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Adela Chirita-Emandi,1,2,* Costela Lacrimioara Serban,2,3,* Corina Paul,4,5,* Nicoleta Andreescu,1,2 Iulian Velea,4,5 Alexandra Mihailescu,1 Vlad Serafim,1,6 Diana-Andreea Tiugan,1 Paul Tutac,1 Cristian Zimbru,1,7 Maria Puiu,1,2 Mihai Dinu Niculescu1,8 1Department of Microscopic Morphology - Genetics, Center of Genomic Medicine, University of Medicine and Pharmacy “Victor Babes”, Timisoara, Romania; 2Regional Center of Medical Genetics Timis, Clinical Emergency Hospital for Children “Louis Turcanu”, Timisoara, Romania; 3Department of Functional Sciences, University of Medicine and Pharmacy “Victor Babes”, Timisoara, Romania; 4Pediatrics Department – Pediatrics Discipline II, University of Medicine and Pharmacy “Victor Babes”, Timisoara, Romania; 5Pediatrics, Endocrinology and Diabetes Department, Clinic II Pediatrics, “Pius Branzeu” Clinical Emergency County Hospital, Timisoara, Romania; 6The National Institute of Research and Development for Biological Sciences, Bucharest, Romania; 7Department of Automation and Applied Informatics, Politehnica University of Timisoara, Timisoara, Romania; 8Advanced Nutrigenomics, Cary, NC 27511, USA*These authors contributed equally to this workCorrespondence: Nicoleta AndreescuDepartment of Microscopic Morphology - Genetics, Center of Genomic Medicine, University of Medicine and Pharmacy “Victor Babes”, Timisoara, RomaniaEmail andreescu.nicoleta@umft.roIntroduction: Insulin resistance plays a major role in metabolic syndrome and is recognized as the most common risk factor for non-alcoholic fatty liver disease (NAFLD). Identifying predictors for insulin resistance could optimize screening and prevention.Purpose: To evaluate the contribution of multiple single nucleotide polymorphisms across genes related to NAFLD and choline metabolism, in predicting insulin resistance in children with obesity.Methods: One hundred fifty-three children with obesity (73 girls), aged 7– 18 years, were evaluated within the NutriGen Study (ClinicalTrials.gov-NCT02837367). Insulin resistance was defined by Homeostatic Model Assessment for insulin-resistance cut-offs that accommodated pubertal and gender differences. Anthropometric, metabolic, intake-related variables, and 55 single nucleotide polymorphisms related to NAFLD and choline metabolism were evaluated. Gene–gene interaction effects were assessed using Multiple Data Reduction Software.Results: Sixty percent (93/153) of participants showed insulin resistance (58.7% of boys, 63% of girls). Children with insulin resistance presented significantly higher values for standardized body mass index, triglycerides, transaminases and plasma choline when compared to those without insulin resistance. Out of 52 single nucleotide polymorphisms analysed, the interaction between genotypes CHDH(rs12676) and PNPLA3(rs738409) predicted insulin resistance. The model presented a 6/10 cross-validation consistency and 0.58 testing accuracy. Plasma choline levels and alanine aminotransferase modulated the gene interaction effect, significantly improving the model.Conclusion: The interaction between genotypes in CHDH and PNPLA3 genes, modulated by choline and alanine aminotransferase levels, predicted insulin-resistance status in children with obesity. If replicated in larger cohorts, these findings could help identify metabolic risk in children with obesity.Keywords: insulin-resistance, obesity, gene–gene interaction, CHDH-PNPLA3, choline, children

ABSTRACT: Objective: Pregnancy-associated osteoporosis (PAO) is a rare condition characterized by back pain and vertebral fracture (VF) during late pregnancy and the early postpartum period.Methods: We report a case of PAO with multiple VFs in a woman treated with low-molecular-weight heparin (LMWH) nadroparine during her second pregnancy and the rapid response to teriparatide treatment.Results: A 29-year-old woman presented for severe back pain and inability to stand upright. The symptoms started shortly after delivery of her second baby. Starting with the second month of pregnancy, she was treated with nordroparine 3,800 UI/day. Magnetic resonance imaging showed multiple thoracic (T) and lumbar (L) VFs. Lateral vertebral assessment (LVA) showed mild T8 and T10 and moderate T12 and L1 VFs. Bone mineral density (BMD) measured using dual-energy X-ray absorptiometry (DXA) revealed very low scores for lumbar spine (T-score, -4.2 SD; Z-score, -3.6 SD) and femoral neck (T-score, -2.1 SD; Z-score, -1.6 SD). Teriparatide treatment 20 μg/day was started plus vitamin D and calcium. The serum concentration of bone formation markers increased significantly after the first month of treatment. After 6 months, the patient reports only occasional back pain and has no limitation to standing. DXA assessment showed a remarkable 12.1 and 3.6% increase in lumbar spine and femoral neck BMD, respectively. No new VFs were apparent on LVA.Conclusion: Prolonged LMWH treatment during pregnancy could cause PAO. Teriparatide treatment is highly efficacious in ameliorating symptoms, increasing spine and femoral neck BMD, and consequently preventing new VFs.Abbreviations: BMD = bone mineral density; DXA = dual-energy X-ray absorptiometry; L = lumbar; LMWH = lowmolecular- weight heparin; PAO = pregnancy-associated osteoporosis; T = thoracic; VF = vertebral fracture

Background and Objectives : The goal of this study was to assess the impact of supplementation with a combination of nutrients on metabolic-dysfunction-associated steatotic liver disease (MASLD)-related liver parameters, and other parameters related to metabolic syndrome in adults with obesity. These measurements included anthropometric and lipid profiling, and FibroScan technology (controlled attenuation parameter (CAP) and transient elastography (TE) values). Materials and Methods: A double-blind, placebo-controlled pilot clinical trial was conducted over a three-month treatment period. Adults with metabolic syndrome and obesity were allocated to receive either a cocktail of nutrients with defined daily dosages (5-MTHF, betaine, alpha-linolenic acid, eicosapentaenoic acid, choline bitartrate, docosahexaenoic acid, and vitamin B12) or a placebo. The participants were evaluated at the start and the end of the three-month treatment period. Results : A total of 155 participants entered the study, comprising 84 in the treatment group and 71 in the placebo group. The administration of the nutritional supplement resulted in a notable reduction in both CAP and TE scores when compared to the placebo group. The treatment group exhibited a mean reduction in CAP of 4% ( p < 0.05) and a mean reduction in TE of 7.8% ( p < 0.05), indicative of a decline in liver fat content and fibrosis. Conclusions : The supplementation over a period of three months led to a significant amelioration of liver fibrosis and steatosis parameters in adults with metabolic syndrome and obesity. These findings suggest that this supplementation regimen could be a beneficial adjunct therapy for improving liver health in adults with obesity-induced MASLD.

