Gene expression and DNA methylation changes in the hypothalamus and hippocampus of adult rats developmentally exposed to bisphenol A or ethinyl estradiol: a CLARITY-BPA consortium study

Bisphenol A (BPA), an endocrine disrupting chemical (EDC), is a ubiquitous pollutant. As part of the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA), we sought to determine whether exposure of Sprague-Dawley rats to 2,500 μg/kg/day BPA (BPA) or 0.5 μg/kg/day ethinyl estradiol (EE) from gestational day 6 through postnatal day 21 induces behavior-relevant gene expression and DNA methylation changes in hippocampus and hypothalamus at adulthood. RNA and DNA were isolated from both regions. Expression of ten genes (Dnmt1, Dnmt3a, Dnmt3b, Esr1, Esr2, Avp, Ar, Oxt, Otr, and Bdnf) presumably altered by early-life BPA/EE exposure was examined. Three genes (Bdnf, Dnmt3b, and Esr1) were studied for DNA methylation changes in their putative 5? promoter regions. Molecular changes in hippocampus were correlated to prior Barnes maze performance, including sniffing correct holes, distance traveled, and velocity. Exposure to BPA and/or EE disrupted patterns of sexually dimorphic gene expression/promoter DNA methylation observed in hippocampus and hypothalamus of controls. In the hippocampus of female offspring, BPA exposure resulted in hypermethylation of the putative 5? promoter region of Bdnf, while EE exposure induced hypomethylation. Bdnf methylation was weakly associated with Bdnf expression in hippocampi of female rats. Hippocampal Bdnf expression in females showed a weak negative association with sniffing correct hole in Barnes maze. Hippocampal expression of Avp, Esr2, Oxt, and Otr was strongly associated with velocity of control rats in Barnes maze. Findings suggest BPA exposure induced non-EE-like gene expression and epigenetic changes in adult rat hippocampi, a region involved in spatial navigation.     

Effects of in utero exposure to Bisphenol A or diethylstilbestrol on the adult male reproductive system

The objective of this study was to determine whether in utero exposure to Bisphenol A (BPA) induced reproductive tract abnormalities in the adult male testis. Using the C57/Bl6 mouse, we examined sex‐organ weights, anogenital distance, and testis histopathology in adult males exposed in utero via oral gavage to sesame oil, 50 µg/kg BPA, 1000 µg/kg BPA, or 2 µg/kg diethylstilbestrol (DES) as a positive control from gestational days 10 to 16. No changes in sperm production or germ cell apoptosis were observed in adult testes after exposure to either chemical. Adult mRNA levels of genes associated with sexual maturation and differentiation, GATA4 and ID2, were significantly lower only in DES‐exposed testes. In summary, the data indicate no gross alterations in spermatogenesis after in utero exposure to BPA or DES. At the molecular level, in utero exposure to DES, but not BPA, leads to decreased mRNA expression of genes associated with Sertoli cell differentiation. Birth Defects Res (Part B) 92:526–533, 2011. © 2011 Wiley Periodicals, Inc.     

Detection of differential DNA methylation in repetitive DNA of mice and humans perinatally exposed to bisphenol A

Developmental exposure to bisphenol A (BPA) has been shown to induce changes in DNA methylation in both mouse and human genic regions; however, the response in repetitive elements and transposons has not been explored. Here we present novel methodology to combine genomic DNA enrichment with RepeatMasker analysis on next-generation sequencing data to determine the effect of perinatal BPA exposure on repetitive DNA at the class, family, subfamily, and individual insertion level in both mouse and human samples. Mice were treated during gestation and lactation to BPA in chow at 0, 50, or 50,000 ng/g levels and total BPA was measured in stratified human fetal liver tissue samples as low (non-detect to 0.83 ng/g), medium (3.5 to 5.79 ng/g), or high (35.44 to 96.76 ng/g). Transposon methylation changes were evident in human classes, families, and subfamilies, with the medium group exhibiting hypomethylation compared to both high and low BPA groups. Mouse repeat classes, families, and subfamilies did not respond to BPA with significantly detectable differential DNA methylation. In human samples, 1251 individual transposon loci were detected as differentially methylated by BPA exposure, but only 19 were detected in mice. Of note, this approach recapitulated the discovery of a previously known mouse environmentally labile metastable epiallele, Cabp^IAP. Thus, by querying repetitive DNA in both mouse and humans, we report the first known transposons in humans that respond to perinatal BPA exposure.     

