Endocrine-Disrupting Chemicals and Hippocampal Development: The Role of Estrogen and Androgen Signaling.

Hormones are important regulators of key processes during fetal brain development. Thus, the developing brain is vulnerable to the action of chemicals that can interfere with endocrine signals. Epidemiological studies have pointed toward sexually dimorphic associations between neurodevelopmental outcomes, such as cognitive abilities, in children and prenatal exposure to endocrine-disrupting chemicals (EDCs). This points toward disruption of sex steroid signaling in the development of neural structures underlying cognitive functions, such as the hippocampus, an essential mediator of learning and memory processes. Indeed, during development, the hippocampus is subjected to the organizational effects of estrogens and androgens, which influence hippocampal cell proliferation, differentiation, dendritic growth, and synaptogenesis in the hippocampal fields of Cornu Ammonis and the dentate gyrus. These early organizational effects correlate with a sexual dimorphism in spatial cognition and are subject to exogenous chemical perturbations. This review summarizes the current knowledge about the organizational effects of estrogens and androgens on the developing hippocampus and the evidence for hippocampal-dependent learning and memory perturbations induced by developmental exposure to EDCs. We conclude that, while it is clear that sex hormone signaling plays a significant role during hippocampal development, a complete picture at the molecular and cellular levels would be needed to establish causative links between the endocrine modes of action exerted by EDCs and the adverse outcomes these chemicals can induce at the organism level.

Exposure to endocrine-disrupting chemicals and implications for neurodevelopment.

Human brain development is a complex multistep process that is partly coordinated by the endocrine system. Any interference with the endocrine system might affect this process and result in deleterious outcomes. Endocrine-disrupting chemicals (EDCs) represent a large group of exogenous chemicals with the capacity of interfering with endocrine functions. In different population-based settings, associations between exposure to EDCs, particularly in prenatal life, and adverse neurodevelopmental outcomes have been demonstrated. These findings are strengthened by numerous experimental studies. Although mechanisms underlying these associations are not entirely delineated, disruption of thyroid hormone and, to a lesser extent, sex hormone signalling have been shown to be involved. Humans are constantly exposed to mixtures of EDCs, and further research combining epidemiological and experimental settings is required to improve our understanding of the link between real-life exposures to these chemicals and their impact on neurodevelopment.

Genotype-dependent epigenetic regulation of DLGAP2 in alcohol use and dependence.

Alcohol misuse is a major public health problem originating from genetic and environmental risk factors. Alterations in the brain epigenome may orchestrate changes in gene expression that lead to alcohol misuse and dependence. Through epigenome-wide association analysis of DNA methylation from human brain tissues, we identified a differentially methylated region, DMR-DLGAP2, associated with alcohol dependence. Methylation within DMR-DLGAP2 was found to be genotype-dependent, allele-specific and associated with reward processing in brain. Methylation at the DMR-DLGAP2 regulated expression of DLGAP2 in vitro, and Dlgap2-deficient mice showed reduced alcohol consumption compared with wild-type controls. These results suggest that DLGAP2 may be an interface for genetic and epigenetic factors controlling alcohol use and dependence.

A Mixture of Endocrine Disrupting Chemicals Associated with Lower Birth Weight in Children Induces Adipogenesis and DNA Methylation Changes in Human Mesenchymal Stem Cells.

Endocrine Disrupting Chemicals (EDCs) are man-made compounds that alter functions of the endocrine system. Environmental mixtures of EDCs might have adverse effects on human health, even though their individual concentrations are below regulatory levels of concerns. However, studies identifying and experimentally testing adverse effects of real-life mixtures are scarce. In this study, we aimed at evaluating an epidemiologically identified EDC mixture in an experimental setting to delineate its cellular and epigenetic effects. The mixture was established using data from the Swedish Environmental Longitudinal Mother and child Asthma and allergy (SELMA) study where it was associated with lower birth weight, an early marker for prenatal metabolic programming. This mixture was then tested for its ability to change metabolic programming of human mesenchymal stem cells. In these cells, we assessed if the mixture induced adipogenesis and genome-wide DNA methylation changes. The mixture increased lipid droplet accumulation already at concentrations corresponding to levels measured in the pregnant women of the SELMA study. Furthermore, we identified differentially methylated regions in genes important for adipogenesis and thermogenesis. This study shows that a mixture reflecting human real-life exposure can induce molecular and cellular changes during development that could underlie adverse outcomes.