This study aimed to evaluate the biostimulant effects of three protein-based gels, GHC 1-B (20% gelatin (GPU-B) obtained by thermal hydrolysis from residual untanned leather and 80% collagen hydrolysates (HCE-B) obtained by alkaline-enzymatic hydrolysis from residual bovine-tanned leather), GHC 2-B (40% keratin hydrolysate (HKU-B) obtained by alkaline-enzymatic hydrolysis from sheep wool + 40% HCE-B + 20% GPU-B), and GHC 3-B (20% GPU-B + 80% hydrolyzed collagen (HPU-B) obtained by thermal and enzymatic hydrolysis from residual untanned leather). A germination study was carried out on pepper and tomato seeds at concentrations of 1%, 3%, and 10%. As a result of the study, it was found that all three protein-based gels showed a stimulatory effect on the tomato seeds at a 1% concentration, where the Gi (germination index) was ˂100%. The GHC 2-B variant had the highest stimulatory effect (Gi-190.23%). Pepper seeds have proven to be more sensitive to the gel's composition. The concentration at which it proved to be non-inhibitory (Gi-88.29%) was 1% in the case of GHC 2-B. It was found that the presence of hydrolyzed keratin in the composition can be a plus compared to the other two protein gels tested due to its composition, which is richer in phytonutrient compounds (e.g., sulfur molecules).

The aim of this paper was to select keratin hydrolysate with bioactive properties by using the enzymatic hydrolysis of wool. Different proteolytic enzymes such as Protamex, Esperase, and Valkerase were used to break keratin molecules in light of bioactive additive preparation. The enzymatic keratin hydrolysates were assessed in terms of the physico-chemical characteristics related to the content of dry substance, total nitrogen, keratin, ash, cysteic sulphur, and cysteine. The influence of enzymatic hydrolysis on molecular weight and amino acid composition was determined by gel permeation chromatography (GPC) and gas chromatography-mass spectrometry (GC-MS) analyses. Antimicrobial activity of keratin hydrolysates was analysed against Fusarium spp., a pathogenic fungus that can decrease the quality of plants. The bioactivity of enzymatic hydrolysates was tested on maize plants and allowed us to select the keratin hydrolysates processed with the Esperase and Valkerase enzymes. The ratio of organised structures of hydrolysate peptides was analysed by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) deconvolution of the amide I band and may explain the difference in their bioactive behaviour. The most important modifications in the ATR spectra of maize leaves in correlation with the experimentally proven performance on maize development by plant length and chlorophyll index quantification were detailed. The potential of enzymatic hydrolysis to design additives with different bioactivity was shown in the case of plant growth stimulation.

Background: Nutritional knowledge assessment is an important component in nutrition research, and a prerequisite for the implementation of many policies and programs aimed at improving eating behavior. In order to generate objective results, validated tools for a given population must be employed. The aim of this study was to determine the validity and reliability of a nutrition knowledge questionnaire for Romanian adults., Methods: Kleimann's version of a General Nutrition Knowledge Questionnaire, was translated and adapted to Romanian language, culture, and cuisine. The final format was developed in several steps and used four components: internal and external reliability were assessed in a general population sample (n1 = 412), respectively in a subgroup (n2 = 46) from Component 1; Component 3 assessed construct validity (n3 = 96) using the "known-groups" method; Component 4 (convergent validity, n4 = 508) tested the association between socio-demographic characteristics and nutrition knowledge., Results: The overall internal reliability was 0.878 and the external reliability was >0.880 in all sections, and overall. Specialists had higher scores than nonspecialists, with a very large effect size. In the general population, females scored higher than males, and middle-aged and older adults scored higher than young adults. Higher scores were associated with higher levels of education. The characteristics of individuals prone to giving wrong answers were: males (beta = 0.170), high school or less (beta = 0.167), and no training in nutrition (beta = 0.154)., Conclusions: The Romanian version of the General Nutrition Knowledge Questionnaire is a reliable and valid tool for measuring nutrition knowledge in adults.

Polyunsaturated fatty acids (PUFAs) play important roles in health and disease. PUFA levels are influenced by nutrition and genetic factors. The relationship between PUFA composition in red blood cells (RBCs) and genetic variations involved in PUFA metabolism has not been investigated in children with obesity. This study evaluated the association between several genetic variations and PUFA levels in RBCs in children with obesity. One hundred ninety-six children with obesity (101 females, 95 males) were evaluated using anthropometric measurements, dietary intakes, plasma and RBC PUFA quantification, blood biochemistry, and 55 single nucleotide polymorphisms within 14 genes. phosphatidylethanolamine N -methyltransferase ( PEMT ) rs1109859 and methylenetetrahydrofolate reductase gene ( MTHFR ) rs4846052 genotypes were associated with PUFA levels in RBCs. PUFA intake did not influence the RBC eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) levels. Higher RBC DHA and EPA levels were observed for PEMT rs1109859 GG and GA genotypes versus the AA genotype. Higher levels of RBC DHA, EPA, arachidonic acid (ARA), and linoleic acid (LA) and were observed for MTHFR rs4846052 TT genotype versus TC and CC genotypes. Genetic variations in PEMT rs1109859 and MTHFR rs4846052 were associated with different PUFA levels in RBC membranes and are estimators for PUFA species in RBCs. Further research is needed to establish whether these genotype-specific alterations are specific to overweight children.