Methylation profiling identified novel differentially methylated markers including OPCML and FLRT2 in prostate cancer

To develop new methods to distinguish indolent from aggressive prostate cancers (PCa), we utilized comprehensive high-throughput array-based relative methylation (CHARM) assay to identify differentially methylated regions (DMRs) throughout the genome, including both CpG island (CGI) and non-CGI regions in PCa patients based on Gleason grade. Initially, 26 samples, including 8 each of low [Gleason score (GS) 6] and high (GS ≥7) grade PCa samples and 10 matched normal prostate tissues, were analyzed as a discovery cohort. We identified 3,567 DMRs between normal and cancer tissues, and 913 DMRs distinguishing low from high-grade cancers. Most of these DMRs were located at CGI shores. The top 5 candidate DMRs from the low vs. high Gleason comparison, including OPCML, ELAVL2, EXT1, IRX5, and FLRT2, were validated by pyrosequencing using the discovery cohort. OPCML and FLRT2 were further validated in an independent cohort consisting of 20 low-Gleason and 33 high-Gleason tissues. We then compared patients with biochemical recurrence (n=70) vs. those without (n=86) in a third cohort, and they showed no difference in methylation at these DMR loci. When GS 3+4 cases and GS 4+3 cases were compared, OPCML-DMR methylation showed a trend of lower methylation in the recurrence group (n=30) than in the no-recurrence (n=52) group. We conclude that whole-genome methylation profiling with CHARM revealed distinct patterns of differential DNA methylation between normal prostate and PCa tissues, as well as between different risk groups of PCa as defined by Gleason scores. A panel of selected DMRs may serve as novel surrogate biomarkers for Gleason score in PCa.     

Bisphenol A exposure modifies DNA methylation of imprint genes in mouse fetal germ cells

Bisphenol A (BPA) is an estrogenic environmental toxin widely used for the production of plastics. Human frequent exposure to this chemical has been proposed to be a potential public health risk. The objective of this study was to assess the effects of BPA on DNA methylation of imprinting genes in fetal mouse germ cell. Pregnant mice were treated with BPA at doses of 0, 40, 80 and 160 μg BPA/kg body weight/day from 0.5 day post coitum. DNA methylation of imprinting genes, Igf2r, Peg3 and H19, was decreased with the increase of BPA concentration in fetal mouse germ cells (p < 0.01).The relative mRNA levels of Nobox were lower in BPA-treated group compared to control (BPA free) in female fetal germ cells, but in male fetal germ cells, a significant higher in Nobox expression was observed in BPA-treated group compared to control. Decreased mRNA expression of specific meiotic genes including Stimulated by Stra8 and Dazl were obtained in the female fetal germ cells. In conclusion, BPA exposure can affect the DNA methylation of imprinting genes in fetal mouse germ cells.     

Inflammatory repercussions in female steroid responsive glands after perinatal exposure to bisphenol A and 17-β estradiol

The mammary gland (MG) and female prostate are plastic reproductive organs which are highly responsive to hormones. Thus, endocrine disruptors, such as bisphenol A (BPA) and exogenous estrogens, negatively affect glandular homeostasis. In addition to previously described alterations, changes in inflammatory markers expression also trigger the development of a microenvironment that contributes to tumor progression. The current work aimed to evaluate the inflammatory responses of the MG and prostate gland to BPA (50 µg/kg) and 17-β estradiol (35 µg/kg) exposure during the perinatal window of susceptibility. The results showed that at 6 months of age there was an increase in the number of phospho-STAT3 (P-STAT3) positive cells in the female prostate from animals perinatally exposed to 50 µg/kg BPA daily. In addition, the number of macrophages increased in these animals in comparison with nonexposed animals, as shown by the F4/80 marker. Despite an increase in the incidence of lobuloalveolar and intraductal hyperplasia, the MG did not show any difference in the expression of the four inflammatory markers evaluated: tumor necrosis factor-α, COX-2, P-STAT3, and F4/80. Analysis of both glands from the same animal led to the conclusion that exposure to endocrine disruptors during the perinatal window of susceptibility leads to different inflammatory responses in different reproductive organs. As the prostate is more susceptible to these inflammatory mechanisms, it is reasonable to affirm that possible neoplastic alterations in this organ are related to changes in the inflammatory pattern of the stroma, a characteristic that is not evident in the MG.     