Bisphenol A Inhibits the Transporter Function of the Blood-Brain Barrier by Directly Interacting with the ABC Transporter Breast Cancer Resistance Protein (BCRP).

The breast cancer resistance protein (BCRP) is an important efflux transporter in the blood-brain barrier (BBB), protecting the brain from a wide range of substances. In this study, we investigated if BCRP function is affected by bisphenol A (BPA), a high production volume chemical used in common consumer products, as well as by bisphenol F (BPF) and bisphenol S (BPS), which are used to substitute BPA. We employed a transwell-based in vitro cell model of iPSC-derived brain microvascular endothelial cells, where BCRP function was assessed by measuring the intracellular accumulation of its substrate Hoechst 33342. Additionally, we used in silico modelling to predict if the bisphenols could directly interact with BCRP. Our results showed that BPA significantly inhibits the transport function of BCRP. Additionally, BPA was predicted to bind to the cavity that is targeted by known BCRP inhibitors. Taken together, our findings demonstrate that BPA inhibits BCRP function in vitro, probably by direct interaction with the transporter. This effect might contribute to BPA's known impact on neurodevelopment.

Role of estrogen receptor beta in neural differentiation of mouse embryonic stem cells.

The ability to propagate mature cells and tissue from pluripotent stem cells offers enormous promise for treating many diseases, including neurodegenerative diseases. Before such cells can be used successfully in neurodegenerative diseases without causing unwanted cell growth and migration, genes regulating growth and migration of neural stem cells need to be well characterized. Estrogen receptor beta (ERß) is essential for migration of neurons and glial cells in the developing mouse brain. To examine whether ERß influences differentiation of mouse embryonic stem cells (mESC) into neural lineages, we compared control and ERß knockout (BERKO) mESCs at defined stages of neural development and examined the effects of an ERß-selective ligand (LY3201) with a combination of global and targeted gene-expression profiling and the expression of key pluripotency markers. We found that ERß was induced in embryoid bodies (EBs) and neural precursor cells (NPCs) during development. Proliferation was higher in BERKO NPCs and was inhibited by LY3201. Neurogenesis was reduced in BERKO ES cells, and oligodendrogliogenesis was enhanced. BERKO EBs expressed higher levels of key ectodermal and neural progenitor markers and lower levels of markers for mesoderm and endoderm lineages. ERß-regulated factors are involved in cell adhesion, axon guidance, and signaling of Notch and GABA receptor pathways, as well as factors important for the differentiation of neuronal precursors into dopaminergic neurons (Engrailed 1) and for the oligodendrocyte fate acquisition (Olig2). Our data suggest that ERß is an important component for differentiation into midbrain neurons as well as for preventing precocious oligodendrogliogenesis.

The ENDpoiNTs Project: Novel Testing Strategies for Endocrine Disruptors Linked to Developmental Neurotoxicity.

Ubiquitous exposure to endocrine-disrupting chemicals (EDCs) has caused serious concerns about the ability of these chemicals to affect neurodevelopment, among others. Since endocrine disruption (ED)-induced developmental neurotoxicity (DNT) is hardly covered by the chemical testing tools that are currently in regulatory use, the Horizon 2020 research and innovation action ENDpoiNTs has been launched to fill the scientific and methodological gaps related to the assessment of this type of chemical toxicity. The ENDpoiNTs project will generate new knowledge about ED-induced DNT and aims to develop and improve in vitro, in vivo, and in silico models pertaining to ED-linked DNT outcomes for chemical testing. This will be achieved by establishing correlative and causal links between known and novel neurodevelopmental endpoints and endocrine pathways through integration of molecular, cellular, and organismal data from in vitro and in vivo models. Based on this knowledge, the project aims to provide adverse outcome pathways (AOPs) for ED-induced DNT and to develop and integrate new testing tools with high relevance for human health into European and international regulatory frameworks.

Alterations in the neuropeptide galanin system in major depressive disorder involve levels of transcripts, methylation, and peptide.

