Karilyn E. Sant

Nutrition and epigenetics: an interplay of dietary methyl donors, one-carbon metabolism and DNA methylation ☆ ☆☆

DNA methylation is the most extensively studied mechanism of epigenetic gene regulation. Increasing evidence indicates that DNA methylation is labile in response to nutritional and environmental influences. Alterations in DNA methylation profiles can lead to changes in gene expression, resulting in diverse phenotypes with the potential for increased disease risk. The primary methyl donor for DNA methylation is S-adenosylmethionine (SAM), a species generated in the cyclical cellular process called one-carbon metabolism. One-carbon metabolism is catalyzed by several enzymes in the presence of dietary micronutrients, including folate, choline, betaine and other B vitamins. For this reason, nutrition status, particularly micronutrient intake, has been a focal point when investigating epigenetic mechanisms. Although animal evidence linking nutrition and DNA methylation is fairly extensive, epidemiological evidence is less comprehensive. This review serves to integrate studies of the animal in vivo with human epidemiological data pertaining to nutritional regulation of DNA methylation and to further identify areas in which current knowledge is limited.

Inhibition of glutathione biosynthesis alters compartmental redox status and the thiol proteome in organogenesis-stage rat conceptuses.

Developmental signals that control growth and differentiation are regulated by environmental factors that generate reactive oxygen species (ROS) and alter steady-state redox environments in tissues and fluids. Protein thiols are selectively oxidized and reduced in distinct spatial and temporal patterns in conjunction with changes in glutathione/glutathione disulfide (GSH/GSSG) and cysteine/cystine (Cys/CySS) redox potentials (E(h)) to regulate developmental signaling. The purpose of this study was to measure compartment-specific thiol redox status in cultured organogenesis-stage rat conceptuses and to evaluate the impact of thiol oxidation on the redox proteome. The visceral yolk sac (VYS) has the highest initial (0 h) total intracellular GSH (GSH+2GSSG) concentration (5.5 mM) and the lowest Eh (-223 mV) as determined by HPLC analysis. Total embryo (EMB) GSH concentrations ranged lower (3.2 mM) and were only slightly more oxidized than the VYS. Total GSH concentrations in yolk sac fluid (YSF) and amniotic fluid (AF) are >500-fold lower than in tissues and are highly oxidized (YSF E(h)=-121 mV and AF E(h)=-49 mV). Steady-state total Cys concentrations (Cys+2CySS) were significantly lower than GSH in tissues but were otherwise equal in VYS and EMB near 0.5 mM. On gestational day 11, total GSH and Cys concentrations in EMB and VYS increase significantly over the 6h time course while E(h) remains relatively constant. The Eh (GSH/GSSG) in YSF and AF become more reduced over time while E(h) (Cys/CySS) become more oxidized. Addition of L-buthionine-S,R-sulfoximine (BS0) to selectively inhibit GSH synthesis and mimic the effects of some GSH-depleting environmental chemicals significantly decreased VYS and EMB GSH and Cys concentrations and increased Eh over the 6h exposure period, showing a greater overall oxidation. In the YSF, BSO caused a significant increase in total Cys concentrations to 1.7 mM but did not significantly change the E(h) for Cys/CySS. A significant net oxidation was seen in the BSO-treated AF compartment after 6 h. Biotinylated iodoacetamide (BIAM) labeling of proteins revealed the significant thiol oxidation of many EMB proteins following BSO treatment. Quantitative changes in the thiol proteome, associated with developmentally relevant pathways, were detected using isotope coded affinity tag (ICAT) labeling and mass spectroscopy. Adaptive pathways were selectively enriched with increased concentrations of proteins involved in mRNA processing (splicesome) and mRNA stabilization (glycolysis, GAPDH), as well as protein synthesis (aminoacyl-tRNA) and protein folding (antigen processing, Hsp70, protein disulfide isomerase). These results show the ability of chemical and environmental modulators to selectively alter compartmental intracellular and extracellular GSH and Cys concentrations and change their corresponding E(h) within the intact viable conceptus. The altered E(h) were also of sufficient magnitude to alter the redox proteome and change relative protein concentrations, suggesting that the mechanistic links through which environmental factors inform and regulate developmental signaling pathways may be discovered using systems developmental biology techniques.

