Objective To investigate the effects of folate deficiency on changes in histone H3 lysine 4 (H3K4) mono-methylation (me1)-marked enhancers and the molecular mechanism underpinning the folate deficiency-induced neural tube defects (NTD). Methods Mouse embryonic stem cells (mESCs) were cultured in the folate-free DMEM medium (folate-deficient group) and the DMEM medium containing 4 mg/L folate (normal control group),respectively.Chromatin immunoprecipitation sequencing (ChIP-seq) was performed for H3K4me1. The mouse model of folate-induced NTD was established,and transcriptome sequencing (RNA-seq) was performed for the brain tissue of fetal mice to reveal the differential expression profiles.The results were validated through real-time quantitative polymerase chain reaction (RT-qPCR).The activity of the differential peak regions of H3K4me1 was verified through the luciferase reporter assay. Results The folate content in the mESCs cultured in the folate-free medium reduced compared with that in the normal control group (P=0.008).The H3K4me1-maked enhancers in the mESCs cultured in the folate-free medium induced significant changes in intronic regions,and these changes were concentrated in metabolic and energy metabolism processes (q=9.56×10^-48,P=1.28×10^-47).The differentially expressed genes harboring H3K4me1-marked enhancers in mESCs were mainly enriched in the Wnt signaling pathway (q=0.004,P=0.004 7).ChIP-qPCR results confirmed that H3K4me1 binding decreased in the differential peak regions of the Ldlrap1 gene (P=0.008),Camta1 gene (P=0.002),and Apc2 gene (P=0.012).The H3K4 demethylase inhibitor T-448 effectively reversed the H3K4me1 binding in the differential peak regions of the aforementioned genes (P=0.01).The results of RNA-seq for the brain tissue of NTD fetal mice showed significant enrichment of the differentially expressed genes in the Wnt signaling pathway (P=1.52×10^-5).The enrichment of differential peak regions of H3K4me1-marked enhancers in Apc2,Ldlrap1,and Camta1 genes in the brain tissue also showed significant changes.The differential peak region in Apc2 exhibited transcription factor activity (P=0.020). Conclusion Folate deficiency may affect changes in H3K4me1-marked enhancers to participate in the regulation of neural tube closure genes,thereby inducing the occurrence of NTD.
Neural Tube Defects/genetics* ; Animals ; Mice ; Folic Acid Deficiency/complications* ; Histones/metabolism* ; Folic Acid/metabolism* ; Methylation ; Mouse Embryonic Stem Cells/metabolism* ; Wnt Signaling Pathway ; Lysine/metabolism* ; Chromatin Immunoprecipitation Sequencing

Folic acid belongs to the group of water-soluble B vitamins and naturally exists in multiple forms in a wide variety of foods such as legumes, vegetables, liver, and milk (Iyer and Tomar, 2009; Lyon et al., 2020). It is involved in many biochemical reactions critical for cell division, such as purine and pyrimidine biosynthesis, DNA/RNA biosynthesis, and amino acid metabolism (Iyer and Tomar, 2009). Mammals cannot synthesize folic acid and thus they must acquire it from food. Although folic acid is ubiquitous in foods, folic acid deficiency still often occurs due to various causes such as unhealthy diet (Hildebrand et al., 2021; Iimura et al., 2022), disease-related malabsorption (Arcot and Shrestha, 2005), medication-related depletion (Arcot and Shrestha, 2005), or vitamin B12 deficiency (Fishman et al., 2000). Folic acid deficiency has been associated with several health problems, such as anemia (Carmel, 2005; Bailey and Caudill, 2012), cancer (Duthie, 1999), cardiovascular diseases (Wald et al., 2002), neural tube defects in newborns (van der Put et al., 2001), neuropsychiatric dysfunction (Shea et al., 2002), depression (Falade et al., 2021), inflammatory diseases (Suzuki and Kunisawa, 2015; Jones et al., 2019), and eye diseases (Sijilmassi, 2019). To prevent folic acid deficiency, its daily intake (400 μg/d) has been recommended for adults in the European Union, and its increased intake (600 μg/d) is advised for women before and during pregnancy (FAO/WHO, 2002; IOM, 2004). The New Zealand government mandated the fortification of non-organic wheat flour with folic acid in July 2021, and the UK government mandated the fortification of non-wholemeal wheat flour with folic acid in September 2021 (Haggarty, 2021).
Adult ; Animals ; Female ; Flour ; Folic Acid/metabolism* ; Folic Acid Deficiency/prevention & control* ; Food, Fortified ; Humans ; Infant, Newborn ; Mammals/metabolism* ; Pregnancy ; Triticum/metabolism*