Major depressive disorder (MDD) is a substantial burden to patients, families, and society, but many patients cannot be treated adequately. Rodent experiments suggest that the neuropeptide galanin (GAL) and its three G protein-coupled receptors, GAL1-3, are involved in mood regulation. To explore the translational potential of these results, we assessed the transcript levels (by quantitative PCR), DNA methylation status (by bisulfite pyrosequencing), and GAL peptide by RIA of the GAL system in postmortem brains from depressed persons who had committed suicide and controls. Transcripts for all four members were detected and showed marked regional variations, GAL and galanin receptor 1 (GALR1) being most abundant. Striking increases in GAL and GALR3 mRNA levels, especially in the noradrenergic locus coeruleus and the dorsal raphe nucleus, in parallel with decreased DNA methylation, were found in both male and female suicide subjects as compared with controls. In contrast, GAL and GALR3 transcript levels were decreased, GALR1 was increased, and DNA methylation was increased in the dorsolateral prefrontal cortex of male suicide subjects, however, there were no changes in the anterior cingulate cortex. Thus, GAL and its receptor GALR3 are differentially methylated and expressed in brains of MDD subjects in a region- and sex-specific manner. Such an epigenetic modification in GALR3, a hyperpolarizing receptor, might contribute to the dysregulation of noradrenergic and serotonergic neurons implicated in the pathogenesis of MDD. Thus, one may speculate that a GAL3 antagonist could have antidepressant properties by disinhibiting the firing of these neurons, resulting in increased release of noradrenaline and serotonin in forebrain areas involved in mood regulation.

A Novel Approach to Chemical Mixture Risk Assessment-Linking Data from Population-Based Epidemiology and Experimental Animal Tests.

Humans are continuously exposed to chemicals with suspected or proven endocrine disrupting chemicals (EDCs). Risk management of EDCs presents a major unmet challenge because the available data for adverse health effects are generated by examining one compound at a time, whereas real-life exposures are to mixtures of chemicals. In this work, we integrate epidemiological and experimental evidence toward a whole mixture strategy for risk assessment. To illustrate, we conduct the following four steps in a case study: (1) identification of single EDCs ("bad actors")-measured in prenatal blood/urine in the SELMA study-that are associated with a shorter anogenital distance (AGD) in baby boys; (2) definition and construction of a "typical" mixture consisting of the "bad actors" identified in Step 1; (3) experimentally testing this mixture in an in vivo animal model to estimate a dose-response relationship and determine a point of departure (i.e., reference dose [RfD]) associated with an adverse health outcome; and (4) use a statistical measure of "sufficient similarity" to compare the experimental RfD (from Step 3) to the exposure measured in the human population and generate a "similar mixture risk indicator" (SMRI). The objective of this exercise is to generate a proof of concept for the systematic integration of epidemiological and experimental evidence with mixture risk assessment strategies. Using a whole mixture approach, we could find a higher rate of pregnant women under risk (13%) when comparing with the data from more traditional models of additivity (3%), or a compound-by-compound strategy (1.6%).

Fluoxetine Affects Differentiation of Midbrain Dopaminergic Neurons In Vitro.

Recent meta-analyses found an association between prenatal exposure to the antidepressant fluoxetine (FLX) and an increased risk of autism in children. This developmental disorder has been related to dysfunctions in the brains' rewards circuitry, which, in turn, has been linked to dysfunctions in dopaminergic (DA) signaling. The present study investigated if FLX affects processes involved in dopaminergic neuronal differentiation. Mouse neuronal precursors were differentiated into midbrain dopaminergic precursor cells (mDPCs) and concomitantly exposed to clinically relevant doses of FLX. Subsequently, dopaminergic precursors were evaluated for expression of differentiation and stemness markers using quantitative polymerase chain reaction. FLX treatment led to increases in early regional specification markers orthodenticle homeobox 2 (Otx2) and homeobox engrailed-1 and -2 (En1 and En2). On the other hand, two transcription factors essential for midbrain dopaminergic (mDA) neurogenesis, LIM homeobox transcription factor 1 α (Lmx1a) and paired-like homeodomain transcription factor 3 (Pitx3) were downregulated by FLX treatment. The stemness marker nestin (Nes) was increased, whereas the neuronal differentiation marker ß3-tubulin (Tubb3) decreased. Additionally, we observed that FLX modulates the expression of several genes associated with autism spectrum disorder and downregulates the estrogen receptors (ERs) α and ß Further investigations using ERß knockout (BERKO) mDPCs showed that FLX had no or even opposite effects on several of the genes analyzed. These findings suggest that FLX affects differentiation of the dopaminergic system by increasing production of dopaminergic precursors, yet decreasing their maturation, partly via interference with the estrogen system.

Effects of Long-Term Alcohol Drinking on the Dopamine D2 Receptor: Gene Expression and Heteroreceptor Complexes in the Striatum in Rats.

