 CpGs in BPA exposure were detected. Therefore, although fur- mouse genome, we were unable to detect statistically sig- ther analysis is necessary, we concluded that under the nificant changes in CpG methylation levels, which might experimental conditions of the present study, the effects be due to the stability of the methylated CpG in the of prenatal BPA exposure on the hippocampal DNA C57BL/6 J mouse hippocampus against small amount of methylation are extremely small. Table 2 The genomic positions of three CpGs showing minimum q-value predicted by using GFDB a b Chr. Position Gene name Methylation level (%) log ratio q-value Cont (n = 6) BPA (n = 6) 5 137,753,995 Trip6 18.21 ± 0.79 26.85 ± 0.67 0.17 0.24 4 35,339,023 9.3kbp up stream of RP23-307 M2.4 29.36 ± 0.34 34.67 ± 0.52 0.07 0.24 X 74,707,008 4.4kbp down stream of RP23-238B14.1 80.53 ± 1.38 65.66 ± 1.35 −0.09 0.24 , Chromosomal nucleotide position of methylated cytosine , The ratio means fold of change (BPA/control) −Log (q−value) Aiba et al. Genes and Environment (2018) 40:12 Page 7 of 8 Additional file different regions of the human brain. Int. J. Environ. Res. Public Health [Internet]. 2017;14:1059. Available from: http://www.mdpi.com/1660-4601/14/9/1059 3. Arase S, Ishii K, Igarashi K, Aisaki K, Yoshio Y, Matsushima A, et al. Endocrine Additional file 1: Table S1. The pup number and sex ratio. Figure S1. disrupter bisphenol a increases in situ estrogen production in the mouse Effect of prenatal BPA exposure on body weight and anogenital distance urogenital sinus. Biol Reprod [Internet]. 2011;84:734–42. Available from: (AGD). Figure S2. A typical MSD-AFLP peak chart after electrophoresis http://www.ncbi.nlm.nih.gov/pubmed/21123812 obtained by a selective primer set. Figure S3. MSRE-PCR analysis of 4. LiuX,MatsushimaA,ShimohigashiM,Shimohigashi Y.Acharacteristic representative CpGs (Chr 4: 35339023 and Chr X: 74707008) showing the back support structure in the bisphenol A-binding pocket in the minimum q-value obtained from the MSD-AFLP data. Table S2. The human nuclear receptor ERRγ. Vanacker J-M, editor. PLoS One [Internet]. KEGG enrichment analysis of the effect on the DNA methylation. 2014;9:e101252. Available from: http://www.ncbi.nlm.nih.gov/pubmed/ (DOCX 289 kb) 5. Sartain CV, Hunt PA. An old culprit but a new story: bisphenol a and Abbreviations “NextGen” bisphenols. Fertil Steril [Internet]. 2016;106:820–6. Available from: AGD: Anogenital distance; BPA: Bisphenol A; DOHaD: Developmental Origins http://www.ncbi.nlm.nih.gov/pubmed/27504789 of Health and Disease; FDR: False discovery rate; GFDB: Genome DNA 6. Krishnan AV, Stathis P, Permuth SF, Tokes L, Feldman D. Bisphenol-a: an Fragment Database; HCA: Hierarchical clustering analysis; KEGG: Kyoto estrogenic substance is released from polycarbonate flasks during Encyclopedia Genes and Genome; MSD-AFLP: Methylated site display- autoclaving. Endocrinology [Internet]. 1993;132:2279–86. Available from: amplified fragment length polymorphism; MSRE-PCR: Methylation-sensitive http://www.ncbi.nlm.nih.gov/pubmed/8504731 restriction enzyme-dependent PCR; PCA: Principal component analysis; 7. Nagel SC, vom Saal FS, Thayer KA, Dhar MG, Boechler M, Welshons WV. UGC: Urogenital complex Relative binding affinity-serum modified access (RBA-SMA) assay predicts the relative in vivo bioactivity of the xenoestrogens bisphenol a and octylphenol. Environ Health Perspect [Internet]. 1997;105:70–6. Available Acknowledgments from: http://www.ncbi.nlm.nih.gov/pubmed/9074884 Dr. Ruby Kuroiwa, for her kind help in English manuscript writing. 8. vom Saal FS, Cooke PS, Buchanan DL, Palanza P, Thayer KA, Nagel SC, et al. A physiologically based approach to the study of bisphenol a and other Funding estrogenic chemicals on the size of reproductive organs, daily sperm This work was supported by JSPS KAKENHI Grant Number 23310044 production, and behavior. Toxicol Ind Health [Internet]. 1998;14:239–60. (Grant-in-Aid for Scientific Research B) to SO, JSPS KAKENHI Grant Number Available from: http://www.ncbi.nlm.nih.gov/pubmed/9460178 15H02830 (Grant-in-Aid for Scientific Research B) to SO, and JSPS KAKENHI 9. Sakaue M, Ohsako S, Ishimura R, Kurosawa S, Kurohmaru M, Hayashi Y, et al. Grant Number 17H06396 (Grant-in-Aid for Scientific Research on Innovation Bisphenol-a affects spermatogenesis in the adult rat even at a low dose. J Areas) to SO. Occup Health [Internet]. 