People with obesity in Romania are often under medical supervision, which is aimed to decrease body weight and treat accompanying metabolic disorders and cardiovascular implications. However, there is limited information regarding the implementation of dietary recommendations in adults with obesity. We aimed to evaluate the prevalence of reaching the recommended intakes of macro- and micro-nutrients in adults with obesity under medical supervision. Individuals with obesity, recruited in the context of a study with a larger scope (NutriGen ClinicalTrials.gov NCT02837367), who were under medical supervision underwent four 24 h recalls in order to assess daily food intakes. Macro- and micro-nutrient intakes were computed, and the prevalence of reaching recommended dietary allowances (RDAs) for each nutrient was calculated. The majority of subjects did not meet the recommended intakes for most nutrients. Energy from fat exceeded the threshold of 35% recommended intake, even in the lowest quartile of energy intake. The micronutrients with less than 5% of individuals reaching the RDAs were vitamin D, vitamin E, fluoride, and omega-3 fatty acids for both males and females, and choline, magnesium, and potassium in females. The burden of inadequate nutrition in individuals with obesity should be acknowledged and properly addressed within efforts to reduce obesity rates and associated disorders.

Few high-performance liquid chromatography⁻tandem mass spectrometry (LC-MS/MS) methods have been developed for the full quantitation of fatty acids from human plasma without derivatization. Therefore, we propose a method that requires fewer sample preparation steps, which can be used for the quantitation of several polyunsaturated fatty acids in human plasma. The method offers rapid, accurate, sensitive, and simultaneous quantification of omega 3 (α-linolenic, eicosapentaenoic, and docosahexaenoic acids) and omega 6 fatty acids (arachidonic and linoleic acids) using high-performance LC-MS/MS. The selected fatty acids were analysed in lipid extracts from both free and total forms. Chromatographic separation was achieved using a reversed phase C18 column with isocratic flow using ammonium acetate for improving negative electrospray ionization (ESI) response. Mass detection was performed in multiple reaction monitoring (MRM) mode, and deuterated internal standards were used for each target compound. The limits of quantification were situated in the low nanomolar range, excepting linoleic acid, for which the limit was in the high nanomolar range. The method was validated according to the U.S. Department of Health and Human Services guidelines, and offers a fast, sensitive, and reliable quantification of selected omega 3 and 6 fatty acids in human plasma.

Polyunsaturated fatty acid metabolism in toddlers is regulated by a complex network of interacting factors. The contribution of maternal genetic and epigenetic makeup to this milieu is not well understood. In a cohort of mothers and toddlers 16 months of age (n = 65 mother-child pairs), we investigated the association between maternal genetic and epigenetic fatty acid desaturase 2 (FADS2) profiles and toddlers' n-6 and n-3 fatty acid metabolism. FADS2 rs174575 variation and DNA methylation status were interrogated in mothers and toddlers, as well as food intake and plasma fatty acid concentrations in toddlers. A multivariate fit model indicated that maternal rs174575 genotype, combined with DNA methylation, can predict α-linolenic acid plasma concentration in all toddlers and arachidonic acid concentrations in boys. Arachidonic acid intake was predictive for its plasma concentration in girls, whereas intake of 3 major n-3 species (eicosapentaenoic, docosapentaenoic, and docosahexaenoic acids) were predictive for their plasma concentrations in boys. FADS2 genotype and DNA methylation in toddlers were not related to plasma concentrations or food intakes, except for CpG8 methylation. Maternal FADS2 methylation was a predictor for the boys' α-linolenic acid intakes. This exploratory study suggests that maternal FADS2 genetic and epigenetic status could be related to toddlers' polyunsaturated fatty acid metabolism., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Maternal transfer of fatty acids is important to fetal brain development. The prenatal environment may differentially affect the substrates supporting declarative memory abilities, as the level of fatty acids transferred across the placenta may be affected by the maternal fatty acid desaturase 2 (FADS2) rs174575 single nucleotide polymorphism. In this study, we hypothesized that toddler and maternal rs174575 genotype and FADS2 promoter methylation would be related to the toddlers' declarative memory performance. Seventy-one 16-month-old toddlers participated in an imitation paradigm designed to test immediate and long-term declarative memory abilities. FADS2 rs174575 genotype was determined and FADS2 promoter methylation was quantified from blood by bisulfite pyrosequencing for the toddlers and their natural mothers. Toddlers of GG mothers at the FADS2 rs174575 single nucleotide polymorphism did not perform as well on memory assessments as toddlers of CC or CG mothers when controlling for plasma α-linolenic acid and child genotype. Toddler methylation status was related to immediate memory performance, whereas maternal methylation status was related to delayed memory performance. Thus, prenatal experience and maternal FADS2 status have a pervasive, long-lasting influence on the brain development of the offspring, but as the postnatal environment becomes more primary, the offsprings' own biology begins to have an effect., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Many animal and human studies indicated that dietary ω-3 fatty acids could have beneficial roles on brain development, memory, and learning. However, the exact mechanisms involved are far from being clearly understood, especially for α-linolenic acid (ALA), which is the precursor for the ω-3 elongation and desaturation pathways. This study investigated the alterations induced by different intakes of flaxseed oil (containing 50% ALA), during gestation and lactation, upon the expression of genes involved in neurogenesis, memory-related molecular processes, and DNA methylation, in the brains of mouse offspring at the end of lactation (postnatal day 19, P19). In addition, DNA methylation status for the same genes was investigated. Maternal flaxseed oil supplementation during lactation increased the expression of Mecp2, Ppp1cc, and Reelin, while decreasing the expression of Ppp1cb and Dnmt3a. Dnmt1 expression was decreased by postnatal flaxseed oil supplementation but this effect was offset by ALA deficiency during gestation. Mecp2 DNA methylation was decreased by maternal ALA deficiency during gestation, with a more robust effect in the lactation-deficient group. In addition, linear regression analysis revealed positive correlations between Mecp2, Reelin, and Ppp1cc, between Gadd45b, Bdnf, and Creb1, and between Egr1 and Dnmt1, respectively. However, there were no correlations, in any gene, between DNA methylation and gene expression. In summary, the interplay between ALA availability during gestation and lactation differentially altered the expression of genes involved in neurogenesis and memory, in the whole brain of the offspring at the end of lactation. The Mecp2 epigenetic status was correlated with ALA availability during gestation. However, the epigenetic status of the genes investigated was not associated with transcript levels, suggesting that either the regulation of these genes is not necessarily under epigenetic control, or that the whole brain model is not adequate for the exploration of epigenetic regulation in the context of this study., (Copyright © 2014 ISDN. Published by Elsevier Ltd. All rights reserved.)