BACKGROUND: Reduced dopamine D2 receptor (D2R) ligand binding has repeatedly been demonstrated in the striatum of humans with alcohol use disorder (AUD). The attenuated D2R binding has been suggested to reflect a reduced D2R density, which in turn has been proposed to drive craving and relapse. However, results from rodent studies addressing the effects of alcohol drinking on D2R density have been inconsistent. METHODS: A validated alcohol drinking model (intermittent access to 20% alcohol) in Wistar rats was used to study the effects of voluntary alcohol drinking (at least 12 weeks) on the D2R in the striatum compared to age-matched alcohol-naïve control rats. Reverse transcriptase quantitative PCR was used to quantify isoform-specific Drd2 gene expression levels. Using bisulfite pyrosequencing, DNA methylation levels of a regulatory region of the Drd2 gene were determined. In situ proximity ligation assay was used to measure densities of D2R receptor complexes: D2R-D2R, adenosine A2A receptor (A2AR)-D2R, and sigma1 receptor (sigma1R)-D2R. RESULTS: Long-term voluntary alcohol drinking significantly reduced mRNA levels of the long D2R isoform in the nucleus accumbens (NAc) but did not alter CpG methylation levels in the analyzed sequence of the Drd2 gene. Alcohol drinking also reduced the striatal density of D2R-D2R homoreceptor complexes, increased the density of A2AR-D2R heteroreceptor complexes in the NAc shell and the dorsal striatum, and decreased the density of sigma1R-D2R heteroreceptor complexes in the dorsal striatum. CONCLUSIONS: The present results on long-term alcohol drinking might reflect reduced D2R levels through reductions in D2R-D2R homoreceptor complexes and gene expression. Furthermore, based on antagonistic interactions between A2AR and D2R, an increased density of A2AR-D2R heteroreceptor complexes might indicate a reduced affinity and signaling of the D2R population within the complex. Hence, both reduced striatal D2R levels and reduced D2R protomer affinity within the striatal A2AR-D2R complex might underlie reduced D2R radioligand binding in humans with AUD. This supports the hypothesis of a hypodopaminergic system in AUD and suggests the A2AR-D2R heteroreceptor complex as a potential novel treatment target.

Hypothalamic-pituitary-adrenal axis dysfunction and illness progression in bipolar disorder.

BACKGROUND: Impaired stress resilience and a dysfunctional hypothalamic-pituitary-adrenal (HPA) axis are suggested to play key roles in the pathophysiology of illness progression in bipolar disorder (BD), but the mechanisms leading to this dysfunction have never been elucidated. This study aimed to examine HPA axis activity and underlying molecular mechanisms in patients with BD and unaffected siblings of BD patients. METHODS: Twenty-four euthymic patients with BD, 18 siblings of BD patients, and 26 healthy controls were recruited for this study. All subjects underwent a dexamethasone suppression test followed by analyses associated with the HPA axis and the glucocorticoid receptor (GR). RESULTS: Patients with BD, particularly those at a late stage of illness, presented increased salivary post-dexamethasone cortisol levels when compared to controls (p = 0.015). Accordingly, these patients presented reduced ex vivo GR responsiveness (p = 0.008) and increased basal protein levels of FK506-binding protein 51 (FKBP51, p = 0.012), a co-chaperone known to desensitize GR, in peripheral blood mononuclear cells. Moreover, BD patients presented increased methylation at the FK506-binding protein 5 (FKBP5) gene. BD siblings presented significantly lower FKBP51 protein levels than BD patients, even though no differences were found in FKBP5 basal mRNA levels. CONCLUSIONS: Our data suggest that the epigenetic modulation of the FKBP5 gene, along with increased FKBP51 levels, is associated with the GR hyporesponsiveness seen in BD patients. Our findings are consistent with the notion that unaffected first-degree relatives of BD patients share biological factors that influence the disorder, and that such changes are more pronounced in the late stages of the illness.

DNA methylation at DLGAP2 and risk for relapse in alcohol dependence during acamprosate treatment.