2001;43:185–90. Available from: http://joi.jlc.jst.go. jp/JST.Journalarchive/joh1996/43.185?from=CrossRef Availability of data and materials 10. Shelby MD. NTP-CERHR monograph on the potential human reproductive and The original data of the MSD-AFLP charts will be available upon request. developmental effects of bisphenol a. NTP CERHR MON [Internet]. 2008;v, vii–ix: 1–64 passim. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19407859 Authors’ contributions 11. Ashby J, Tinwell H, Haseman J. Lack of effects for low dose levels of SO conceived and designed experiment; TA, TS, and AH performed bisphenol a and diethylstilbestrol on the prostate gland of CF1 mice experiments; SS and HY made GFDB system; TA and TM analyzed the data exposed in utero. Regul Toxicol Pharmacol [Internet]. 1999;30:156–66. with help by WF; TA and SO wrote the manuscript. All authors have read Available from: http://www.ncbi.nlm.nih.gov/pubmed/10536110 and approved the final manuscript. 12. Ashby J, Tinwell H, Lefevre PA, Joiner R, Haseman J. The effect on sperm production in adult Sprague-Dawley rats exposed by gavage to bisphenol a Ethics approval and consent to participate between postnatal days 91-97. Toxicol Sci [Internet]. 2003;74:129–38. Not applicable. Available from: http://www.ncbi.nlm.nih.gov/pubmed/12773777 13. Cagen SZ, Waechter JMJ, Dimond SS, Breslin WJ, Butala JH, Jekat FW, et al. Competing interests Normal reproductive organ development in CF-1 mice following prenatal The authors declare that they have no competing interest. exposure to bisphenol a. Toxicol Sci [Internet]. 1999;50:36–44. Available from: 10445751 14. Ema M, Fujii S, Furukawa M, Kiguchi M, Ikka T, Harazono A. Rat two- Publisher’sNote generation reproductive toxicity study of bisphenol a. Reprod Toxicol Springer Nature remains neutral with regard to jurisdictional claims in [Internet]. 2001;15:505–23. Available from: http://www.ncbi.nlm.nih.gov/ published maps and institutional affiliations. pubmed/11780958 15. 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Does the prenatal bisphenol A exposure alter DNA methylation levels in the mouse hippocampus?: An analysis using a high-sensitivity methylome technique

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Abstract

Background: There is still considerable debate about the effects of exposure to bisphenol A (BPA) an endocrine disrupter at low doses. Recently, many studies using animal models have shown that prenatal BPA exposure induces behavioral and neuronal disorders due to epigenetic changes in the brain. However, striking evidence of epigenomic changes has to be shown. Methods: To investigate whether low-dose BPA exposure in the fetal stage can alter CpG methylation levels in the central nervous system, the hippocampus of the inbred C57BL/6 J mouse as the target tissue was collected to detect alterations in CpG methylation levels using a highly sensitive method of genome-wide DNA methylation analysis, methylated site display–amplified fragment length polymorphism (MSD-AFLP). Results: BPA showed the sex-hormone like effects on male reproductive organs. Although we examined the methylation levels of 43,840 CpG sites in the control and BPA (200 μg/kg/day)-treated group (6 mice per group), we found no statistically significant changes in methylation levels in any CpG sites. Conclusions: At least under the experimental condition in this study, it is considered that the effect of low-dose BPA exposure during the fetal stage on hippocampal DNA methylation levels is extremely small. Keywords: Bisphenol A, Hippocampus, DNA methylation, Epigenetics Background study [6]. Subsequently, in experimental animal stu- Bisphenol A (BPA), an endocrine disruptor, has still been the dies, exposureofpregnantanimals to BPA ata low focusofattentionofscientistsinthe fieldofenvironmental doses (2–20 μg/kg) altered the development of male health since the last century. Even today, many researchers reproductive organs such as the prostate [7–9]. It was in field of hygiene and environmental health science are also reported that the sperm count decreased