Effect alleles (alleles with a polymorphism that is associated with the effect being measured) in a small number of single-nucleotide polymorphisms (SNPs) are known to influence the dietary requirement for choline. In this study, we examined a much larger number of SNPs (n=200) in 10 genes related to choline metabolism for associations with development of organ dysfunction (liver or muscle) when 79 humans were fed a low-choline diet. We confirmed that effect alleles in SNPs such as the C allele of PEMT rs12325817 increase the risk of developing organ dysfunction in women when they consume a diet low in choline, and we identified novel effect alleles, such as the C allele of CHKA SNP rs7928739, that alter dietary choline requirements. When fed a low-choline diet, some people presented with muscle damage rather than liver damage; several effect alleles in SLC44A1 (rs7873937, G allele; rs2771040, G; rs6479313, G; rs16924529, A; and rs3199966, C) and one in CHKB (rs1557502, A) were more common in these individuals. This suggests that pathways related to choline metabolism are more important for normal muscle function than previously thought. In European, Mexican, and Asian Americans, and in individuals of African descent, we examined the prevalence of the effect alleles in SNPs that alter choline requirement and found that they are differentially distributed among people of different ethnic and racial backgrounds. Overall, our study has identified novel genetic variants that modulate choline requirements and suggests that the dietary requirement for choline may be different across racial and ethnic groups.-Da Costa, K.-A., Corbin, K. D., Niculescu, M. D., Galanko, J. A., Zeisel, S. H. Identification of new genetic polymorphisms that alter the dietary requirement for choline and vary in their distribution across ethnic and racial groups., (© FASEB.)

Background: Tumor necrosis factor-α (TNF-α) plays a central role in the molecular pathogenesis of periodontal disease. However, the epigenetic regulation attributable to microbial and inflammatory signals at the biofilm-gingival interface are poorly understood. In this study, the DNA methylation alteration within the TNFA promoter in human gingival biopsies from different stages of periodontal disease is investigated and the regulatory mechanism of TNFA transcription by DNA methylation is explored., Methods: Gingival biopsies were obtained from 17 patients with chronic periodontitis (CP) and 18 periodontally healthy individuals. Another 11 individuals participated in an experimentally induced gingivitis study, and gingival biopsies were collected at the baseline, induction, and resolution phase. To confirm that TNFA promoter methylation modulated TNFA transcription, THP.1 cells were treated with a DNA methyltransferase inhibitor, 5-Aza-2-deoxycytidine (5-Aza-2dC), and an RAW294.7 cell line transfected with a TNFA promoter-specific luciferase reporter system with or without methylation was used., Results: In gingival biopsies from individuals with severe CP, two individual cytosine-guanine dinucleotides (CpG sites) within the TNFA promoter (at -163 and -161 bp) displayed increased methylation in CP samples compared to those with gingival health (16.1% ± 5.1% versus 11.0% ± 4.6%, P = 0.02 and 19.8% ± 4.1% versus 15.4% ± 3.6%, P = 0.04, respectively). The methylation level at -163 bp was inversely associated with the transcription level of TNFA (P = 0.018). However, no significant difference in the TNFA promoter methylation pattern was observed in samples biopsied during the induction or resolution phase of experimentally induced gingivitis, which represented a reversible periodontal lesion. THP.1 cells treated with 5-Aza-2dC demonstrated a time-dependent increase in TNFA messenger level. It was also found that the luciferase activity decreased 2.6-fold in a construct containing an in vitro methylated TNFA promoter when compared to the unmethylated insert (P = 0.03)., Conclusion: Although the biopsy samples represented a mixed cell population, the change in promoter methylation status in chronic periodontal disease suggested that DNA methylation may be an important regulatory mechanism in controlling TNFA transcriptional expression in periodontal disease.

Social and behavioral research in public health is often intimately tied to profound, but frequently neglected, biological influences from underlying genetic, environmental, and epigenetic events. The dynamic interplay between the life, social, and behavioral sciences often remains underappreciated and underutilized in addressing complex diseases and disorders and in developing effective remediation strategies. Using a case-study format, we present examples as to how the inclusion of genetic, environmental, and epigenetic data can augment social and behavioral health research by expanding the parameters of such studies, adding specificity to phenotypic assessments, and providing additional internal control in comparative studies. We highlight the important roles of gene-environment interactions and epigenetics as sources of phenotypic change and as a bridge between the life and social and behavioral sciences in the development of robust interdisciplinary analyses.

Previous studies indicated that the intake of α-linolenic acid (ALA) can alter the concentration of both ω-6 and ω-3 fatty acids in both mother and offspring, with consequences on postnatal brain development. This study describes the association between maternal ALA availability during gestation and lactation, and alterations in the Fads2 DNA methylation in both maternal and offspring livers, at the end of lactation period. Both Fads2 promoter and intron 1 DNA methylation were increased in the groups receiving postnatal flaxseed oil containing 50% ALA (mothers or pups), while bivariate analysis indicated a significant association of the Fads2 epigenetic status in the liver between each mother and its offspring. In addition, Fads2 expression was negatively correlated with promoter methylation at the individual level in maternal livers (P<0.05). This study also indicated that the interplay between ALA availability during gestation and lactation can differentially alter the expression of desaturases and elongases involved in ω-6 and ω-3 metabolic pathways. In summary, when considering the perinatal dietary ALA requirements in mice, both gestation and lactation periods should be considered as having distinct roles in modulating the metabolism of ω-6 and ω-3 fatty acids in maternal mouse livers.

Multiple cues from the environment of our indirect and immediate ancestors, which often persist throughout the prenatal period and adulthood, are shaping our phenotypes through either direct, parent-to-child influences, or transgenerational inheritance. These effects are due to gene-environment interactions, which are intended to be a predictive tool and a mechanism of quick adaptation to the environment, as compared with genetic variations that are inherited over many generations. In certain circumstances the influences induced by the gene-environment interactions can have deleterious effects upon the health status, in the context of a radical change in the environment that does not fit with the predicted conditions, via epigenetic alterations. Conversely the best fit to the expected environment might have a delayed aging process and a longer life span. This review will touch upon the Developmental Origins of Health and Disease (DoHAD) concept, while discussing recent advances in the understanding of metabolic and cognitive disruptions, with a focus on epigenetic factors, their transgenerational effects, and the consequences they might have upon the onset of chronic disease and premature exitus.