An expression microarray approach for the identification of metastable epialleles in the mouse genome

Genetic loci displaying environmentally responsive epigenetic marks, termed metastable epialleles, offer a solution to the paradox presented by genetically identical yet phenotypically distinct individuals. The murine viable yellow agouti (A^vy) metastable epiallele exhibits stochastic DNA methylation and histone modifications associated with coat color variation in isogenic individuals. The distribution of A^vy variable expressivity shifts following maternal nutritional and environmental exposures. To characterize additional murine metastable epialleles, we utilized genome-wide expression arrays (N = 10 male individuals, 3 tissues per individual) and identified candidates displaying large variability in gene expression among individuals (V_i = inter-individual variance), concomitant with a low variability in gene expression across tissues from the three germ layers (V_t = inter-tissue variance), two features characteristic of the A^vy metastable epiallele. The CpG island in the promoter of Dnajb1 and two contraoriented ERV class II repeats in Glcci1 were validated to display underlying stochasticity in methylation patterns common to metastable epialleles. Furthermore, liver DNA methylation in mice exposed in utero to 50 mg bisphenol A (BPA)/kg diet (N = 91) or a control diet (N = 79) confirmed environmental lability at validated candidate genes. Significant effects of exposure on mean CpG methylation were observed at the Glcci1 Repeat 1 locus (p &lt; 0.0001). Significant effects of BPA also were observed at the first and fifth CpG sites studied in Glcci1 Repeat 2 (p &lt; 0.0001 and p = 0.004, respectively). BPA did not affect methylation in the promoter of Dnajb1 (p = 0.59). The characterization of metastable epialleles in humans is crucial for the development of novel screening and therapeutic targets for human disease prevention.     

MTSS1 hypermethylation is associated with prostate cancer progression

This study was conducted to evaluate the influence of DNA methylation of metastasis suppressor 1 (MTSS1) on prostate cancer (PCa) progression. Forty-nine paired PCa tissue samples and normal tissue samples from The Cancer Genome Atlas were analyzed. Methylome analysis, CpG island arrays and Hierarchical clustering were used to analyze methylation profiles of PCa tissues. MTSS1 methylation level was detected by methylation-specific PCR. Relative messenger RNA and the expression level of MTSS1 protein were identified by quantitative real-time PCR (qRT-PCR) and western blot analysis. The migration, invasion, proliferation, and cell cycle were detected separately by wound-healing assay, transwell chamber assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and flow cytometry. The roles of MTSS1 in PCa progression were demonstrated in vivo by tumor formation assays in nude mice. MTSS1 expression was decreased in PCa tissues in comparison with paired adjacent normal prostate tissues. Compared to the methylation of MTSS1 in normal prostate tissues based on the MethHC website, the MTSS1 in PCa tissues was hypermethylated. The expression of MTSS1 detected by qRT-PCR and western blot analysis was found to be downregulated in PCa cells and tissues. The reduced expression of MTSS1 by small interfering RNA-MTSS1 was recovered by 5-aza-2′-deoxycytidine treatment. Besides, MTSS1 demethylation inhibited migration, invasion, and proliferation of PCa cells, and induced cell cycle to be arrested at G0/G1 phase. Furthermore, it was shown by tumor xenograft assay that MTSS1 inhibited the growth of tumor in vivo. Hypermethylated MTSS1 promoted PCa cells migration, invasion, and proliferation, and suppressed cell cycle arrest at the G0/G1 phase.     