DNA Methylation Screening and Analysis

DNA methylation is an epigenetic form of gene regulation that is universally important throughout the life course, especially during in utero and postnatal development. DNA methylation aids in cell cycle regulation and cellular differentiation processes. Previous studies have demonstrated that DNA methylation profiles may be altered by diet and the environment, and that these profiles are especially vulnerable during development. Thus, it is important to understand the role of DNA methylation in developmental governance and subsequent disease progression. A variety of molecular methods exist to assay for global, gene-specific, and epigenome-wide methylation. Here we describe these methods and discuss their relative strengths and limitations.

Mono-2-ethylhexyl phthalate (MEHP) alters histiotrophic nutrition pathways and epigenetic processes in the developing conceptus.

Histiotrophic nutrition pathways (HNPs) are processes by which the organogenesis-stage conceptus obtains nutrients, amino acids, vitamins and cofactors required for protein biosynthesis and metabolic activities. Nutrients are captured from the maternal milieu as whole proteins and cargoes via receptor-mediated endocytosis in the visceral yolk sac (VYS), degraded by lysosomal proteolysis and delivered to the developing embryo (EMB). Several nutrients obtained by HNPs are required substrates for one-carbon (C1) metabolism and supply methyl groups required for epigenetic processes, including DNA and histone methylation. Increased availability of methyl donors has been associated with reduced risk for neural tube defects (NTDs). Here, we show that mono-2-ethylhexyl phthalate (MEHP) treatment (100 or 250μM) alters HNPs, C1 metabolism and epigenetic programming in the organogenesis-stage conceptus. Specifically, 3-h MEHP treatment of mouse EMBs in whole culture resulted in dose-dependent reduction of HNP activity in the conceptus. To observe nutrient consequences of decreased HNP function, C1 components and substrates and epigenetic outcomes were quantified at 24h. Treatment with 100-μM MEHP resulted in decreased dietary methyl donor concentrations, while treatment with 100- or 250-μM MEHP resulted in dose-dependent elevated C1 products and substrates. In MEHP-treated EMBs with NTDs, H3K4 methylation was significantly increased, while no effects were seen in treated VYS. DNA methylation was reduced in MEHP-treated EMB with and without NTDs. This research suggests that environmental toxicants such as MEHP decrease embryonic nutrition in a time-dependent manner and that epigenetic consequences of HNP disruption may be exacerbated in EMB with NTDs.

Novel Epigenetic Biomarkers Mediating Bisphenol A Exposure and Metabolic Phenotypes in Female Mice

There is compelling evidence that epigenetic modifications link developmental environmental insults to adult disease susceptibility. Animal studies have associated perinatal bisphenol A (BPA) exposure to altered DNA methylation, but these studies are often limited to candidate gene and global non-loci-specific approaches. By using an epigenome-wide discovery platform, we elucidated epigenetic alterations in liver tissue from adult mice offspring (10 months) following perinatal BPA exposure at human physiologically relevant doses (50-ng, 50-μg, and 50-mg BPA/kg diet). Biological pathway analysis identified an enrichment of significant differentially methylated regions in metabolic pathways among females. Furthermore, through the use of top enriched biological pathways, 4 candidate genes were chosen to assess DNA methylation as a mediating factor linking the association of perinatal BPA exposure to metabolic phenotypes previously observed in female offspring. DNA methylation status at Janus kinase-2 (Jak-2), retinoid X receptor (Rxr), regulatory factor x-associated protein (Rfxap), and transmembrane protein 238 (Tmem238) was used within a mediational regression analysis. DNA methylation in all four of the candidate genes was identified as a mediator in the mechanistic pathway of developmental BPA exposure and female-specific energy expenditure, body weight, and body fat phenotypes. Data generated from this study are crucial for deciphering the mechanistic role of epigenetics in the pathogenesis of chronic disease and the development of epigenetic-based prevention and therapeutic strategies for complex human disease.

Inhibition of proteolysis in histiotrophic nutrition pathways alters DNA methylation and one-carbon metabolism in the organogenesis-stage rat conceptus.