Inorganic arsenic (iAs) is a complete transplacental carcinogen in mice. Previous studies have demonstrated that in utero exposure to iAs promotes cancer in adult mouse offspring, possibly acting through epigenetic mechanisms. Humans and rodents enzymatically convert iAs to its methylated metabolites. This reaction requires S-adenosylmethionine (SAM) as methyl group donor. SAM is also required for DNA methylation. Supplementation with folate, a major dietary source of methyl groups for SAM synthesis, has been shown to modify iAs metabolism and the adverse effects of iAs exposure. However, effects of gestational folate supplementation on iAs metabolism and fetal DNA methylation have never been thoroughly examined. In the present study, pregnant CD1 mice were fed control (i.e. normal folate, or 2.2 mg/kg) or high folate diet (11 mg/kg) from gestational day (GD) 5 to 18 and drank water with 0 or 85 ppm of As (as arsenite) from GD8 to 18. The exposure to iAs significantly decreased body weight of GD18 fetuses and increased both SAM and S-adenosylhomocysteine (SAH) concentrations in fetal livers. High folate intake lowered the burden of total arsenic in maternal livers but did not prevent the effects of iAs exposure on fetal weight or hepatic SAM and SAH concentrations. In fact, combined folate-iAs exposure caused further significant body weight reduction. Notably, iAs exposure alone had little effect on DNA methylation in fetal livers. In contrast, the combined folate-iAs exposure changed the CpG island methylation in 2,931 genes, including genes known to be imprinted. Most of these genes were associated with neurodevelopment, cancer, cell cycle, and signaling networks. The canonical Wnt-signaling pathway, which regulates fetal development, was among the most affected biological pathways. Taken together, our results suggest that a combined in utero exposure to iAs and a high folate intake may adversely influence DNA methylation profiles and weight of fetuses, compromising fetal development and possibly increasing the risk for early-onset of disease in offspring., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Abstract The rapid progress in nutritional epigenetics allowed for a much better understanding of the mechanisms involved in gene-nutrient interactions and the roles that nutrition has in transgenerational inheritance of acquired epigenetic traits. Studies indicated that a considerable number of nutrients or diet types are capable of inducing epimutations. In parallel, the rapid development of genome-wide DNA methylation detection methods allowed for a broader image on how nutrition impacts the epigenetic status in human and animal models. But this increased complexity in the epigenetic field and also brought important challenges that need resolution, or it suggests that some of the initial epigenetic paradigms have to be revisited or reconsidered. The aim of this review is to discuss the inherent challenges that need to be resolved, from both practical and theoretical aspects, stemming from the rapid progress in the field of nutritional epigenetics, with a focus on DNA methylation. Because such challenges are present at every stage of study development, the review systematically discusses the most common issues relevant to DNA methylation in a nutritional context. Various types of challenges and potential bias generators are discussed within study design, sample quality, detection methods, data processing, and statistical and bioinformatic analysis. Additional aspects to be considered include epigenetic heterogeneity of treatment groups, the role of genomic variability in introducing measurement bias and errors in interpretation of changes, and issues related to the final interpretation of results and in assigning functional significance. It is also posited that all these issues will be largely resolved within the next decade.

Within the last two decades, significant progress has been made in understanding the importance of epigenetic mechanisms in the regulation of gene expression as a consequence of gene-environment interactions. Nutrition, among many other environmental factors, is a key player that can induce epigenetic changes not only in the directly exposed organisms but also in subsequent generations through the transgenerational inheritance of epigenetic traits. This article aims to provide insights into the usefulness of the mouse model for epigenetic studies involving nutrition as well as the inherent limitations when compared with epigenetic phenomena in humans. Mice are one of the most versatile models for nutrition and epigenetic studies because of several features, such as short life-span, relative low cost for generating samples, the existence of well-characterized genetically engineered lines, the detailed sequencing of genomes, and the relative similarity of their metabolic processes to human metabolism. However, several limitations have to be acknowledged, such as the different location of genes on the chromosomes (and hence possibly different consequences of some epigenetic alterations), differences in the epigenetic patterns established during late embryogenesis, and possible epigenetic differences associated with cellular senescence caused by the different structure of telomeres when compared with humans. All these aspects have to be carefully analyzed when deciding whether a mouse model should be considered for a study in nutrition and epigenetics. Consequently, the results obtained from mouse studies should be carefully interpreted regarding their relevance to humans.

The availability of ω-3 polyunsaturated fatty acids is essential for perinatal brain development. While the roles of docosahexaenoic acid (the most abundant ω-3 species) were extensively described, less is known about the role of α-linolenic acid (ALA), which is the initial molecular species undergoing elongation and desaturation within the ω-3 pathways. This study describes the association between maternal ALA availability during gestation and lactation, and alterations in hippocampal development (dentate gyrus) in the mouse male offspring, at the end of lactation (postnatal day 19, P19). Postnatal ALA supplementation increased cell proliferation (36% more proliferating cells compared to a control group) and early neuronal differentiation, while postnatal ALA deficiency increased cellular apoptosis within the dentate gyrus of suckling pups (61% more apoptotic cells compared to a control group). However, maternal ALA deficiency during gestation prevented the increased neurogenesis induced by postnatal supplementation. Fatty acid analysis revealed that ALA supplementation increased the concentration of the ω-3 species in the maternal liver and serum, but not in the brain of the offspring, excepting for ALA itself. Interestingly, ALA supplementation also increased the concentration of dihomo γ-linolenic acid (a ω-6 species) in the P19 brains, but not in maternal livers or serum. In conclusion, postnatal ALA supplementation enhances neurogenesis in the dentate gyrus of the offspring at postnatal day 19, but its beneficial effects are offset by maternal ALA deficiency during gestation. These results suggest that ALA is required in both fetal and postnatal stages of brain development., (Copyright © 2011 ISDN. Published by Elsevier Ltd. All rights reserved.)