Prenatal exposure to bisphenol A promotes angiogenesis and alters steroid-mediated responses in the mammary glands of cycling rats

Prenatal exposure to BPA disturbs mammary gland histoarchitecture and increases the carcinogenic susceptibility to chemical challenges administered long after BPA exposure. Our aim was to assess the effect of prenatal BPA exposure on mammary gland angiogenesis and steroid hormone pathways in virgin cycling rats. Pregnant Wistar rats were exposed to either 25 or 250 g/kg/day (25 and 250 BPA, respectively) or to vehicle. Female offspring were autopsied on postnatal day (PND) 50 or 110. Ovarian steroid serum levels, the expression of steroid receptors and their co-regulators SRC-3 and SMRT in the mammary gland, and angiogenesis were evaluated. At PND 50, all BPA-treated animals had lower serum levels of progesterone, while estradiol levels remained unchanged. The higher dose of BPA increased mammary ERα and decreased SRC-3 expression at PND 50 and PND 110. SMRT protein levels were similar among groups at PND 50, whereas at PND 110, animals exposed to 250 BPA showed a lower SMRT expression. Interestingly, in the control and 25 BPA groups, SMRT increased from PND 50 to PND 110. At PND 50, an increased vascular area associated with higher VEGF expression was observed in the 250 BPA-treated rats. At PND 110, the vascular area was still increased, but VEGF expression was similar to that of control rats. The present results demonstrate that prenatal exposure to BPA alters the endocrine environment of the mammary gland and its angiogenic process. Increased angiogenesis and altered steroid hormone signals could explain the higher frequency of pre-neoplastic lesions found later in life. This article is part of a Special Issue entitled 'Endocrine disruptors'.     

Selenium-methionine and chromium-methionine supplementation of sheep around parturition: impacts on dam and offspring performance

To examine the effects of maternal energy restriction along with selenium-methionine (Se-Met) and chromium-methionine (Cr-Met) supplementation on performance of pregnant sheep and their offspring, the following treatments were allotted randomly to 40 multiparous Baluchi ewes (53.9 ± 1.15 kg of body weight [BW]) from 5 weeks prior to 5 weeks after parturition: (1) Control diet (60% and 100% of NRC energy requirements in pre- and post-partum, respectively); (2) Control diet plus 5 mg Se-Met/kg dry matter (DM); (3) Control diet plus 3 mg Cr-Met/kg DM and (4) Control diet plus 5 mg Se-Met and 3 mg Cr-Met/kg DM (Se–Cr-Met) of concentrate diet. The results indicated that Cr-Met alone or in combination with Se-Met increased average DM intake of ewes. In addition, Group Cr-Met had higher average BW than the Control (p < 0.05). Se-Met and/or Cr-Met supplementation led to decreased average serum cholesterol of the ewes (p < 0.05). Groups Cr-Met and Se–Cr-Met displayed decreased average serum malondialdehyde compared to the Control (p < 0.05). At 24 h post-partum, Group Se–Cr-Met had a greater serum Se content than the Control (p = 0.006). Compared with the Control, the Se concentration in milk was significantly increased from 30 to 138 µg/l and 197 µg/l in Groups Se-Met and Se–Cr-Met, respectively (p < 0.01), which proved that Se-Met supplementation can increase the Se concentration of ewe milk. Furthermore, feeding Cr-Met may attenuate BW loss post-partum and Se-Met and/or Cr-Met supplements may ameliorate oxidative stress condition in ewes around parturition.     

Diagnosis and prognosis potential of four gene promoter hypermethylation in prostate cancer