BACKGROUND: Alcohol use disorders are prevalent mental disorders with significant health implications. Epigenetic alterations may play a role in their pathogenesis, as DNA methylation at several genes has been associated with these disorders. We have previously shown that methylation in the DLGAP2 gene, coding for a synaptic density protein, is associated with alcohol dependence. In this study, we aimed to examine the association between DLGAP2 methylation and treatment response among patients undergoing acamprosate treatment. METHODS: 102 patients under acamprosate treatment were included. DNA methylation analysis at DLGAP2 was performed by bisulfite pyrosequencing at the start and after 3-month treatment. Treatment outcomes were having a relapse during the treatment and severity of craving at the end of three months. Cox proportional hazard and linear regression models were performed. RESULTS: Patients whose methylation levels were decreased during the treatment showed an increased risk for relapse within three months in comparison to the ones without methylation change (hazard ratio [HR]=2.44; 95% confidence interval [CI]=1.04, 5.73; p=0.04). For the same group, a positive association for the severity of craving was observed, yet statistical significance was not reached (ß=2.97; 95% CI=-0.41, 6.34; p=0.08). CONCLUSION: We demonstrate that patients whose DLGAP2 methylation levels decrease during acamprosate treatment are more likely to relapse compared to the ones without changes. This is in line with our previous findings showing that DLGAP2 methylation is lower in alcohol dependent subjects compared to controls, and might suggest a role for changes in DLGAP2 methylation in treatment response.

New approach methods to assess developmental and adult neurotoxicity for regulatory use: a PARC work package 5 project.

In the European regulatory context, rodent in vivo studies are the predominant source of neurotoxicity information. Although they form a cornerstone of neurotoxicological assessments, they are costly and the topic of ethical debate. While the public expects chemicals and products to be safe for the developing and mature nervous systems, considerable numbers of chemicals in commerce have not, or only to a limited extent, been assessed for their potential to cause neurotoxicity. As such, there is a societal push toward the replacement of animal models with in vitro or alternative methods. New approach methods (NAMs) can contribute to the regulatory knowledge base, increase chemical safety, and modernize chemical hazard and risk assessment. Provided they reach an acceptable level of regulatory relevance and reliability, NAMs may be considered as replacements for specific in vivo studies. The European Partnership for the Assessment of Risks from Chemicals (PARC) addresses challenges to the development and implementation of NAMs in chemical risk assessment. In collaboration with regulatory agencies, Project 5.2.1e (Neurotoxicity) aims to develop and evaluate NAMs for developmental neurotoxicity (DNT) and adult neurotoxicity (ANT) and to understand the applicability domain of specific NAMs for the detection of endocrine disruption and epigenetic perturbation. To speed up assay time and reduce costs, we identify early indicators of later-onset effects. Ultimately, we will assemble second-generation developmental neurotoxicity and first-generation adult neurotoxicity test batteries, both of which aim to provide regulatory hazard and risk assessors and industry stakeholders with robust, speedy, lower-cost, and informative next-generation hazard and risk assessment tools.

Epigenetics of methylmercury.

PURPOSE OF REVIEW: Methylmercury (MeHg) is neurotoxic at high levels and particularly affects the developing brain. One proposed mechanism of MeHg neurotoxicity is alteration of the epigenetic programming. In this review, we summarise the experimental and epidemiological literature on MeHg-associated epigenetic changes. RECENT FINDINGS: Experimental and epidemiological studies have identified changes in DNA methylation following in utero exposure to MeHg, and some of the changes appear to be persistent. A few studies have evaluated associations between MeHg-related changes in DNA methylation and neurodevelopmental outcomes. Experimental studies reveal changes in histone modifications after MeHg exposure, but we lack epidemiological studies supporting such changes in humans. Experimental and epidemiological studies have identified microRNA-related changes associated with MeHg; however, more research is needed to conclude if these changes lead to persistent and toxic effects. SUMMARY: MeHg appears to interfere with epigenetic processes, potentially leading to persistent changes. However, observed associations of mercury with epigenetic changes are as of yet of unknown relevance to neurodevelopmental outcomes.

Prenatal exposure to phthalates and gender-specific play behavior at seven years of age in the SELMA study.