Levels of prostaglandin E(2) and the prostaglandin-endoperoxide synthase-2 (PTGS2, or COX-2) increase in actively progressing periodontal lesions, but decrease in chronic disease. We hypothesized that chronic inflammation is associated with altered DNA methylation levels within the PTGS2 promoter, with effects on COX-2 mRNA expression. PTGS2 promoter methylation levels from periodontally inflamed gingival biopsies showed a 5.06-fold increase as compared with non-inflamed samples (p = 0.03), and the odds of methylation in a CpG site in the inflamed gingival group is 4.46 times higher than in the same site in the non-inflamed group (p = 0.016). The level of methylation at -458 bp was inversely associated with transcriptional levels of PTGS2 (RT-PCR) (p = 0.01). Analysis of the data suggests that, in chronically inflamed tissues, there is a hypermethylation pattern of the PTGS2 promoter in association with a lower level of PTGS2 transcription, consistent with a dampening of COX-2 expression in chronic periodontitis. These findings suggest that the chronic persistence of the biofilm and inflammation may be associated with epigenetic changes in local tissues at the biofilm-gingival interface.

Maternal choline availability is essential for fetal neurogenesis. Choline deprivation (CD) causes hypomethylation of specific CpG islands in genes controlling cell cycling in fetal hippocampus. We now report that, in C57BL/6 mice, CD during gestational days 12-17 also altered methylation of the histone H3 in E17 fetal hippocampi. In the ventricular and subventricular zones, monomethyl-lysine 9 of H3 (H3K9me1) was decreased by 25% (P<0.01), and in the pyramidal layer, dimethyl-lysine 9 of H3 (H3K9me2) was decreased by 37% (P<0.05). These changes were region specific and were not observed in whole-brain preparations. Also, the same effects of CD on H3 methylation were observed in E14 neural progenitor cells (NPCs) in culture. Changes in G9a histone methyltransferase might mediate altered H3K9me2,1. Gene expression of G9a was decreased by 80% in CD NPCs (P<0.001). In CD, H3 was hypomethylated upstream of the RE1 binding site in the calbindin 1 promoter, and 1 CpG site within the calbindin1 promoter was hypermethylated. REST binding to RE1 (recruits G9a) was decreased by 45% (P<0.01) in CD. These changes resulted in increased expression of calbindin 1 in CD (260%; P<0.05). Thus, CD modulates histone methylation in NPCs, and this could underlie the observed changes in neurogenesis.

The importance of maternal nutrition for fetal brain development is increasingly recognized. Previous studies have suggested that maternal obesity or maternal exposure to obesogenic diets may permanently alter brain structure and function in the offspring. To test whether maternal exposure to a high-fat diet, prior and during gestation, alters fetal hippocampal development, we fed 8-week old C57BL/6 females with a high-fat diet (60% calories from fat) for 10 weeks prior to matting and 17 days after. Fetal brains at embryonic day E17 were used to determine developmental changes in the hippocampus. We report that maternal exposure to the high-fat diet induced small for gestational age (SGA) status and fetal resorption. The proliferation of neural progenitors was increased in the neuroepithelium from hippocampus and cortex in fetuses from mothers fed the high-fat diet when compared to controls, but decreased within the dentate gyrus (DG). Apoptosis in the hippocampus was decreased (Ammon's Horn and fimbria). The differentiation of calretinin-positive neurons within the DG was also decreased. These data indicate that, under the influence of a maternal high-fat diet administered prior and during gestation, fetal hippocampal development is altered at embryonic day 17, as indicated by region-specific changes in proliferation of neural precursors, decreased apoptosis, and by decreased neuronal differentiation within the dentate gyrus.

We report the case of a 42-year-old woman who was admitted to the hospital for fever, chills, nausea, vomiting, fatigue, myalgia, and general muscle weakness. All these symptoms had occurred 3 weeks after the ingestion of inadequately cooked pork meat, subsequently confirmed to be infested with Trichinella spiralis. Laboratory results showed mild leukocytosis, inflammation, and mild liver and muscle cytolytic syndrome, all suggestive of trichinellosis. Echocardiography showed apical hypokinesis and an apical mass (likely a thrombus). The immunologic assessment for the presence of Trichinella antigens was positive. The outcome was favorable after treatment with an anticoagulant, an antiaggregant, prednisone, and mebendazole. Follow-up controls showed the absence of any symptoms and thrombus, with only mild electrocardiogram modifications still present.

Diethanolamine (DEA) is a common ingredient of personal care products. Dermal administration of DEA diminishes hepatic stores of the essential nutrient choline and alters brain development. We previously reported that 80 mg/kg/day of DEA during pregnancy in mice reduced neurogenesis and increased apoptosis in the fetal hippocampus. This study was designed to establish the dose-response relationships for this effect of DEA. Timed-pregnant C57BL/6 mouse dams were dosed dermally from gestation day 7-17 with DEA at 0 (controls), 5, 40, 60, and 80 mg/kg body/day. Fetuses (embryonic day 17 [E17]) from dams treated dermally with 80 mg/kg body/day DEA had decreased neural progenitor cell mitosis at the ventricular surface of the ventricular zone (hippocampus, 54.1 +/- 5.5%; cortex, 58.9 +/- 6.8%; compared to controls; p < 0.01). Also, this dose of DEA to dams increased rates of apoptosis in E17 fetal hippocampus (to 177.2 +/- 21.5% of control; measured using activated caspase-3; p < 0.01). This dose of DEA resulted in accumulation of DEA and its metabolites in liver and in plasma. At doses of DEA less than 80 mg/kg body/day to dams, there were no differences between treated and control groups. In a small group of human subjects, dermal treatment for 1 month with a commercially available skin lotion containing 1.8 mg DEA per gram resulted in detectable plasma concentrations of DEA and dimethyldiethanolamine, but these were far below those concentrations associated with perturbed brain development in the mouse.

Choline is an essential nutrient for humans, though some of the requirement can be met by endogenous synthesis catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). Premenopausal women are relatively resistant to choline deficiency compared with postmenopausal women and men. Studies in animals suggest that estrogen treatment can increase PEMT activity. In this study we investigated whether the PEMT gene is regulated by estrogen. PEMT transcription was increased in a dose-dependent manner when primary mouse and human hepatocytes were treated with 17-beta-estradiol for 24 h. This increased message was associated with an increase in protein expression and enzyme activity. In addition, we report a region that contains a perfect estrogen response element (ERE) approximately 7.5 kb from the transcription start site corresponding to transcript variants NM&#95;007169 and NM-008819 of the human and murine PEMT genes, respectively, three imperfect EREs in evolutionarily conserved regions and multiple imperfect EREs in nonconserved regions in the putative promoter regions. We predict that both the mouse and human PEMT genes have three unique transcription start sites, which are indicative of either multiple promoters and/or alternative splicing. This study is the first to explore the underlying mechanism of why dietary requirements for choline vary with estrogen status in humans.

