Developmental exposures to common environmental contaminants, DEHP and lead, alter adult brain and blood hydroxymethylation in mice

Introduction: The developing epigenome changes rapidly, potentially making it more sensitive to toxicant exposures. DNA modifications, including methylation and hydroxymethylation, are important parts of the epigenome that may be affected by environmental exposures. However, most studies do not differentiate between these two DNA modifications, possibly masking significant effects.Methods: To investigate the relationship between DNA hydroxymethylation and developmental exposure to common contaminants, a collaborative, NIEHS-sponsored consortium, TaRGET II, initiated longitudinal mouse studies of developmental exposure to human-relevant levels of the phthalate plasticizer di(2-ethylhexyl) phthalate (DEHP), and the metal lead (Pb). Exposures to 25 mg DEHP/kg of food (approximately 5 mg DEHP/kg body weight) or 32 ppm Pb-acetate in drinking water were administered to nulliparous adult female mice. Exposure began 2 weeks before breeding and continued throughout pregnancy and lactation, until offspring were 21 days old. At 5 months, perinatally exposed offspring blood and cortex tissue were collected, for a total of 25 male mice and 17 female mice (n = 5–7 per tissue and exposure). DNA was extracted and hydroxymethylation was measured using hydroxymethylated DNA immunoprecipitation sequencing (hMeDIP-seq). Differential peak and pathway analysis was conducted comparing across exposure groups, tissue types, and animal sex, using an FDR cutoff of 0.15.Results: DEHP-exposed females had two genomic regions with lower hydroxymethylation in blood and no differences in cortex hydroxymethylation. For DEHP-exposed males, ten regions in blood (six higher and four lower) and 246 regions (242 higher and four lower) and four pathways in cortex were identified. Pb-exposed females had no statistically significant differences in blood or cortex hydroxymethylation compared to controls. Pb-exposed males, however, had 385 regions (all higher) and six pathways altered in cortex, but no differential hydroxymethylation was identified in blood.Discussion: Overall, perinatal exposure to human-relevant levels of two common toxicants showed differences in adult DNA hydroxymethylation that was specific to sex, exposure type, and tissue, but male cortex was most susceptible to hydroxymethylation differences by exposure. Future assessments should focus on understanding if these findings indicate potential biomarkers of exposure or are related to functional long-term health effects

Developmental Exposures to Phthalates and Phthalate Mixtures and Life-Course Metabolic Outcomes: Using a Mouse Model to Inform Human Studies and Elucidate Mechanisms

Nearly 40 percent of US adults and 20 percent of US children are obese. Given obesity’s multiple dangerous comorbidities, this presents a significant concern for public health. A growing body of evidence suggests that exposures to environmental chemicals may be contributing to the obesity epidemic. Such chemicals have been termed “obesogens” and among them are phthalates, endocrine disrupting chemicals (EDCs) that are present in food packaging, children’s toys, and personal care products. Exposures to phthalates during development have been linked to adverse metabolic health outcomes in both animal and human studies, but findings from human studies are less consistent. One possible reason is humans are co-exposed to many phthalates, and these mixture exposures are difficult to interpret. Additionally, the vast majority of animal studies to date have focused on examining metabolic impacts of diethylhexyl phthalate (DEHP), despite the recent introduction of newer phthalates on the market to replace it, including diisononyl phthalate (DINP). Furthermore, mechanisms linking developmental exposures and later-life health outcomes, such as epigenetic reprogramming via DNA methylation, are still poorly understood. The overall objective of this dissertation was to utilize an animal model of perinatal phthalate exposures to investigate long-term metabolic impacts in a manner that would inform human studies and infer underlying mechanisms. We incorporated exposures to three individual phthalates (DEHP, DINP, and dibutyl phthalate (DBP)), as well as two phthalate mixtures (DEHP+DINP and DEHP+DINP+DBP). We then took phenotypic and molecular measurements on the offspring at two time points: at weaning on postnatal day 21 (PND21) at the end of the exposure period and at 10 months of age, >9 months after exposure had ceased. In Aim 1, we investigated early-life metabolic phenotypes by measuring body weight and relative liver weights and examined biomarkers of whole-genome DNA methylation alterations at PND21. In Aim 2, we evaluated metabolic phenotypes longitudinally at two and eight months of age to determine whether developmental exposures to phthalates influenced metabolism across the life course. Finally, in Aim 3, we measured the transcriptome and DNA methylation in liver and white adipose tissue (WAT) at both PND21 and 10 months to elucidate a molecular mechanism. We found that developmental exposures to individual phthalates and phthalate mixtures were associated with increased body weights in males and females in early postnatal life. Females, but not males, perinatally exposed to DINP-only and a mixture of DEHP+DINP also had increased relative liver weights at PND21. We also observed a sex-specific effect on tail DNA methylation at repetitive elements in mice exposed to individual phthalates and phthalate mixtures, indicating a sexually dimorphic effect on the epigenome. Developmental exposures to DEHP-only and DINP-only resulted in increased body fat percentage and glucose intolerance, respectively, across the life course. However, we did not observe longitudinal adverse metabolic impacts in mice perinatally exposed to phthalate mixtures, suggesting a potential adaptive response in these mice. In females perinatally exposed to DINP, we identified several persistently up-regulated PPAR target genes in the liver that could lead to increased fatty acid synthesis. Fatty acid synthase (Fasn) also exhibited increased promoter region DNA methylation at both PND21 and 10 months of age, implicating a role for epigenetic reprogramming. Taken together, the work here demonstrates short-term and long-term metabolic impacts following perinatal exposures to phthalates, and presents a new potential mechanism describing the underlying biology in the liver.PHDToxicologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/151581/1/kneier_1.pd