Objective: This study aimed to investigate the effects of Methods: In this study, 0.1% DMG was supplemented in 20% casein diets that were either folate-sufficient (20C) or folate-deficient (20CFD). Blood and liver of rats were subjected to assays of Hcy and its metabolites. Hcy and its related metabolite concentrations were determined using a liquid chromatographic system. Results: Folate deprivation significantly increased pHcy concentration in rats fed 20C diet (from 14.19 ± 0.39 μmol/L to 28.49 ± 0.50 μmol/L; Conclusion DMG supplementation exhibited hypohomocysteinemic effects under folate-sufficient conditions. By contrast, the combination of folate deficiency and DMG supplementation has deleterious effect on pHcy concentration.
Animals ; Biomarkers/metabolism* ; Chromatography, Liquid ; Diet ; Dietary Supplements ; Folic Acid Deficiency/metabolism* ; Homocysteine/metabolism* ; Liver/metabolism* ; Male ; Random Allocation ; Rats ; Rats, Wistar ; Sarcosine/metabolism*

OBJECTIVETo study the effects of maternal folate deficiency on fetal growth and development and the methylation profiles of insulin-like growth factor system in the offspring rats.

METHODSTwenty-two Sprague-Dawley female rats were randomly assigned to two groups: a folate deficient group (n=12) and a control group (n=10). They were fed with folate deficient and normal diet respectively. Dams were mated after 2 weeks of feeding. Eight female rats from each group were pregnant. On the 20th day of gestation, the fetuses were delivered by caesarean section. Thirty-two fetal rats from each group were randomly selected and the body length and weight were measured. Eight fetal rats from each group were randomly selected and ELISA was used to measure the level of folate content, IGF-1 and IGFBP-3 in the fetal brain and liver. Three fetal rats from each group were randomly selected and methylated DNA immunoprecipitation sequencing (MeDIP-Seq) was used to detect the methylation level of insulin-like growth factor system in the fetal brain and liver. ELISA was used to measure the level of IGF-1 and IGFBP-3 in the maternal serum from both groups.

RESULTSThe mean fetal length and weight were lower in the folate deficient group than in the control group (P<0.05). The levels of IGF-1 and IGFBP-3 in the maternal serum, as well as folate content and IGFBP-3 in the fetal brain and liver were significantly lower in the folate deficient group than in the control group (P<0.05). The methylation levels of IGF-1R, IGF-2R, IGFBP-2, IGFBP-5, IGFBP-6 and IGFBP-7 in the fetal brain were higher in the folate deficient group than in the control group (P<0.05). The methylation levels of IGF-1R, IGF-2R, IGFBP-3 and IGFBP-5 in the fetal liver were higher in the folate deficient group than in the control group. The methylation of IGF-2 gene showed a significant reduction in the folate deficient group (P<0.05).

CONCLUSIONSMaternal folate deficiency may cause retardation of growth and development of the offspring, which is possibly associated with the changes of methylation profiles of insulin-like growth factors.

Animals ; Brain ; metabolism ; DNA Methylation ; Female ; Fetal Development ; Fetus ; metabolism ; Folic Acid Deficiency ; metabolism ; Insulin-Like Growth Factor Binding Protein 3 ; blood ; Insulin-Like Growth Factor I ; analysis ; Liver ; metabolism ; Rats ; Rats, Sprague-Dawley