Background: Some humans fed a low-choline diet develop hepatosteatosis, liver and muscle damage, and lymphocyte apoptosis. The risk of developing such organ dysfunction is increased by the presence of single-nucleotide polymorphisms (SNPs) in genes involved in folate and choline metabolism., Objective: We investigated whether these changes that occur in the expression of many genes when humans are fed a low-choline diet differ between subjects who develop organ dysfunction and those who do not. We also investigated whether expression changes were dependent on the presence of the SNPs of interest., Design: Thirty-three subjects aged 20-67 y were fed for 10 d a baseline diet containing the recommended adequate intake of choline. They then were fed a low-choline diet for up to 42 d or until they developed organ dysfunction. Blood was collected at the end of each phase, and peripheral lymphocytes were isolated and used for genotyping and for gene expression profiling with the use of microarray hybridization., Results: Feeding a low-choline diet changed the expression of 259 genes, and the profiles of subjects who developed and those who did not develop signs of organ dysfunction differed. Group clustering and gene ontology analyses found that the diet-induced changes in gene expression profiles were significantly influenced by the SNPs of interest and that the gene expression phenotype of the variant gene carriers differed significantly even with the baseline diet., Conclusion: These findings support our hypothesis that a person's susceptibility to organ dysfunction when fed a low-choline diet is modulated by specific SNPs in genes involved in folate and choline metabolism.

Human and animal studies suggest that dietary soy isoflavones reduce cancer risk, ameliorate postmenopausal syndrome and decrease bone resorption in postmenopausal women. The capacity to form the metabolite equol from daidzein is suggested as an important modulator of response to isoflavones; this capacity depends on gut colonization with appropriate bacteria. We administered a dietary supplement containing high-dose purified soy isoflavones (genistein, 558 mg/day; daidzein, 296 mg/day; and glycitein, 44 mg/day) to 30 postmenopausal women for 84 days and collected peripheral lymphocytes at timed intervals. Using microarray analysis, we determined whether changes in gene expression associated with this treatment support existing hypotheses as to isoflavones' mechanisms of action. Expression of a large number of genes was altered by isoflavone treatment, including induction of genes associated with cyclic adenosine 3',5'-monophosphate (cAMP) signaling and cell differentiation and decreased expression of genes associated with cyclin-dependent kinase activity and cell division. We report that isoflavone treatment in subjects who have the capacity to produce equol differentially affects gene expression as compared with nonproducers, supporting the plausibility of the importance of equol production. In general, isoflavones had a stronger effect on some putative estrogen-responsive genes in equol producers than in nonproducers. Our study suggests that, in humans, isoflavone changes are related to increased cell differentiation, increased cAMP signaling and G-protein-coupled protein metabolism and increased steroid hormone receptor activity and have some estrogen agonist effects; equol-production status is likely to be an important modulator of responses to isoflavones.

Over a four-year period, we collected clinical and biochemical data from five Amish children who were homozygous for missense mutations in 5,10-methylenetetrahydrofolate reductase (MTHFR c.1129C>T). The four oldest patients had irreversible brain damage prior to diagnosis. The youngest child, diagnosed and started on betaine therapy as a newborn, is healthy at her present age of three years. We compared biochemical data among four groups: 16 control subjects, eight heterozygous parents, and five affected children (for the latter group, both before and during treatment with betaine anhydrous). Plasma amino acid concentrations were used to estimate changes in cerebral methionine uptake resulting from betaine therapy. In all affected children, treatment with betaine (534+/-222 mg/kg/day) increased plasma S-adenosylmethionine, improved markers of tissue methyltransferase activity, and resulted in a threefold increase of calculated brain methionine uptake. Betaine therapy did not normalize plasma total homocysteine, nor did it correct cerebral 5-methyltetrahydrofolate deficiency. We conclude that when the 5-methyltetrahydrofolate content of brain tissue is low, dietary betaine sufficient to increase brain methionine uptake may compensate for impaired cerebral methionine recycling. To effectively support the metabolic requirements of rapid brain growth, a large dose of betaine should be started early in life.

Diethanolamine (DEA) is a widely used ingredient in many consumer products and in a number of industrial applications. It has been previously reported that dermal administration of DEA to mice diminished hepatic stores of choline and altered brain development in the fetus. The aim of this study was to use mouse neural precursor cells in vitro to assess the mechanism underlying the effects of DEA. Cells exposed to DEA treatment (3mM) proliferated less (by 5-bromo-2-deoxyuridine incorporation) at 48 h (24% of control [CT]), and had increased apoptosis at 72 h (308% of CT). Uptake of choline into cells was reduced by DEA treatment (to 52% of CT), resulting in diminished intracellular concentrations of choline and phosphocholine (55 and 12% of CT, respectively). When choline concentration in the growth medium was increased threefold (to 210 microM), the effects of DEA exposure on cell proliferation and apoptosis were prevented, however, intracellular phosphocholine concentrations remained low. In choline kinase assays, we observed that DEA can be phosphorylated to phospho-DEA at the expense of choline. Thus, the effects of DEA are likely mediated by inhibition of choline transport into neural precursor cells and by altered metabolism of choline. Our study suggests that prenatal exposure to DEA may have a detrimental effect on brain development.

Background: Whereas deficiency of the essential nutrient choline is associated with DNA damage and apoptosis in cell and rodent models, it has not been shown in humans., Objective: The objective was to ascertain whether lymphocytes from choline-deficient humans had greater DNA damage and apoptosis than did those from choline-sufficient humans., Design: Fifty-one men and women aged 18-70 y were fed a diet containing the recommended adequate intake of choline (control) for 10 d. They then were fed a choline-deficient diet for up to 42 d before repletion with 138-550 mg choline/d. Blood was collected at the end of each phase, and peripheral lymphocytes were isolated. DNA damage and apoptosis were then assessed by activation of caspase-3, terminal deoxynucleotide transferase-mediated dUTP nick end-labeling, and single-cell gel electrophoresis (COMET) assays., Results: All subjects fed the choline-deficient diet had lymphocyte DNA damage, as assessed by COMET assay, twice that found when they were fed the control diet. The subjects who developed organ dysfunction (liver or muscle) when fed the choline-deficient diet had significantly more apoptotic lymphocytes, as assessed by the activated caspase-3 assay, than when fed the control diet., Conclusions: A choline-deficient diet increased DNA damage in humans. Subjects in whom these diets induced liver or muscle dysfunction also had higher rates of apoptosis in their peripheral lymphocytes than did subjects who did not develop organ dysfunction. Assessment of DNA damage and apoptosis in lymphocytes appears to be a clinically useful measure in humans (such as those receiving parenteral nutrition) in whom choline deficiency is suspected.