in adult investigating exposure level in human population [1, 2], and male rats even at a low dose of BPA exposure reporting studies focusing on mechanistic aspects [3–5]. An (20 μg/kg/day) [9]. This dose was comparable to the initial study found that BPA monomer released from a poly- maximum exposure levels (375–857 ng/kg/day) for in- carbonate dish has an estrogenic activity in a cell culture fants [10]. Subsequently, similar experiments on a low-dose of BPA exposure were performed by re- search groups associated with some industrial com- panies [11–13]. However, they failed to reproduce the * Correspondence: ohsako@m.u-tokyo.ac.jp above described results. Furthermore, the rat two- Toshiki Aiba and Seiichiroh Ohsako contributed equally to this work. 1 generation and three-generation reproductive toxicity Laboratory of Environmental Health Science, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, studies carried out by independent groups did not 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654, Japan detect changes in the reproductive organs by BPA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Aiba et al. Genes and Environment (2018) 40:12 Page 2 of 8 diet throughout the experiments [14, 15]. These in- Methods consistent conclusions brought the story of BPA into Reagents complex. The reagents and materials used in this study were as After a while, with the focus of studies shifting away follows. BPA and corn oil were purchased from Wako from reproductive toxicity, a number of studies started to Pure Chemical Industries (Osaka, Japan). T4 DNA ligase show that low-dose BPA exposure in the perinatal period and restriction enzymes Hpa II, Msp I, Sbf I were from affected behavioral and brain development [16–18]. In New England Biolabs (MA, USA). AllPrep DNA/RNA particular, the effects of low-dose BPA exposure on the mini kit was from Qiagen (Hilden, Germany). Oligonu- hippocampus were observed in many studies. BPA was cleotides were from Operon (Alameda, Calif., USA) and found to decreased hippocampal spine number as induced streptavidin-coated magnetic beads (Dynabeads M-280 by estrogen in the African green monkey (Chlorocebus Streptavidin) were from Dynal (Oslo, Norway). TITAN- aethiops sabaeus)[19], inhibit acetylcholine production in IUM Taq DNA polymerase was from Takara Bio mice [20], and reduce the spine density in the mouse CA1 (Kusatsu, Japan) and LightCycler® 480 SYBR Green I region [21], suggesting that perinatal BPA exposure may Master was from Roche (Diagnostics GmbH, Mannheim, cause developmental disturbance. Germany). POP-7™ Polymer, GeneScan™ 500 LIZ® Size Such effects of BPA can be regarded as the Developmental Standard and BigDye® Terminator v3.1 Cycle Sequencing Origins of Health and Disease (DOHaD), that is fetal/neo- Kit were from ThermoFisher Scientific Inc., (San Diego, natal environmental factors are the causes of diseases after CA, USA). maturation [22–24]. It is hypothesized that exposure to en- vironmental factors changes epigenomic states, such as Animals and treatments DNA methylation levels, which remain after birth Pregnant C57BL/6 J mice were purchased from CLEA [25, 26]. Furthermore, many challenging studies re- Japan (Tokyo, Japan). The mice were bred at temperature ported that gestational exposure to environmental of 23 °C, in 12/12 h light/dark cycle (animal facility, 08: 00– chemicals, such as vinclozolin and arsenite, causes in 20: 00). Laboratory rodent feed (Lab MR stock, Nosan, epigenetic transgenerational changes in rodents [27– Yokohama, Japan) and distilled water were provided ad libi- 29]. Now the chemical-induced epigenomic alteration tum. BPA in vehicle (corn oil) or vehicle only was orally ad- is extremely controversial issue not only in DOHaD ministered for 12 consecutive days at a dose of 200 μg/kg/ studies but also in general biology concerning evolution day from the 6th to 17th days of gestation (GD); (Fig. 1). [30]. In our series of environmental epigenetics studies, Pup mice were culled to 5 males and 3 females on postnatal we found that a single and low dose of 2,3,7,8-tetrachloro- day 9 (PND 9) and maintained under the same conditions. dibenzo-p-dioxin induced hypomethylation of CpGs in Body weight and anogenital distance (AGD) were measured Cyp1a1 gene promoter region in mouse liver [31]. This from PND 0 to PND 49. Male (PND 84) and female (PND hypomethylation was induced by adulthood exposure, but 87) mice were killed by cervical dislocation. Body weight also by in utero exposure [32]. Thus, we convinced that a and weights of some tissues [liver, heart, kidney, urogenital low level of environmental pollutants causes the alterna- complex (UGC), testes, and uterus] were measured. The tion of DNA methylation status. hippocampus was dissected on ice, stored at − 80 °C, and Indeed, several animal studies have shown that used for DNA methylation analysis. The animal experiment prenatal exposure to BPA causes epigenetic changes protocol was approved by the Animal Care and Use Com- [33–38]. However, there are only a few studies using mittee of the University of Tokyo (I-P11–015). a sufficient number of inbred animals for comparison and/or by genome-wide DNA methylation analysis. MSD-AFLP Recently, we have developed a technique called meth- MSD-AFLP analysis was performed in accordance with ylated site display-amplified fragment length poly- our previous report [39]. Briefly, genomic DNA was iso- morphism (MSD-AFLP) analysis, which is a sensitive lated from the male pup hippocampus using the AllPrep and affordable method of CpG methylation profile DNA/RNA Mini Kit. Genomic DNA (100 ng) was analysis [39]. MSD-AFLP analysis is cost-effective be- digested with the primary restriction enzymes Sbf I and cause multiple samples can be analyzed at the same Msp I, and then ligated using adaptor A (Upper, b-TCC time. MSD-AFLP analysis is also a very sensitive tech- GAC TGG TAT CAA CGC AGA GTA CTA GAG TTG nique because it can detect slight changes in DNA CA; Lower, p-ACT CTA GTA CTC TGC GTT GAT methylation levels. Therefore, in this study, a low-dose ACC AGT CGG A; here, b indicates 5′-biotiylation and BPA (200 μg/kg/day) was administered to pregnant p, 5′-phospholation). The ligated DNA fragment was B57BL/6 J mice and the changes of DNA methylation digested with methylation-sensitive Hpa II. After the level in the hippocampus of offspring were detected by ligation reaction of adaptor B (Upper, AAT GGC TAC MSD-AFLP analysis. ACG AAC TCG GTT CAT GAC C; Lower, p-CGT Aiba et al. Genes and Environment (2018) 40:12 Page 3 of 8 Daily dose of BPA (0 or 200 µg/kg/day) PND 84 (male) GD6 GD17 PND 9 PND 49 PND 87 (female) Gestation Birth Culling Dissection (Cont, dam=5; BPA, dam=6) Fig. 1 Experimental design of prenatal BPA exposure to mice. Vehicle (corn oil) or BPA was orally administered to female C57BL/6 J mice during gestation at a dose of 200 μg/kg/day daily by gavage from GD 6 to GD 17. Pups were eliminated to be of the same number per mother on PND 9. Body weight and AGD were measured from PND 0 to PND 49. Male (PND 84) and female (PND 87) mice were sacrificed by cervical dislocation GTC ATG AAC CGA GTT CGT GTA GCC ATT), pre- p-value less than 0.05 was considered statistically PCR was performed using adapter-specific primers (For- significant. The significance of differences in methyla- ward, AAT GGC TAC ACG AAC TCG GTT CAT GAC tion level between groups was analyzed by adjusted ACG G; Reverse, TCC GAC TGG TAT CAA CGC p-value (q-value, false discovery rate (FDR)) calculated AGA) to amplify only the genomic DNA fragments that with Benjamini–Hochberg (BH) correction after Student’s have methylated CpG at the end. The pre-PCR amplicon t-test using R statistical software. Statistical probabilities of (MSD library) was subjected to selective PCR using 256 FDRless than0.05were considered significant. Hierarchical primer sets (Forward, f-AAT GGC TAC ACG AAC clustering analysis (HCA) and principal component analysis TCG GTT CAT GAC AII INN; Reverse, AGA GTA (PCA) of methylation patterns were performed using CTA GAG TTG CAG GNN; here, f means 5′-6-carbo- Euclidean distance, and the unweighted pair grouping xyfluorescein (6-FAM) conjugation; I, inosine). The method using arithmetic mean (UPGMA) was carried out. product of selective PCR was electrophoresed using a The Kyoto Encyclopedia Genes and Genome (KEGG) en- capillary sequencer to obtain an AFLP chart. richment analysis was performed using Gene Set Enrichment Analysis (GSEA) software. GFDB analysis