Genome-Wide DNA Methylation Profiles of Neurodevelopmental Disorder Genes in Mouse Placenta and Fetal Brain Following Prenatal Exposure to Polychlorinated Biphenyls

Background Polychlorinated biphenyls (PCBs) are developmental neurotoxicants implicated as environmental risk factors for neurodevelopmental disorders (NDD), including autism spectrum disorders (ASD). Objective We examined the effects of prenatal exposure to a human-relevant mixture of PCBs on the DNA methylome of fetal mouse brain and placenta to determine if there was a shared subset of differentially methylated regions (DMRs). Methods A PCB mixture formulated to model the 12 most abundant congeners detected in the serum of pregnant women from a prospective high-risk ASD cohort was administered to female mice prior to and during pregnancy. Whole-genome bisulfite sequencing (WGBS) was performed to assess genome-wide DNA methylation profiles of placenta and brain on gestational day 18. Results We found thousands of significant (empirical p < 0.05) DMRs distinguishing placentas and brains from PCB-exposed embryos from sex-matched vehicle controls. In both placenta and brain, PCB-associated DMRs were significantly ( p < 0.005) enriched for functions related to neurodevelopment, cellular adhesion, and cellular signaling, and significantly (Odds Ratio > 2.4, q < 0.003) enriched for bivalent chromatin marks. The placenta and brain PCB DMRs overlapped significantly (Z-score = 4.5, p = 0.0001) by genomic coordinate and mapped to a shared subset of genes significantly ( q < 0.05) enriched for Wnt signaling, Slit/Robo signaling, and genes differentially expressed in multiple NDD/ASD models. The placenta and brain DMRs also significantly ( q < 0.05) overlapped by genomic coordinate with brain samples from humans with Rett syndrome and Dup15q syndrome. Discussion These results demonstrate that placenta can be used as a surrogate for embryonic brain DNA methylation changes over genes relevant to NDD/ASD in a mouse model of prenatal PCB exposure

Placenta and fetal brain share a neurodevelopmental disorder DNA methylation profile in a mouse model of prenatal PCB exposure.

Polychlorinated biphenyls (PCBs) are developmental neurotoxicants implicated as environmental risk factors for neurodevelopmental disorders (NDDs). Here, we report the effects of prenatal exposure to a human-relevant mixture of PCBs on the DNA methylation profiles of mouse placenta and fetal brain. Thousands of differentially methylated regions (DMRs) distinguish placenta and fetal brain from PCB-exposed mice from sex-matched vehicle controls. In both placenta and fetal brain, PCB-associated DMRs are enriched for functions related to neurodevelopment and cellular signaling and enriched within regions of bivalent chromatin. The placenta and brain PCB DMRs overlap significantly and map to a shared subset of genes enriched for Wnt signaling, Slit/Robo signaling, and genes differentially expressed in NDD models. The consensus PCB DMRs also significantly overlap with DMRs from human NDD brain and placenta. These results demonstrate that PCB-exposed placenta contains a subset of DMRs that overlap fetal brain DMRs relevant to an NDD

Wilson Disease: Intersecting DNA Methylation and Histone Acetylation Regulation of Gene Expression in a Mouse Model of Hepatic Copper Accumulation.