Choline is derived not only from the diet, but also from de novo synthesis. It is important for methyl-group metabolism, the formation of membranes, kidney function, and neurotransmission. When deprived of dietary choline, most adult men and postmenopausal women develop signs of organ dysfunction (fatty liver or muscle damage) and have a decreased capacity to convert homocysteine to methionine. Choline is critical during fetal development, when it influences stem cell proliferation and apoptosis, thereby altering brain structure and function (memory is permanently enhanced in rodents exposed to choline during the latter part of gestation).

The availability of choline during critical periods of fetal development alters hippocampal development and affects memory function throughout life. Choline deficiency during fetal development reduces proliferation and migration of neuronal precursor cells in the mouse fetal hippocampus and these changes are associated with modifications in the protein levels of some cell cycle regulators and early differentiation markers. We fed C57 BL/6 mouse dams diets deficient or normal in choline content from days 12 to 17 of pregnancy, and then collected fetal brains on embryonic day 17. Using laser-capture micro-dissection we harvested cells from the ventricular and subventricular zones of Ammon's horn and from the prime germinal zone of the dentate gyrus (hippocampus). In the ventricular and subventricular zones from the choline-deficient group, we observed increased protein levels for kinase-associated phosphatase (Kap) and for p15(INK4b) (two cell cycle inhibitors). In the dentate gyrus, we observed increased levels of calretinin (an early marker of neuronal differentiation). In fetal brain from mothers fed a choline-deficient diet, DNA global methylation was decreased in the ventricular and subventricular zones of Ammon's horn. We also observed decreased gene-specific DNA methylation of the gene (Cdkn3) that encodes for Kap, correlating with increased expression of this protein. This was not the case for p15(INK4b) or calretinin (Cdkn2b and Calb2, respectively). These data suggest that choline deficiency-induced changes in gene methylation could mediate the expression of a cell cycle regulator and thereby alter brain development.

Choline is an essential nutrient and an important methyl donor. Choline deficiency alters fetal development of the hippocampus in rodents and these changes are associated with decreased memory function lasting throughout life. Also, choline deficiency alters global and gene-specific DNA methylation in several models. This gene expression profiling study describes changes in cortical neural precursor cells from embryonic day 14 mice, after 48 h of exposure to a choline-deficient medium. Using Significance Analysis of Microarrays, we found the expression of 1003 genes to be significantly changed (from a total of 16,000 total genes spotted on the array), with a false discovery rate below 5%. A total of 846 genes were overexpressed while 157 were underexpressed. Classification by gene ontology revealed that 331 of these genes modulate cell proliferation, apoptosis, neuronal and glial differentiation, methyl metabolism, and calcium-binding protein classes. Twenty-seven genes that had changed expression have previously been reported to be regulated by promoter or intron methylation. These findings support our previous work suggesting that choline deficiency decreases the proliferation of neural precursors and possibly increases premature neuronal differentiation and apoptosis.

Choline is an important methyl donor and a component of membrane phospholipids. In this study, we tested the hypothesis that choline availability can modulate cell proliferation and the methylation of genes that regulate cell cycling. In several other model systems, hypomethylation of cytosine bases that are followed by a guanosine (CpG) sites in the promoter region of a gene is associated with increased gene expression. We found that in choline-deficient IMR-32 neuroblastoma cells, the promoter of the cyclin-dependent kinase inhibitor 3 gene (CDKN3) was hypomethylated. This change was associated with increased expression of CDKN3 and increased levels of its gene product, kinase-associated phosphatase (KAP), which inhibits the G(1)/S transition of the cell cycle by dephosphorylating cyclin-dependent kinases. Choline deficiency also reduced global DNA methylation. The percentage of cells that accumulated bromodeoxyuridine (proportional to cell proliferation) was 1.8 times lower in the choline-deficient cells than in the control cells. Phosphorylated retinoblastoma (p110) levels were 3 times lower in the choline-deficient cells than in control cells. These findings suggest that the mechanism whereby choline deficiency inhibits cell proliferation involves hypomethylation of key genes regulating cell cycling. This may be a mechanism for our previously reported observation that stem cell proliferation in hippocampus neuroepithelium is decreased in choline-deficient rat and mouse fetuses.

DNA methylation influences the expression of some genes and depends upon the availability of methyl groups from S-adenosylmethionine (SAM). Dietary methyl groups derive from foods that contain methionine, one-carbon units and choline (or the choline metabolite betaine). Humans ingest approximately 50 mmol of methyl groups per day; 60% of them are derived from choline. Transmethylation metabolic pathways closely interconnect choline, methionine, methyltetrahydrofolate (methyl-THF) and vitamins B-6 and B-12. The pathways intersect at the formation of methionine from homocysteine. Perturbing the metabolism of one of these pathways results in compensatory changes in the others. For example, methionine can be formed from homocysteine using methyl groups from methyl-THF, or using methyl groups from betaine that are derived from choline. Similarly, methyl-THF can be formed from one-carbon units derived from serine or from the methyl groups of choline via dimethylglycine, and choline can be synthesized de novo using methyl groups derived from methionine (via SAM). When animals and humans are deprived of choline, they use more methyl-THF to remethylate homocysteine in the liver and increase dietary folate requirements. Conversely, when they are deprived of folate, they use more methyl groups from choline, increasing the dietary requirement for choline. The availability of transgenic and knockout mice has made possible additional studies that demonstrate the interrelationship of these methyl sources. In summary, as we consider dietary requirements and possible effects on DNA methylation, it is important to realize that methionine, methyl-THF and choline can be fungible sources of methyl groups, and the design of our studies should reflect this.