The genomic position was predicted from the AFLP Results peak chart using the Genome DNA Fragment Database Reproductive outcome (GFDB) system. GFDB can simulate the MSD-AFLP Prenatal BPA exposure did not significantly affect litter size procedure of genomic DNA cleavage with any restriction or sex ratio compared with controls (Additional file 1:Table enzyme or any selective PCR [39]. S1). In both sexes, the BPA-exposed group showed no effect on body weight or AGD from birth to PND 49 (Additional MSRE–PCR file 1:FigureS1).However,BPA exposure significantly Methylation-sensitive restriction-enzyme-dependent PCR decreased the UGC weight of male on PND 84 (Table 1). (MSRE-PCR) analysis of two CpGs was carried out as de- There were no significant differences in other tissues. scribed in our previous study {Amenya, 2016 #432}. The following primers were used in this study: Chr 4: 35339023 (Forward, TCA CTC TTC ACC TGC AGG Table 1 Effects of prenatal BPA exposure on tissue weight of AAG; Reverse, GCT GTC ACT CTG TGC TCT TCT) mice at dissection and Chr X: 74707008 (Forward, CTG CTT TGC TGC Control BPA TCA GAG TTT; Reverse, CCC GAG TCC TGA GAT Male (n = 24) Female (n = 14) Male (n = 19) Female (n = 12) TAA AGG). Purified genomic DNA (100 ng) was divided Liver 4.340 ± 0.063 5.703 ± 0.284 4.231 ± 0.066 5.183 ± 0.196 into two portions. One portion was digested with Hpa II and the other was not digested. Both were subjected to UGC 1.030 ± 0.015 N/A 0.941 ± 0.026* N/A quantitative PCR analysis using Light Cycler 480 and the Testis 0.764 ± 0.012 N/A 0.743 ± 0.014 N/A methylation level of CpG was calculated as the ratio of Uterus N/A 0.686 ± 0.036 N/A 0.567 ± 0.083 target copy number from nondigested DNA to that from Heart 0.576 ± 0.014 0.514 ± 0.011 0.573 ± 0.013 0.501 ± 0.012 Hpa II-digested DNA. kidney 1.138 ± 0.017 1.158 ± 0.019 1.138 ± 0.014 1.146 ± 0.017 , Tissue weight data was expressed as percentage per body weight Statistical analysis N/A, not available The significance of differences in the tissue weight and Data were expressed as means ± SE. Statistically significant difference MSRE-PCR measurement analyzed by Student’s t-test. between means from control group was analyzed by t-test (*: p < 0.05) Aiba et al. Genes and Environment (2018) 40:12 Page 4 of 8 Overall effect of BPA exposure on CpG methylation shown in MSD-AFLP analysis were detected, neither of the We investigated the effects of prenatal BPA exposure on two CpGs showed significant difference in the MSRE-PCR the CpG methylation profile of the hippocampus in male analysis (Additional file 1:FigureS3). pups by MSD–AFLP analysis (Additional file 1:FigureS2). A total of 43,840 CpG data were obtained by using 256 se- The KEGG enrichment analysis of CpG methylation level lective primer sets. The mean methylation levels were 59.5 The enrichment analysis of CpG methylation level on ± 0.1% in the control group and 59.0 ± 0.1% in the BPA- the intragenic region and promoter region (5 kb exposed group (p = 0.22); (Fig. 2). There was no significant upstream from TSS) was performed to examine if statis- difference between control and BPA samples in the HCA tical difference is detected in KEGG pathway (Additional (Fig. 3). Moreover, PCA showed no clusters and large vari- file 1: Table S2). The hypermethylated CpGs in BPA ex- ances of both control and BPA groups (Fig. 4). These obser- posure were tended to highly enrich in the pathway of vations suggest that prenatal BPA exposure hardly affects N-Glycan Biosynthesis (NES = 1.600; FDR = 0.77). On DNA methylation in the mouse hippocampus. the other hands, the hypomethylated CpGs in BPA exposure were tended to highly enrich in the pathway of Statistical analysis for possibility of BPA-induced change Histidine Metabolism (NES = 1.602; FDR = 0.917). How- in CpG methylation ever, the minimum FDR was 0.776, no pathway with sig- We analyzed the significance of differences in methylation nificantly different methylation level was detected level between groups by q-value calculated with BH correc- according to the criteria FDR < 0.05. tion after Student’s t-test using all CpGs data determined by MSD-AFLP to explore possibility of BPA-induced Discussion changes. However, we detected no CpGs showing less than In