Epigenetic changes frequently occur in human colorectal cancer. Genomic global hypomethylation, gene promoter region hypermethylation, histone modifications, and alteration of miRNA patterns are major epigenetic changes in colorectal cancer. Loss of imprinting(LOI) is associated with colorectal neoplasia. Folate deficiency may cause colorectal carcinogenesis by inducing gene-specific hypermethylation and genomic global hypomethylation. HDAC inhibitors and demethylating agents have been approved by the FDA for myelodysplastic syndrome and leukemia treatment. Non-coding RNA is regarded as another kind of epigenetic marker in colorectal cancer. This review is mainly focused on DNA methylation, histone modification, and microRNA changes in colorectal cancer.
Colorectal Neoplasms ; genetics ; metabolism ; CpG Islands ; genetics ; DNA Methylation ; Epigenesis, Genetic ; Folic Acid Deficiency ; genetics ; Genomic Imprinting ; Histone Deacetylase Inhibitors ; metabolism ; Histone Deacetylases ; metabolism ; Histones ; metabolism ; Humans ; MicroRNAs ; genetics ; metabolism ; Promoter Regions, Genetic ; genetics ; RNA, Untranslated ; genetics ; metabolism

Brain ; metabolism ; pathology ; Child, Preschool ; Chromatography, High Pressure Liquid ; Diagnosis, Differential ; Folate Receptor 1 ; genetics ; metabolism ; Folic Acid ; blood ; cerebrospinal fluid ; metabolism ; Folic Acid Deficiency ; diagnosis ; drug therapy ; etiology ; Humans ; Infant ; Leucovorin ; therapeutic use ; Malnutrition ; complications ; diagnosis ; Tetrahydrofolates ; cerebrospinal fluid ; metabolism

OBJECTIVETo explore the interaction of folate deficiency and aberration of DNA methyltransferase 1 (DNMT1) in the progression of cervix carcinogenesis.

METHODSAll clinical samples were collected from 80 patients with cervix squamous cell carcinoma (SCC), 105 patients with cervical intraepithelial neoplasm (CINI, n = 52; CINII/III, n = 53) and 53 patients with cervix inflammation (CI). The participants were diagnosed by histology at Shanxi Province Tumor Hospital and Second Hospital of Shanxi Medical University during the period of September 2009 to May 2010. Meanwhile, cervical cancer cell lines Caski and C33A were treated with different concentration of folate. Radioimmunoassay (RIA), Western blotting and real-time PCR were used to detect the levels of serum folate, the expression of DNMT1 protein and mRNA, respectively. The data were analyzed by Student t test, ANOVA, chi-square test and Spearman correlation using SPSS statistical package. The correlation strength between factors and cervical canceration was calculated by OR and 95%CI value. Interaction effect was evaluated by the application of additive effect model.

RESULTSThe levels of serum folate (median inter-quartile range) were (2.66 ± 1.82), (2.83 ± 2.23), (3.17 ± 1.91) and (3.21 ± 1.74) ng/ml, the levels of DNMT1 protein (x(-) ± s) were 2.28 ± 0.55, 1.84 ± 0.37, 1.33 ± 0.38 and 0.92 ± 0.29, the Ct-ratio (Ct value of DNMT1/Ct value of β-actin) of DNMT1 mRNA (x(-) ± s) were 1.26 ± 0.13, 1.27 ± 0.12, 1.27 ± 0.12 and 1.33 ± 0.11 in the group of SCC, CINII/III, CINIand CI, respectively. The results showed that the serum folate levels were descended, and the expression levels of DNMT1 protein (χ(2)(tend) = 50.80, P < 0.05) and mRNA (χ(2)(tend) = 17.63, P < 0.05) were increased steadily with the severity of the cervix lesions. Moreover, our results revealed that there was an additive interaction between folate deficiency and high-expression of DNMT1 protein related to the risk of CIN and SCC. And it showed that the relative excess risk of interaction (RERI), attributable proportion of interaction (API) and synergy index(S) was 0.27, 0.14 and 1.40 in CINI group, 0.47, 0.19, 1.46 in CINII/III group, 1.60, 0.31, 1.61 in SCC group, respectively. It was found that folate was able to reduce the proliferation of Caski and C33A cells (r values were 0.954 and 0.969, all P values < 0.05), with 11.4% and 13.6% of growth inhibition at the concentration of 10 µg/ml, 64.8% and 49.4% at 1000 µg/ml in Caski and C33A cells, respectively. The result showed there was an inverse correlation between the levels of folate and DNMT1 protein (r values were -0.859 and -0.914, all P values < 0.05), with 1.96 and 1.92 of expression levels at the concentration of 10 µg/ml, and 1.60 and 1.38 at 1000 µg/ml in Caski and C33A cells, respectively. At folate concentration of 1000 µg/ml, the expression of DNMT1 protein or mRNA was higher in Caski cell than in C33A cell (t values were -4.22 and 3.50, all P values < 0.05).