Background & aimsThe pathogenesis of Wilson disease (WD) involves hepatic and brain copper accumulation resulting from pathogenic variants affecting the ATP7B gene and downstream epigenetic and metabolic mechanisms. Prior methylome investigations in human WD liver and blood and in the Jackson Laboratory (Bar Harbor, ME) C3He-Atp7btx-j/J (tx-j) WD mouse model revealed an epigenetic signature of WD, including changes in histone deacetylase (HDAC) 5. We tested the hypothesis that histone acetylation is altered with respect to copper overload and aberrant DNA methylation in WD.MethodsWe investigated class IIa HDAC4 and HDAC5 and H3K9/H3K27 histone acetylation in tx-j mouse livers compared with C3HeB/FeJ (C3H) control in response to 3 treatments: 60% kcal fat diet, D-penicillamine (copper chelator), and choline (methyl group donor). Experiments with copper-loaded hepatoma G2 cells were conducted to validate in vivo studies.ResultsIn 9-week tx-j mice, HDAC5 levels increased significantly after 8 days of a 60% kcal fat diet compared with chow. In 24-week tx-j mice, HDAC4/5 levels were reduced 5- to 10-fold compared with C3H, likely through mechanisms involving HDAC phosphorylation. HDAC4/5 levels were affected by disease progression and accompanied by increased acetylation. D-penicillamine and choline partially restored HDAC4/5 and H3K9ac/H3K27ac to C3H levels. Integrated RNA and chromatin immunoprecipitation sequencing analyses revealed genes regulating energy metabolism and cellular stress/development, which, in turn, were regulated by histone acetylation in tx-j mice compared with C3H mice, with Pparα and Pparγ among the most relevant targets.ConclusionsThese results suggest dietary modulation of class IIa HDAC4/5, and subsequent H3K9/H3K27 acetylation/deacetylation can regulate gene expression in key metabolic pathways in the pathogenesis of WD

Networks of placental DNA methylation correlate with maternal serum PCB concentrations and child neurodevelopment.

BackgroundGestational exposure to polychlorinated biphenyls (PCBs) has been associated with elevated risk for neurodevelopmental disorders. Placental epigenetics may serve as a potential mechanism of risk or marker of altered placental function. Prior studies have associated differential placental DNA methylation with maternal PCB exposure or with increased risk of autism spectrum disorder (ASD). However, sequencing-based placental methylomes have not previously been tested for simultaneous associations with maternal PCB levels and child neurodevelopmental outcomes.ObjectivesWe aimed to identify placental DNA methylation patterns associated with maternal PCB levels and child neurodevelopmental outcomes in the high-risk ASD MARBLES cohort.MethodsWe measured 209 PCB congeners in 104 maternal serum samples collected at delivery. We identified networks of DNA methylation from 147 placenta samples using the Comethyl R package, which performs weighted gene correlation network analysis for whole genome bisulfite sequencing data. We tested placental DNA methylation modules for association with maternal serum PCB levels, child neurodevelopment, and other participant traits.ResultsPCBs 153 + 168, 170, 180 + 193, and 187 were detected in over 50% of maternal serum samples and were highly correlated with one another. Consistent with previous findings, maternal age was the strongest predictor of serum PCB levels, alongside year of sample collection, pre-pregnancy BMI, and polyunsaturated fatty acid levels. Twenty seven modules of placental DNA methylation were identified, including five which significantly correlated with one or more PCBs, and four which correlated with child neurodevelopment. Two modules associated with maternal PCB levels as well as child neurodevelopment, and mapped to CSMD1 and AUTS2, genes previously implicated in ASD and identified as differentially methylated regions in mouse brain and placenta following gestational PCB exposure.ConclusionsPlacental DNA co-methylation modules were associated with maternal PCBs and child neurodevelopment. Methylation of CSMD1 and AUTS2 could be markers of altered placental function and/or ASD risk following maternal PCB exposure

The role of intestine in metabolic dysregulation in murine Wilson disease.

