Liyuan Deng

High intake of folic acid disrupts embryonic development in mice

BACKGROUND Folic acid fortification and supplementation has increased folate intake and blood folate concentrations and successfully reduced the incidence of neural tube defects. However, the developmental consequences of high folate intake are unknown. This study investigated the impact of high folate intake, alone or with methylenetetrahydrofolate reductase (MTHFR) deficiency, on embryonic and placental development in mice. METHODS Mthfr +/+ or +/- pregnant mice on a control diet (CD; recommended intake of folic acid for rodents) or folic acid-supplemented diet (FASD; 20-fold higher than the recommended intake) were examined for embryonic loss, delay, and defects at 10.5 and 14.5 days post coitum (dpc); 10.5-dpc placenta, and 14.5-dpc embryo hearts were studied histologically. RESULTS Total plasma folate was 10-fold higher in FASD compared to CD mice; plasma homocysteine levels were not affected by diet. At 10.5 dpc, the FASD was associated with embryonic delay and growth retardation, and may confer susceptibility to embryonic defects. The FASD did not adversely affect 10.5-dpc placental development. At 14.5 dpc, embryos from the FASD Mthfr +/+ group were delayed and the FASD was associated with thinner ventricular walls in embryonic hearts. There was a significant interaction between maternal MTHFR deficiency and a high folate diet for several developmental outcomes. CONCLUSIONS Our study suggests that high folate intake may have adverse effects on fetal mouse development and that maternal MTHFR deficiency may improve or rescue some of the adverse outcomes. These findings underscore the need for additional studies on the potential negative impact of high folate intake during pregnancy.

Moderately high intake of folic acid has a negative impact on mouse embryonic development

BACKGROUND The incidence of neural tube defects has diminished considerably since the implementation of food fortification with folic acid (FA). However, the impact of excess FA intake, particularly during pregnancy, requires investigation. In a recent study, we reported that a diet supplemented with 20-fold higher FA than the recommended intake for rodents had adverse effects on embryonic mouse development at embryonic days (E)10.5 and 14.5. In this report, we examined developmental outcomes in E14.5 embryos after administering a diet supplemented with 10-fold higher FA than recommended to pregnant mice with and without a mild deficiency of methylenetetrahydrofolate reductase (MTHFR). METHODS Pregnant mice with or without a deficiency in MTHFR were fed a control diet (recommended FA intake of 2 mg/kg diet for rodents) or an FA-supplemented diet (FASD; 10-fold higher than the recommended intake [20 mg/kg diet]). At E14.5, mice were examined for embryonic loss and growth retardation, and hearts were assessed for defects and for ventricular wall thickness. RESULTS Maternal FA supplementation was associated with embryonic loss, embryonic delays, a higher incidence of ventricular septal defects, and thinner left and right ventricular walls, compared to mothers fed control diet. CONCLUSIONS Our work suggests that even moderately high levels of FA supplementation may adversely affect fetal mouse development. Additional studies are warranted to evaluate the impact of high folate intake in pregnant women. Birth Defects Research (Part A), 2013.

Steatosis in Mice Is Associated with Gender, Folate Intake, and Expression of Genes of One-Carbon Metabolism

Disrupted choline metabolism may affect hepatic lipid metabolism and lead to steatosis. Because folate and the choline metabolite betaine independently serve as methyl donors for homocysteine (Hcy) remethylation to methionine, we assessed the impact of folate deficiency on steatosis, choline metabolism, and expression of 9 genes involved in folate-mediated one-carbon metabolism. Liver histology, choline metabolites, and mRNA and protein expression were examined in mice fed control (CD; 2 mg/kg folic acid) or folate-deficient diets (FD; 0.3 mg/kg folic acid) for 12 mo. Females fed CD were not steatotic (0/6), whereas males were mildly to moderately steatotic (5/6). Steatosis was observed in FD-fed males and females; it was more severe and more frequent in males (7/7) than in females (4/10) (P = 0.005). Hepatic betaine was lower in males (P = 0.014) and FD-fed mice (P < 0.001) and negatively correlated with steatosis severity in mice fed CD (r = -0.87; P = 0.001). Gender differences in the expression of 6 enzymes may contribute to increased steatosis susceptibility in males. Males relied more on betaine-dependent (folate-independent) Hcy remethylation [72% more betaine-Hcy methyltransferase (P < 0.001) and 28% less folate-dependent methionine synthase (MTR) (P < 0.001)]. FD-fed mice of both genders appeared to shift to betaine-dependent remethylation by reducing MTR expression 70% (P < 0.001) and increasing betaine demand; there was a correlation between MTR expression and betaine levels (r = 0.50; P = 0.031). Our work demonstrates that chronic folate insufficiency leads to steatosis in mice. Increased utilization of betaine for Hcy remethylation in males and in both genders during folate deficiency may lead to steatosis by disrupting choline metabolism.