this study we investigated whether prenatal low-dose 0.05 of q-value of FDR. Figure 5 is a volcanic plot of q-value BPA exposure causes epigenomic changes in the cerebral of FDR and fold of changes by BPA exposure. We found hippocampus. Our study showed that the weight of the only three CpGs shows the lowest q-value of FDR (q = 0.24, male UGC consisting of three tissues, i.e., the prostate, −log(q-value) = 0.620) out of 43,840 CpGs. The genomic seminal vesicle, and urethra, was significantly reduced. positions of three CpGs predicted by using the GFDB sys- Although this observation is in contrast to a previous re- tem were shown in Table 2. Using the genomic DNA sam- port on prostate development [8], our experiment also ples, in order to examine if fold of changes detected by showed sex-hormone-like activity in pups with maternal MSD-AFLP are significant, we performed MSRE-PCR ana- BPA exposure. Therefore, we focused on the hippocam- lysis. Since another Hpa II site is located on immediately pus, which is the part of the central nervous system that nearby the CpG (Chr 5: 137753995), it was impossible to is thought to be most strongly affected by BPA, as design primers that are amplifiable in MSRE-qPCR. There- shown by recent studies, and performed MSD–AFLP fore, we examined the remaining only two CpGs (Chr 4: analysis which is a new method that can detect genome- 35339023 and Chr X: 74707008). Although similar trends wide CpG methylation level changes with a difference in of difference between control and BPA in both CpGs the methylation level of less than 5% and a variation rate of the methylation level of less than 10% in an inter- tissue comparison experiment [39]. However, in this study, no statistically significant differences in methylation level between the control group and the BPA-exposed BPA (59.0 ± ± 0.1%) group were detected in all CpGs (43,840) analyzed in the Control (59.5 ± ± 0.1%) hippocampus. The effects of BPA exposure on CpG methylation level in genomic DNA in the brain and gonads have been re- Control ported. BPA at a low dose (20 μg/kg) was administered to BPA pregnant ICR/Jcl mice and the methylation level changes in the forebrain of the fetus was analyzed by the genome- wide restriction landmark genomic scanning (RLGS) method [37], which showed that the methylation levels of 48 CpGs were altered. A low dose BPA (10 μg/kg) 0 20 40 60 80 100 injection to pups of Sprague-Dawley (SD) rat has also been Methylation level (%) reported to lead to the hypomethylation of the phospho- Fig. 2 Histogram of methylation levels of all CpGs detected by diesterase type 4 gene in the prostate and increase suscepti- MSD-AFLP analysis. Blue and red lines indicate the mean methylation bility to 7,12-dimethylbenz[a]anthracene (DMBA)-induced levels in the control and BPA-exposed groups prostate carcinogenesis [35]. On the other hand, in utero Number of CpG-site Aiba et al. Genes and Environment (2018) 40:12 Page 5 of 8 Control BPA Fig. 3 Hierarchical clustering analysis (HCA) of the effects of BPA. HCA of normalized methylation patterns of each sample using Euclidean distance and weighted pair grouping method using arithmetic mean (UPGMA) and lactational exposure of BPA (0.05, 7.5, 30 and 120 mg/ excretion rate (ADME) vary depending on the rat strain. kg/day, by oral) to the mother rats of inbred Fisher 344 strain Therefore, differences in ADME values among strains may did not result in the DMBA-induced carcinogenesis of the be reflected in blood BPA level and may have affected the ex- prostate [40]. The urinary excretion rate of BPA were 21% perimental results. Additionally, there is an argument that and 70%, and the fecal excretion rates of BPA were 42% and closed colonies such as ICR mice and SD rats show genetic 50% in SD and Fisher 344 rats, respectively [41]. Further- diversity within colonies, resulting in false positives data that more, the absorption, distribution, metabolic rate, and occasionally occur due to genetic diversity that may arise even by simply dividing pups into two groups [42]. Thus, it is preferable to use inbred strain animals to exclude genetic variation among individuals in the analysis of epigenomic changes. As an example, when using inbred Avy mice, it has been reported