BACKGROUND: The clinical manifestations of Wilson disease (WD) are related to copper accumulation in the liver and the brain, but little is known about other tissue involvement regarding metabolic changes in WD. In vitro studies suggested that the loss of intestinal ATP7B affects metabolic dysregulation in WD. We tested this hypothesis by evaluating the gut microbiota and lipidome in 2 mouse models of WD and by characterizing a new mouse model with a targeted deletion of Atp7b in the intestine. METHODS: Cecal content 16S sequencing and untargeted hepatic and plasma lipidome analyses in the Jackson Laboratory toxic-milk and the Atp7b null global knockout mouse models of WD were profiled and integrated. Intestine-specific Atp7b knockout mice (Atp7bΔIEC) were generated and characterized using targeted lipidome analysis following a high-fat diet challenge. RESULTS: Gut microbiota diversity was reduced in animal models of WD. Comparative prediction analysis revealed amino acid, carbohydrate, and lipid metabolism functions to be dysregulated in the WD gut microbial metagenome. Liver and plasma lipidomic profiles showed dysregulated triglyceride and diglyceride, phospholipid, and sphingolipid metabolism in WD models. However, Atp7bΔIEC mice did not show gut microbiome differences compared to wild type. When challenged with a high-fat diet, Atp7bΔIEC mice exhibited profound alterations to fatty acid desaturation and sphingolipid metabolism pathways as well as altered APOB48 distribution in intestinal epithelial cells. CONCLUSIONS: Gut microbiome and lipidome underlie systemic metabolic manifestations in murine WD. Intestine-specific ATP7B deficiency affected both intestinal and systemic response to a high-fat challenge but not the microbiome profile, at least at early stages. WD is a systemic disease in which intestinal-specific ATP7B loss and diet influence the phenotype and the lipidome profile

Tissue and sex-specific programming of DNA methylation by perinatal lead exposure: implications for environmental epigenetics studies

Early developmental environment can influence long-term health through reprogramming of the epigenome. Human environmental epigenetics studies rely on surrogate tissues, such as blood, to assess the effects of environment on disease-relevant but inaccessible target tissues. However, the extent to which environment-induced epigenetic changes are conserved between these tissues is unclear. A better understanding of this conservation is imperative for effective design and interpretation of human environmental epigenetics studies. The Toxicant Exposures and Responses by Genomic and Epigenomic Regulators of Transcription (TaRGET II) consortium was established by the National Institute of Environmental Health Sciences to address the utility of surrogate tissues as proxies for toxicant-induced epigenetic changes in target tissues. We and others have recently reported that perinatal exposure to lead (Pb) is associated with adverse metabolic outcomes. Here, we investigated the sex-specific effects of perinatal exposure to a human environmentally relevant level of Pb on DNA methylation in paired liver and blood samples from adult mice using enhanced reduced-representation bisulphite sequencing. Although Pb exposure ceased at 3 weeks of age, we observed thousands of sex-specific differentially methylated cytosines in the blood and liver of Pb-exposed animals at 5 months of age, including 44 genomically imprinted loci. We observed significant tissue overlap in the genes mapping to differentially methylated cytosines. A small but significant subset of Pb-altered genes exhibit basal sex differences in gene expression in the mouse liver. Collectively, these data identify potential molecular targets for Pb-induced metabolic diseases, and inform the design of more robust human environmental epigenomics studies

Sex disparate gut microbiome and metabolome perturbations precede disease progression in a mouse model of Rett syndrome.

Rett syndrome (RTT) is a regressive neurodevelopmental disorder in girls, characterized by multisystem complications including gut dysbiosis and altered metabolism. While RTT is known to be caused by mutations in the X-linked gene MECP2, the intermediate molecular pathways of progressive disease phenotypes are unknown. Mecp2 deficient rodents used to model RTT pathophysiology in most prior studies have been male. Thus, we utilized a patient-relevant mouse model of RTT to longitudinally profile the gut microbiome and metabolome across disease progression in both sexes. Fecal metabolites were altered in Mecp2e1 mutant females before onset of neuromotor phenotypes and correlated with lipid deficiencies in brain, results not observed in males. Females also displayed altered gut microbial communities and an inflammatory profile that were more consistent with RTT patients than males. These findings identify new molecular pathways of RTT disease progression and demonstrate the relevance of further study in female Mecp2 animal models