CONCLUSIONOur finding indicated that the low levels of serum folate and high-expression of DNMT1 protein or mRNA seemed to be associated with high risk of cervical cancer and cervix precancerous lesion. Sufficient folate is able to effectively inhibit the growth of cervical cancer cells in vitro, and would counteract transcriptional and posttranscriptional aberration of DNMT1. It suggested that there might be a synergistic action between folate deficiency and aberration of DNMT1 in the progression of cervix carcinogenesis.

Adult ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Line, Tumor ; Cell Transformation, Neoplastic ; metabolism ; pathology ; Cervical Intraepithelial Neoplasia ; metabolism ; pathology ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; Female ; Folic Acid ; blood ; Folic Acid Deficiency ; metabolism ; Humans ; Middle Aged ; RNA, Messenger ; genetics ; Uterine Cervical Neoplasms ; metabolism ; pathology

OBJECTIVEThis study examined the effects of maternal deficiency of folic acid during pregnancy on pulmonary development and protein A (SP-A) expression in newborn rats in order to explore the possible mechanism of lung developmental disorders.

METHODSThirty-six adult Sprague-Dawley female rats were randomly assigned into two groups: control and study (n=18). The study and the control groups were fed with fodder containing folic acid or not respectively. Two weeks later, the female rats in the two groups copulated with normal male rats. Newborn rats were sacrificed at 1, 7 and 14 days after birth (8 pups at each time point). Lung sections were stained with hematoxylin and eosin for histological examination. SP-A expression of protein and mRNA were determined by immunohistochemistry and real-time quantitative RT-PCR, respectively.

RESULTSThe newborn rats from the study group showed damaged lung tissue structures. The mean optical density of type II cells with positive expression of SP-A decreased significantly from 1 to 14 days in newborn rats of the study group compared with the control newborn rats (P<0.05). The real-time quantitative RT-PCR showed that the expression of lung SP-A mRNA also decreased significantly from 1 to 14 days in newborn rats of the study group compared with control newborn rats (P<0.05).

CONCLUSIONSMaternal deficiency of folic acid during pregnancy can decrease the expression of SP-A in lung tissues of newborn rats, which might lead to the disorder of lung development maturation.

Animals ; Animals, Newborn ; Female ; Folic Acid Deficiency ; metabolism ; Immunohistochemistry ; Lung ; embryology ; Male ; Pregnancy ; Pregnancy Complications ; metabolism ; Pulmonary Surfactant-Associated Protein A ; analysis ; genetics ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo construct the folic acid deficient model in zebrafish and observe the abnormal cardiac phenotypes, to find the optimal period for supplementing folic acid that can most effectively prevent the heart malformation induced by folic acid deficiency, and to investigate the possible mechanisms by which folic acid deficiency induces malformations of heart.

METHODThe folic acid deficient zebrafish model was constructed by using both the folic acid antagonist methotrexate (MTX) and knocking-down dhfr (dihydrofolate reductase gene). Exogenous tetrahydrofolic acid rescue experiment was performed. Folic acid was given to folic acid deficient groups in different periods. The percent of cardiac malformation, the cardiac phenotypes, the heart rate and the ventricular shortening fraction (VSF) were recorded. The out flow tract (OFT) was observed by using fluorescein micro-angiography. Whole-mount in situ hybridization and real-time PCR were performed to detect vmhc, amhc, tbx5 and nppa expressions.