that prenatal BPA exposure (50-mg BPA/kg diet) decreased the CpG methylation levels of the transposon sequence in Agouti by about 10% [33]. Agouti determines body hair color and the inserted transposon methylation negatively regulates gene expression. Therefore, they con- cluded that the yellow hair of Avy mice changed into brown due to this hypomethylation. However, they did not study whether there was any alteration in other genes. Control Using inbred BALB/c mice, there was a report that BPA prenatal exposure of BPA (200 μg/kg/day, by oral) hyper- methylated the transcriptional regulatory region of the hippocampal neurotrophic factor (BDNF) of the males and consequently decreased the mRNA expression level [36]. Additionally, they also analyzed using free DNA in Fig. 4 Principal component analysis (PCA) using MSD-AFLP data. blood and interestingly showed that brain BDNF methyla- Control, red circle (n = 6); BPA, blue circle (n = 6). PCA showed large tion levels correlate with those of blood DNA [36]. The variances of both control and BPA groups with no clusters levels of 17β-estradiol and testosterone and the ratio of Aiba et al. Genes and Environment (2018) 40:12 Page 6 of 8 1.4 q = 0.05 1.2 1.0 Chr 4: 35339023 Chr 5: 137753995 Chr X: 74707008 0.8 0.6 0.4 q = 0.5 0.2 -0.5 -0.3 -0.1 0.1 0.3 0.5 Log (fold of change) Fig. 5 Effects of BPA on CpG methylation profile in hippocampus. Volcanic plot showing the difference in methylation level between control group and BPA-exposed group. The logarithmic value of the fold of change (BPA/control) is presented in the x-axis, and the logarithmic value of the q-value is presented in the y-axis. Three CpGs which show less than 0.24 of q-value are indicated by arrows these two sex hormones were different between mouse environmental chemicals. If further analysis is performed strains [43]. The difference in the endocrine system be- with a wider coverage, it may be possible to detect signifi- tween C57BL/6 J and CBA/Lac mice appears to affect the cant BPA-specific methylation level alterations even in the activity of endocrine disruptors, such as BPA. In addition, C57BL/6 J hippocampus. although 17β-estradiol had been used to positive control in the studies of effect for prenatal BPA exposure of SD rat offspring on increase DMBA-induced prostate carcino- Conclusions genesis [35], neonatal exposure of 17β-estradiol had no ef- In this study, we investigated whether BPA alters CpG fects on mutagenicity of DMBA in reproductive tissues of methylation levels in the mouse hippocampus. Despite adult Big Blue transgenic mice [44]. Thus, the difference the use of MSD–AFLP analysis, which is a high- in genetic background may change the effects of BPA. Al- precision and highly sensitive analytical method, no though we analyzed 43,840 CpGs in this study, which are changes in methylation levels as the effect of low-dose approximately 0.3% out of the total 20,000,000 CpGs in BPA exposure were detected. Therefore, although fur- mouse genome, we were unable to detect statistically sig- ther analysis is necessary, we concluded that under the nificant changes in CpG methylation levels, which might experimental conditions of the present study, the effects be due to the stability of the methylated CpG in the of prenatal BPA exposure on the hippocampal DNA C57BL/6 J mouse hippocampus against small amount of methylation are extremely small. Table 2 The genomic positions of three CpGs showing minimum q-value predicted by using GFDB a b Chr. Position Gene name Methylation level (%) log ratio q-value Cont (n = 6) BPA (n = 6) 5 137,753,995 Trip6 18.21 ± 0.79 26.85 ± 0.67 0.17 0.24 4 35,339,023 9.3kbp up stream of RP23-307 M2.4 29.36 ± 0.34 34.67 ± 0.52 0.07 0.24 X 74,707,008 4.4kbp down stream of RP23-238B14.1 80.53 ± 1.38 65.66 ± 1.35 −0.09 0.24 , Chromosomal nucleotide position of methylated cytosine , The ratio means fold of change (BPA/control) −Log (q−value) Aiba et al. Genes and Environment (2018) 40:12 Page 7 of 8 Additional file different regions of the human brain. Int. J. Environ. Res. Public Health [Internet]. 