The current prostate special antigen (PSA) test causes the overtreatment of indolent prostate cancer (PCa). It also increases the risk of delayed treatment of aggressive PCa. DNA methylation aberrations are important events for gene expression dysregulation during tumorigenesis and have been suggested as novel candidate biomarkers for PCa. This may improve the diagnosis and prognosis of PCa. This study assessed the differential methylation and messenger RNA (mRNA) expression between normal and PCa samples. Correlation between promoter methylation and mRNA expression was estimated using Pearson's correlation coefficients. Moreover, the diagnostic potential of candidate methylation markers was estimated by the receiver operating characteristic (ROC) curve using continuous beta values. Survival and Cox analysis was performed to evaluate the prognostic potential of the candidate methylation markers. A total of 359 hypermethylated sites 3435 hypomethylation sites, 483 upregulated genes, and 1341 downregulated genes were identified from The Cancer Genome Atlas database. Furthermore, 17 hypermethylated sites (covering 13 genes), including known genes associated with hypermethylation in PCa (e.g., AOX1 and C1orf114), showed high discrimination between adjacent normal tissues and PCa samples with the area under the ROC curve from 0.88 to 0.94. Notably, ANXA2, FGFR2, HAAO, and KCNE3 were identified as valuable prognostic markers of PCa through the Kaplan–Meier analysis. Using gene methylation as a continuous variable, four promoter hypermethylation was significantly associated with disease-free survival in univariate Cox regression and multivariate Cox regression. This study identified four novel diagnostic and prognostic markers for PCa. The markers provide important strategies for improving the timely diagnosis and prognosis of PCa.     

Neonatal exposure to bisphenol A advances pubertal development in female rats

Neonatal exposure to bisphenol A (BPA) is hypothesized to advance pubertal development. However, the effects of neonatal BPA exposure on pubertal development has not been described. In this study, female Sprague‐Dawley rats were exposed to 0.05, 0.5, 5, or 10 mg·kg^?1·day^?1 BPA, or corn oil vehicle alone from postnatal day 1 (PND1) to PND10 via subcutaneous injection. We evaluated day of vaginal opening (DVO), ovarian morphology, serum hormone concentrations, and hypothalamic expression of Gnrh1 and Kiss1 in female rats at PND35. DVO was significantly advanced in rats exposed to 5 and 10 mg·kg^?1·day^?1 BPA. Serum hormone concentrations increased as BPA dose increased. Additionally, hypothalamic Gnrh1 and Kiss1 expression were increased with BPA exposure; rats exposed to 10 mg·kg^?1·day^?1 BPA had significantly upregulated hypothalamic Gnrh1 and Kiss1 expressions in terms of both messenger RNA and protein levels. Our results suggest that exposure to a 10 mg·kg^?1·day^?1 dose of BPA might advance pubertal development significantly. In addition, within the range of 0 to 10 mg·kg^?1·day^?1, neonatal exposure to BPA may affect pubertal development in a dose‐dependent manner.     

Stat3 is a candidate epigenetic biomarker of perinatal Bisphenol A exposure associated with murine hepatic tumors with implications for human health

Bisphenol A (BPA) is an endocrine disrupting chemical (EDC) that has been implicated as a potential carcinogen and epigenotoxicant. We have previously reported dose-dependent incidence of hepatic tumors in 10-month-old isogenic mice perinatally exposed to BPA. Here, we evaluated DNA methylation at 3 candidate genes (Esr1, Il-6st, and Stat3) in liver tissue of BPA-exposed mice euthanized at 2 time points: post-natal day 22 (PND22; n = 147) or 10-months of age (n = 78, including n = 18 with hepatic tumors). Additionally, DNA methylation profiles were analyzed at human homologs of murine candidate genes in human fetal liver samples (n = 50) with known liver tissue BPA levels. Candidate genes were chosen based on reported expression changes in both rodent and human hepatocellular carcinoma (HCC). Regions for bisulfite sequencing were chosen by mining whole genome next generation sequencing methylation datasets of both mice and human liver samples with known perinatal BPA exposures. One of 3 candidate genes, Stat3, displayed dose-dependent DNA methylation changes in both 10-month mice with liver tumors as compared to those without liver tumors and 3-week sibling mice from the same exposure study, implicating Stat3 as a potential epigenetic biomarker of both early life BPA exposure and adult disease in mice. DNA methylation profiles within STAT3 varied with liver tissue BPA level in human fetal liver samples as well, suggesting STAT3 may be a translationally relevant candidate biomarker. These data implicate Stat3 as a potential early life biomarker of adult murine liver tumor risk following early BPA exposure with early evidence of relevance to human health.