Increasing Maternal or Post-Weaning Folic Acid Alters Gene Expression and Moderately Changes Behavior in the Offspring

et al. (2014) Increasing Maternal or Post-Weaning Folic Acid Alters Gene Expression and Moderately Changes Behavior in the Offspring. PLoS ONE 9(7): e101674. doi:10.1371/journal.pone.0101674 Increasing Maternal or Post-Weaning Folic Acid Alters Gene Expression and Moderately Changes Behavior in the Offspring Subit Barua 0 Kathryn K. Chadman 0 Salomon Kuizon 0 Diego Buenaventura 0 Nathan W. Stapley 0 Felicia Ruocco 0 Umme Begum 0 Sara R. Guariglia 0 W. Ted Brown 0 Mohammed A. Junaid 0 Cheryl S. Rosenfeld, University of Missouri, United States of America 0 1 Department of Developmental Biochemistry, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York, United States of America, 2 Department of Developmental Neurobiology, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York, United States of America, 3 Department of Environmental Health Sciences, Columbia University, New York, United States of America, 4 Department of Human Genetics, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York, United States of America, 5 Graduate Center and College of Staten Island, City University of New York , Staten Island, New York , United States of America Background: Studies have indicated that altered maternal micronutrients and vitamins influence the development of newborns and altered nutrient exposure throughout the lifetime may have potential health effects and increased susceptibility to chronic diseases. In recent years, folic acid (FA) exposure has significantly increased as a result of mandatory FA fortification and supplementation during pregnancy. Since FA modulates DNA methylation and affects gene expression, we investigated whether the amount of FA ingested during gestation alters gene expression in the newborn cerebral hemisphere, and if the increased exposure to FA during gestation and throughout the lifetime alters behavior in C57BL/6J mice. Methods: Dams were fed FA either at 0.4 mg or 4 mg/kg diet throughout the pregnancy and the resulting pups were maintained on the diet throughout experimentation. Newborn pups brain cerebral hemispheres were used for microarray analysis. To confirm alteration of several genes, quantitative RT-PCR (qRT-PCR) and Western blot analyses were performed. In addition, various behavior assessments were conducted on neonatal and adult offspring. Results: Results from microarray analysis suggest that the higher dose of FA supplementation during gestation alters the expression of a number of genes in the newborns' cerebral hemispheres, including many involved in development. QRTPCR confirmed alterations of nine genes including down-regulation of Cpn2, Htr4, Zfp353, Vgll2 and up-regulation of Xist, Nkx6-3, Leprel1, Nfix, Slc17a7. The alterations in the expression of Slc17a7 and Vgll2 were confirmed at the protein level. Pups exposed to the higher dose of FA exhibited increased ultrasonic vocalizations, greater anxiety-like behavior and hyperactivity. These findings suggest that although FA plays a significant role in mammalian cellular machinery, there may be a loss of benefit from higher amounts of FA. Unregulated high FA supplementation during pregnancy and throughout the life course may have lasting effects, with alterations in brain development resulting in changes in behavior. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. Gene expression data files are available from NCBI GEO database with accession number GSE45607. Funding: This study was supported by grant 12-FY12-170 from March of Dimes Research Foundation to KKC WTB MAJ and Institutional Funds from the New York State Office for People with Developmental Disabilities to KKC and MAJ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Maternal nutrition during the peri- or post-conception period is strongly related to fetal development and the risk of noncommunicable diseases. Micronutrients including vitamins and minerals are major intrauterine environmental factors that regulate the fetal genome machinery and human reproductive health [1,2]. Since the early 1970s, the vitamin folic acid (FA) has received considerable attention because of its important role in alleviating neural tube defects (NTDs) [3]. In 1998, mandatory FA fortification of breakfast cereals and grains was introduced by several countries [4]. These guidelines are credited with a significant reduction in NTDs in infants [5]. Intrauterine and early life exposure to FA has increased well beyond the minimum necessary requirements as a result of the additive levels from fortified foodstuffs, prescriptions by physicians and over-thecounter prenatal vitamins, as well as energy drinks [6]. Studies suggest that folate concentrations in the serum (57%) and red blood cells (136%) of pregnant women were much higher than the reported dietary folate intake (28%) [7], and concerns have been raised about the interference of excess unmetabolized FA [8,9]. Several epidemiological reports have suggested that excess FA increases the risk of asthma and type 2 diabetes in newborns [10 12], and evidence of unmetabolized FA has been found in fetal cord blood [13]. While studies have found an important role of maternal FA in the proper closure of the neural tube, there have been no published reports yet on whether higher FA supplementation during pregnancy and throughout life may impact brain development. A previous studies from our laboratory has showed that FA supplementation significantly dysregulates gene expression in human lymphoblastoid cell lines [14], and our recent study in a mouse model has shown that gestational FA induces substantial alterations in methylation patterns of several genes in the cerebral hemispheres of the offspring [15]. Studies in laboratory animals have shown adverse effects including tumors and birth defects in offspring exposed to higher FA concentrations, and suppression of thyroid function with motivational and spatial memory deficits in adolescence [6,1622]. The current study had two objectives: first, to determine if the amount of maternal FA supplementation during gestation modulates gene expression in the developing cerebral hemispheres and consequently affects neonatal behavior, and second, to explore if gestational and lifelong exposure to excess FA alters the behavior of the adult. We found that higher maternal FA supplementation caused large-scale alterations in gene expression in the newborns cerebral hemispheres. Additionally we found moderate alterations in behavior in both the newborn and the adult as a result of higher FA exposure. To our knowledge, this is the first study of its (...truncated)