2017;14:1059. Available from: http://www.mdpi.com/1660-4601/14/9/1059 3. Arase S, Ishii K, Igarashi K, Aisaki K, Yoshio Y, Matsushima A, et al. Endocrine Additional file 1: Table S1. The pup number and sex ratio. Figure S1. disrupter bisphenol a increases in situ estrogen production in the mouse Effect of prenatal BPA exposure on body weight and anogenital distance urogenital sinus. Biol Reprod [Internet]. 2011;84:734–42. Available from: (AGD). Figure S2. A typical MSD-AFLP peak chart after electrophoresis http://www.ncbi.nlm.nih.gov/pubmed/21123812 obtained by a selective primer set. Figure S3. MSRE-PCR analysis of 4. LiuX,MatsushimaA,ShimohigashiM,Shimohigashi Y.Acharacteristic representative CpGs (Chr 4: 35339023 and Chr X: 74707008) showing the back support structure in the bisphenol A-binding pocket in the minimum q-value obtained from the MSD-AFLP data. Table S2. The human nuclear receptor ERRγ. Vanacker J-M, editor. PLoS One [Internet]. KEGG enrichment analysis of the effect on the DNA methylation. 2014;9:e101252. Available from: http://www.ncbi.nlm.nih.gov/pubmed/ (DOCX 289 kb) 5. Sartain CV, Hunt PA. An old culprit but a new story: bisphenol a and Abbreviations “NextGen” bisphenols. Fertil Steril [Internet]. 2016;106:820–6. Available from: AGD: Anogenital distance; BPA: Bisphenol A; DOHaD: Developmental Origins http://www.ncbi.nlm.nih.gov/pubmed/27504789 of Health and Disease; FDR: False discovery rate; GFDB: Genome DNA 6. Krishnan AV, Stathis P, Permuth SF, Tokes L, Feldman D. Bisphenol-a: an Fragment Database; HCA: Hierarchical clustering analysis; KEGG: Kyoto estrogenic substance is released from polycarbonate flasks during Encyclopedia Genes and Genome; MSD-AFLP: Methylated site display- autoclaving. Endocrinology [Internet]. 1993;132:2279–86. Available from: amplified fragment length polymorphism; MSRE-PCR: Methylation-sensitive http://www.ncbi.nlm.nih.gov/pubmed/8504731 restriction enzyme-dependent PCR; PCA: Principal component analysis; 7. Nagel SC, vom Saal FS, Thayer KA, Dhar MG, Boechler M, Welshons WV. UGC: Urogenital complex Relative binding affinity-serum modified access (RBA-SMA) assay predicts the relative in vivo bioactivity of the xenoestrogens bisphenol a and octylphenol. Environ Health Perspect [Internet]. 1997;105:70–6. Available Acknowledgments from: http://www.ncbi.nlm.nih.gov/pubmed/9074884 Dr. Ruby Kuroiwa, for her kind help in English manuscript writing. 8. vom Saal FS, Cooke PS, Buchanan DL, Palanza P, Thayer KA, Nagel SC, et al. A physiologically based approach to the study of bisphenol a and other Funding estrogenic chemicals on the size of reproductive organs, daily sperm This work was supported by JSPS KAKENHI Grant Number 23310044 production, and behavior. Toxicol Ind Health [Internet]. 1998;14:239–60. (Grant-in-Aid for Scientific Research B) to SO, JSPS KAKENHI Grant Number Available from: http://www.ncbi.nlm.nih.gov/pubmed/9460178 15H02830 (Grant-in-Aid for Scientific Research B) to SO, and JSPS KAKENHI 9. Sakaue M, Ohsako S, Ishimura R, Kurosawa S, Kurohmaru M, Hayashi Y, et al. Grant Number 17H06396 (Grant-in-Aid for Scientific Research on Innovation Bisphenol-a affects spermatogenesis in the adult rat even at a low dose. J Areas) to SO. Occup Health [Internet]. 2001;43:185–90. Available from: http://joi.jlc.jst.go. jp/JST.Journalarchive/joh1996/43.185?from=CrossRef Availability of data and materials 10. Shelby MD. NTP-CERHR monograph on the potential human reproductive and The original data of the MSD-AFLP charts will be available upon request. developmental effects of bisphenol a. NTP CERHR MON [Internet]. 2008;v, vii–ix: 1–64 passim. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19407859 Authors’ contributions 11. Ashby J, Tinwell H, Haseman J. Lack of effects for low dose levels of SO conceived and designed experiment; TA, TS, and AH performed bisphenol a and diethylstilbestrol on the prostate gland of CF1 mice experiments; SS and HY made GFDB system; TA and TM analyzed the data exposed in utero. Regul Toxicol Pharmacol [Internet]. 1999;30:156–66. with help by WF; TA and SO wrote the manuscript. All authors have read Available from: http://www.ncbi.nlm.nih.gov/pubmed/10536110 and approved the final manuscript. 12. Ashby J, Tinwell H, Lefevre PA, Joiner R, Haseman J. 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Genes and EnvironmentSpringer Journals

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