Epigenetic modifications, including DNA methylation, contribute to the transcriptional regulation of developmental genes that control growth and differentiation during embryogenesis. The methyl donor, S-adenosylmethionine (SAM), is biosynthesized from methionine and adenosine triphosphate by methionine adenosyltransferase 2a (Mat2a) in the one-carbon (C1) metabolism pathway. SAM biosynthesis requires a steady supply of nutrients, vitamins and cofactors obtained by the developing conceptus through histiotrophic nutrition pathways (HNPs). The visceral yolk sac (VYS) captures proteins and their substrate cargos by receptor-mediated endocytosis and degrades them using lysosomal proteases. We hypothesize that leupeptin, a protease inhibitor, reduces the availability of methionine and C1 substrates, restricting SAM biosynthesis and altering patterns of DNA methylation. Rat conceptuses were exposed to 50 and 100 μM leupeptin in whole embryo culture for periods of 26 h from gestational day (GD) 10 or 6 h on GD11. After 6 h on GD11, the 100-μM leupeptin treatment significantly decreased methionine in embryo (EMB) and VYS, reduced Mat2a protein levels and inhibited Mat2a specific activity, all of which produced a significant 52% reduction of SAM in the VYS. The 50- and 100-μM leupeptin treatments significantly decreased global methylation levels by 6%-9% in EMB and by 11%-15% in VYS following both 6- and 26-h exposure periods. This study demonstrates that HNP disruption alters C1 activity and significantly reduces global DNA methylation during organogenesis. Because epigenetic reprogramming is crucial for normal differentiation and growth, these findings suggest a possible mechanism through which nutrients and environmental factors may alter early developmental regulation.

Ethanol Attenuates Histiotrophic Nutrition Pathways and Alters the Intracellular Redox Environment and Thiol Proteome during Rat Organogenesis

Ethanol (EtOH) is a reactive oxygen-generating teratogen involved in the etiology of structural and functional developmental defects. Embryonic nutrition, redox environment, and changes in the thiol proteome following EtOH exposures (1.56.0 mg/ml) were studied in rat whole embryo culture. Glutathione (GSH) and cysteine (Cys) concentrations with their respective intracellular redox potentials (Eh) were determined using high-performance liquid chromatography. EtOH reduced GSH and Cys concentrations in embryo (EMB) and visceral yolk sac (VYS) tissues, and also in yolk sac and amniotic fluids. These changes produced greater oxidation as indicated by increasingly positive Eh values. EtOH reduced histiotrophic nutrition pathway activities as measured by the clearance of fluorescin isothiocyanate (FITC)-albumin from culture media. A significant decrease in total FITC clearance was observed at all concentrations, reaching approximately 50% at the highest dose. EtOH-induced changes to the thiol proteome were measured in EMBs and VYSs using isotope-coded affinity tags. Decreased concentrations for specific proteins from cytoskeletal dynamics and endocytosis pathways (α-actinin, α-tubulin, cubilin, and actin-related protein 2); nuclear translocation (Ran and RanBP1); and maintenance of receptor-mediated endocytosis (cubilin) were observed. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis also identified a decrease in ribosomal proteins in both EMB and VYS. Results show that EtOH interferes with nutrient uptake to reduce availability of amino acids and micronutrients required by the conceptus. Intracellular antioxidants such as GSH and Cys are depleted following EtOH and Eh values increase. Thiol proteome analysis in the EMB and VYS show selectively altered actin/cytoskeleton, endocytosis, ribosome biogenesis and function, nuclear transport, and stress-related responses.

Mono-2-ethylhexyl phthalate disrupts neurulation and modifies the embryonic redox environment and gene expression.

Mono-2-ethylhexl phthalate (MEHP) is the primary metabolite of di-2-ethylhexyl phthalate (DEHP), a ubiquitous contaminant in plastics. This study sought to determine how structural defects caused by MEHP in mouse whole embryo culture were related to temporal and spatial patterns of redox state and gene expression. MEHP reduced morphology scores along with increased incidence of neural tube defects. Glutathione (GSH) and cysteine (Cys) concentrations fluctuated spatially and temporally in embryo (EMB) and visceral yolk sac (VYS) across the 24h culture. Redox potentials (Eh) for GSSG/GSH were increased by MEHP in EMB (12h) but not in VYS. CySS/CyS Eh in EMB and VYS were significantly increased at 3h and 24h, respectively. Gene expression at 6h showed that MEHP induced selective alterations in EMB and VYS for oxidative phosphorylation and energy metabolism pathways. Overall, MEHP affects neurulation, alters Eh, and spatially alters the expression of metabolic genes in the early organogenesis-stage mouse conceptus.

Epigenome-wide DNA methylation analysis implicates neuronal and inflammatory signaling pathways in adult murine hepatic tumorigenesis following perinatal exposure to bisphenol A.