RESULTAbout (78.00 ± 3.74)% embryos in MTX treated group and (68.00 ± 6.32)% embryos in dhfr knocking-down group had heart malformations, including the abnormal cardiac shapes, the hypogenesis of OFT and the reduced heart rate and VSF. Giving exogenous tetrahydrofolic acid rescued the above abnormalities. Given the folic acid on 8 - 12 hours post-fertilization (hpf), both the MTX treated group (20.20% ± 3.77%) and dhfr knocking-down group (43.40% ± 4.51%) showed the most significantly reduced percent of cardiac malformation and the most obviously improved cardiac development. In folic acid deficient group, the expressions of tbx5 and nppa were reduced while the expressions of vmhc and amhc appeared normal. After being given folic acid to MTX treated group and dhfr knocking-down group, the expressions of tbx5 and nppa were increased.

CONCLUSIONSThe synthesis of tetrahydrofolic acid was decreased in our folic acid deficient model. Giving folic acid in the middle period, which is the early developmental stage, can best prevent the abnormal developments of hearts induced by folic acid deficiency. Folic acid deficiency did not disrupt the differentiations of myosins in ventricle and atrium. The cardiac malformations caused by folic acid deficiency were related with the reduced expressions of tbx5 and nppa.

Animals ; Atrial Natriuretic Factor ; metabolism ; Cell Differentiation ; drug effects ; Folic Acid ; metabolism ; Folic Acid Deficiency ; genetics ; metabolism ; Gene Knockdown Techniques ; Heart ; drug effects ; embryology ; growth & development ; T-Box Domain Proteins ; metabolism ; Zebrafish ; embryology ; genetics

Elevated plasma homocysteine ( Hcy) is a risk factor for cognitive dysfunction and Alzheimer disease, although the mechanism is still unknown. Both folate and betaine, a choline metabolite, play essential roles in the remethylation of Hcy to methionine. Choline deficiency may be associated with low folate status and high plasma Hcy. Alterations in DNA methylation also have established critical roles for methylation in development of the nervous system. This study was un-dertaken to assess the effect of choline and folate deficiency on Hcy metabolism and genomic DNA methylation status of the liver and brain. Groups of adult male Sprague Dawley rats were fed on a control, choline-deficient ( CD) , folate-deficient ( FD) or choline/folate-deficient ( CFD) diets for 8 weeks. FD resulted in a significantly lower hepatic folate ( 23%)(p < 0.001) and brain folate ( 69%)(p < 0.05) compared to the control group. However, plasma and brain folate remained unaltered by CD and hepatic folate reduced to 85% of the control by CD ( p < 0.05) . Plasma Hcy was signi-ficantly increased by FD ( 18.34 +/- 1.62 micrometer) and CFD ( 19.35 +/-3.62 micrometer) compared to the control ( 6.29 +/-0.60 micrometer) ( p < 0.001) , but remained unaltered by CD. FD depressed S-adenosylmethionine ( SAM) by 59% ( p < 0.001) and ele-vated S-adenosylhomocysteine ( SAH) by 47% in liver compared to the control group ( p < 0.001) . In contrast, brain SAM levels remained unaltered in CD, FD and CFD rats. Genomic DNA methylation status was reduced by FD in liver ( p< 0.05) . Genomic DNA hypomethylation was also observed in brain by CD, FD and CFD although it was not signifi-cantly different from the control group. Genomic DNA methylation status was correlated with folate stores in liver ( r = - 0.397, p < 0.05) and brain ( r = - 0.390, p < 0.05) , respectively. In conclusion, our data demonstrated that genomic DNA methylation and SAM level were reduced by folate deficiency in liver, but not in brain, and correlated with folate concentration in the tissue. The fact that folate deficiency had differential effects on SAM, SAH and genomic DNA methylation in liver and brain suggests that the Hcy metabolism and DNA methylation are regulated in tissue-specific ways.
Adult ; Alzheimer Disease ; Animals ; Betaine ; Brain ; Choline ; Choline Deficiency ; Diet ; DNA Methylation* ; DNA* ; Folic Acid ; Homocysteine* ; Humans ; Liver ; Male ; Metabolism ; Methionine ; Methylation ; Nervous System ; Plasma* ; Rats* ; Rats, Sprague-Dawley ; Risk Factors ; S-Adenosylhomocysteine ; S-Adenosylmethionine