BACKGROUND: A growing body of evidence shows that prenatal exposure to phthalates affects child development. Since many phthalates have been shown to alter endocrine signaling, they may influence reproductive development, neurodevelopment, and child behavior. Indeed, a few studies reported associations between prenatal phthalate exposure and gender-specific play behavior. However, evidence for this relationship is limited, and previous findings are based on single phthalates, while human exposure entails mixtures of chemicals. OBJECTIVE: We aimed to investigate the associations between prenatal exposure to single phthalates, as well as a phthalate mixture, and gender-specific play behavior. METHODS: A total of 715 mother-child pairs from the Swedish Environmental Longitudinal, Mother and Child, Asthma and Allergy (SELMA) study were included. In the median week 10 of pregnancy, phthalate metabolites were measured in urine. Gender-specific play behavior was measured with Preschool Activities Inventory at the age of seven years. Linear and weighted quantile sum regressions were used; data was stratified by sex. Models were adjusted for child and maternal age, maternal education, parental attitudes toward play behavior, and urinary creatinine concentration. RESULTS: For boys, single compound analyses revealed negative associations of prenatal exposure to di-isononyl phthalate (DINP) concentrations with masculine (ß = -1.44; 95% CI = -2.72, -0.16) and composite (ß = -1.43; 95% CI = -2.72, -0.13) scores. Suggestive associations were also observed with a mixture approach identifying DINP as the main contributor of the association of decreased masculine play. Among girls, higher urinary concentrations of 2,4-methyl-7-oxyooctyl-oxycarbonyl-cyclohexane carboxylic acid (MOiNCH) was associated with decreased feminine (ß = -1.59; 95% CI = -2.62, -0.57) and masculine scores (ß = -1.22; 95% CI = -2.14, -0.29), whereas the mixture analyses did not yield conclusive results for girls. CONCLUSION: Our findings suggest associations of prenatal exposure to DINP with decreased masculine play behavior in boys while the results for girls were not fully conclusive.

Present and future challenges for the investigation of transgenerational epigenetic inheritance.

Epigenetic pathways are essential in different biological processes and in phenotype-environment interactions in response to different stressors and they can induce phenotypic plasticity. They encompass several processes that are mitotically and, in some cases, meiotically heritable, so they can be transferred to subsequent generations via the germline. Transgenerational Epigenetic Inheritance (TEI) describes the phenomenon that phenotypic traits, such as changes in fertility, metabolic function, or behavior, induced by environmental factors (e.g., parental care, pathogens, pollutants, climate change), can be transferred to offspring generations via epigenetic mechanisms. Investigations on TEI contribute to deciphering the role of epigenetic mechanisms in adaptation, adversity, and evolution. However, molecular mechanisms underlying the transmission of epigenetic changes between generations, and the downstream chain of events leading to persistent phenotypic changes, remain unclear. Therefore, inter-, (transmission of information between parental and offspring generation via direct exposure) and transgenerational (transmission of information through several generations with disappearance of the triggering factor) consequences of epigenetic modifications remain major issues in the field of modern biology. In this article, we review and describe the major gaps and issues still encountered in the TEI field: the general challenges faced in epigenetic research; deciphering the key epigenetic mechanisms in inheritance processes; identifying the relevant drivers for TEI and implement a collaborative and multi-disciplinary approach to study TEI. Finally, we provide suggestions on how to overcome these challenges and ultimately be able to identify the specific contribution of epigenetics in transgenerational inheritance and use the correct tools for environmental science investigation and biomarkers identification.

A human-relevant mixture of endocrine disrupting chemicals induces changes in hippocampal DNA methylation correlating with hyperactive behavior in male mice.

Humans are ubiquitously exposed to endocrine disrupting chemicals (EDCs), substances that interfere with endogenous hormonal signaling. Exposure during early development is of particular concern due to the programming role of hormones during this period. A previous epidemiological study has shown association between prenatal co-exposure to 8 EDCs (Mixture N1) and language delay in children, suggesting an effect of this mixture on neurodevelopment. Furthermore, in utero exposure to Mixture N1 altered gene expression and behavior in adult mice. In this study, we investigated whether epigenetic mechanisms could underlie the long term effects of Mixture N1 on gene expression and behavior. To this end, we analyzed DNA methylation at regulatory regions of genes whose expression was affected by Mixture N1 in the hippocampus of in utero exposed mice using bisulfite-pyrosequencing. We show that Mixture N1 decreases DNA methylation in males at three genes that are part of the hypothalamus-pituitary-adrenal (HPA) axis: Nr3c1, Nr3c2, and Crhr1, coding for the glucocorticoid receptor, the mineralocorticoid receptor, and the corticotropin releasing hormone receptor 1, respectively. Furthermore, we show that the decrease in Nr3c1 methylation correlates with increased gene expression, and that Nr3c1, Nr3c2, and Crhr1 methylation correlates with hyperactivity and reduction in social behavior. These findings indicate that an EDC mixture corresponding to a human exposure scenario induces epigenetic changes, and thus programming effects, on the HPA axis that are reflected in the behavioral phenotypes of the adult male offspring.