Developmental exposure to the endocrine‐active compound bisphenol A (BPA) has been linked to epigenotoxic and potential carcinogenic effects in rodent liver, prostate, and mammary glands. A dose‐dependent increase in hepatic tumors in 10‐month mice perinatally exposed to one of three doses of BPA (50 ng, 50 µg, or 50 mg BPA/kg chow) was previously reported. These tumors represent early‐onset disease and lack classical sexual dimorphism in incidence. Here, adult epigenome‐wide liver DNA methylation profiles to identify gene promoters associated with perinatal BPA exposure and disease in 10‐month mice with and without liver tumors were investigated. Mice with hepatic tumors showed 12,822 (1.8%) probes with differential methylation as compared with non‐tumor animals, of which 8,656 (67.5%) were hypomethylated. A significant enrichment of differential methylation in Gene Ontology (GO) terms and biological processes related to morphogenesis and development, and epigenomic alteration were observed. Pathway enrichment revealed a predominance of hypermethylated neuronal signaling pathways linked to energy regulation and metabolic function, supporting metabolic consequences in the liver via BPA‐induced disruption of neuronal signaling pathways. Hypothesis‐driven pathway analysis revealed mouse and human genes linked to BPA exposure related to intracellular Jak/STAT and MAPK signaling pathways. Taken together, these findings are indicators of the relevance of the hepatic tumor phenotype seen in BPA‐exposed mice to human health. This work demonstrated that epigenome‐wide discovery experiments in animal models were effective tools for identification and understanding of paralagous epimutations salient to human disease. Environ. Mol. Mutagen. 57:435–446, 2016.

Amino acid starvation induced by protease inhibition produces differential alterations in redox status and the thiol proteome in organogenesis-stage rat embryos and visceral yolk sacs

The process of embryonic nutrition in rodent conceptuses during organogenesis has been shown to involve a dominant histiotrophic mechanism where essential developmental substrates and micronutrients are supplied as whole maternal proteins or cargoes associated with proteins. The histiotrophic nutrition pathways (HNP) responsible for uptake and initial processing of proteins across maternal-conceptal interfaces involve uptake via receptor mediated endocytosis and protein degradation via lysosomal proteolysis. Chemical inhibition of either process can lead to growth deficits and malformation in the embryo (EMB), but selective inhibition of either HNP component will elicit a different subset of developmental perturbations. In vitro, whole embryo culture exposure of GD10 or GD11 rat conceptuses to the natural protease inhibitor, leupeptin, leads to significant reductions in all measured embryonic growth parameters as well as a myriad of other effects. Leupeptin doses of 10 μM or 20 μM over a 26-h period (GD10-GD11) and 50 μM over a 3 h pulse period produced significant decreases in the clearance of FITC-albumin from culture media. The near complete loss of acid soluble fluorescence and increased total visceral yolk sac (VYS) protein content confirmed the selective inhibition of proteolysis. Inhibition of lysosomal proteolysis thus deprives the developing EMB of essential nutrient amino acids producing conditions akin to amino acid starvation, but may also cause direct effects on pathways critical for normal growth and differentiation. Following leupeptin exposure for 26 or 6 h, total glutathione (GSH) concentrations dropped significantly in the VYS, but only slightly in yolk sac (YSF) and amniotic (AF) fluids. Cys concentrations increased in VYS and EMB, but dropped in YSF and AF fluids. Redox potentials (Eh) for the glutathione disulfide (GSSG)/glutathione (GSH) redox couple trended significantly toward the positive, confirming the net oxidation of conceptual tissues following leupeptin treatment. Analysis of the thiol proteome showed few alterations to specific pathways mapped to the Kyoto Encyclopedia of Genes and Genomes Pathway database, but did reveal significant increases in concentrations of proteins associated with glycolysis/gluconeogenesis in the VYS and decreased concentrations proteins associated with ribosome biogenesis and function in the EMB. A subset of proteins elevated by >2-23-fold in the VYS were identified as serum (blood) proteins and represent the maternal-side proteins captured by the VYS and which are not degraded in the lysosomes as a result of leupeptins inhibitory action. The observed constellation of proteins decreased in the EMB by leupeptin represent proteins from several adaptive pathways that are commonly altered in responses to amino acid starvation. These studies show clear differential responses to protease inhibition in VYS and EMB during organogenesis and suggest the possibility for additional roles of redox regulation, cellular adaptations and metabolic insufficiency caused by protease inhibition.