Low Dietary Folate Initiates Intestinal Tumors in Mice, with Altered Expression of G2-M Checkpoint Regulators Polo-Like Kinase 1 and Cell Division Cycle 25c

Clinical reports have suggested that low dietary folate increases risk for colorectal cancer. Animal studies for investigation of folate and tumorigenesis have used carcinogen induction or mice with germ-line mutations. We have developed a new spontaneous tumor model in which mice, with or without a null allele in a key folate-metabolizing enzyme, methylenetetrahydrofolate reductase (Mthfr), develop intestinal tumors due to low dietary folate alone. On folate-deficient diets, 12.5% of Mthfr(+/+) mice and 28.1% of Mthfr(+/-) mice developed tumors; mice on control diets were negative. Dietary and genotype effects on tumor development were significant. To investigate mechanisms of folate-dependent tumorigenesis, we examined levels of DNA damage and gene expression of two genes involved in DNA damage response and G(2)-M checkpoint regulation, polo-like kinase 1 (Plk1) and cell division cycle 25c (Cdc25c). Folate deficiency increased DNA damage and decreased expression of both genes (assessed by quantitative reverse transcription-PCR and immunofluorescence) in normal intestine compared with levels in mice on control diets. An immunofluorescence assay for CDC25c activity (phosphorylated CDC2) also found CDC25c activity to be decreased in folate-deficient normal intestine. In tumors, however, Plk1 and Cdc25c mRNA were found to be higher (11- and 3-fold, respectively) compared with normal intestine from folate-deficient mice; immunofluorescence studies of PLK1, CDC25c, and phosphorylated CDC2 supported these findings. Our data suggest that folate deficiency can initiate tumor development, that Mthfr mutation can enhance this phenomenon, and that altered expression of Plk1 and Cdc25c may contribute to folate-dependent intestinal tumorigenesis.

High folic acid consumption leads to pseudo-MTHFR deficiency, altered lipid metabolism, and liver injury in mice

Background: Increased consumption of folic acid is prevalent, leading to concerns about negative consequences. The effects of folic acid on the liver, the primary organ for folate metabolism, are largely unknown. Methylenetetrahydrofolate reductase (MTHFR) provides methyl donors for S-adenosylmethionine (SAM) synthesis and methylation reactions. Objective: Our goal was to investigate the impact of high folic acid intake on liver disease and methyl metabolism. Design: Folic acid–supplemented diet (FASD, 10-fold higher than recommended) and control diet were fed to male Mthfr+/+ and Mthfr+/− mice for 6 mo to assess gene-nutrient interactions. Liver pathology, folate and choline metabolites, and gene expression in folate and lipid pathways were examined. Results: Liver and spleen weights were higher and hematologic profiles were altered in FASD-fed mice. Liver histology revealed unusually large, degenerating cells in FASD Mthfr+/− mice, consistent with nonalcoholic fatty liver disease. High folic acid inhibited MTHFR activity in vitro, and MTHFR protein was reduced in FASD-fed mice. 5-Methyltetrahydrofolate, SAM, and SAM/S-adenosylhomocysteine ratios were lower in FASD and Mthfr+/− livers. Choline metabolites, including phosphatidylcholine, were reduced due to genotype and/or diet in an attempt to restore methylation capacity through choline/betaine-dependent SAM synthesis. Expression changes in genes of one-carbon and lipid metabolism were particularly significant in FASD Mthfr+/− mice. The latter changes, which included higher nuclear sterol regulatory element-binding protein 1, higher Srepb2 messenger RNA (mRNA), lower farnesoid X receptor (Nr1h4) mRNA, and lower Cyp7a1 mRNA, would lead to greater lipogenesis and reduced cholesterol catabolism into bile. Conclusions: We suggest that high folic acid consumption reduces MTHFR protein and activity levels, creating a pseudo-MTHFR deficiency. This deficiency results in hepatocyte degeneration, suggesting a 2-hit mechanism whereby mutant hepatocytes cannot accommodate the lipid disturbances and altered membrane integrity arising from changes in phospholipid/lipid metabolism. These preliminary findings may have clinical implications for individuals consuming high-dose folic acid supplements, particularly those who are MTHFR deficient.

Methylenetetrahydrofolate reductase deficiency and low dietary folate reduce tumorigenesis in Apcmin/+ mice

Background: Clinical studies suggest that mild methylenetetrahydrofolate reductase (MTHFR) deficiency and high dietary folate may reduce the risk for colorectal cancer. There is concern, however, that high folate intake (a consequence of food fortification) may enhance tumour growth in individuals with pre-existing tumours or genetic predisposition to tumorigenesis. Aim: To determine if Mthfr deficiency and low dietary folate influence tumorigenesis in mice genetically predisposed to form numerous intestinal adenomas (Apcmin/+). Methods: Male Apcmin/+ mice were mated with Mthfr+/− and/or Mthfr+/+ females. Diets with variable folate content were administered either pre-natally or at weaning; tumours were counted in offspring at 10 weeks of age. Plasma homocysteine and levels of apoptosis, DNA methylation and nucleotide ratios (dUTP:dTTP) in normal (pre-neoplastic) intestine were measured. Results: Apcmin/+ mice fed high folate diets from weaning developed more adenomas than those fed the folic acid-deficient diet (FADD) or the control diet (CD); Mthfr deficiency did not affect adenoma number. However, when the FADD and CD were administered to dams prior to conception, throughout pregnancy and continued in offspring post-weaning, Apcmin/+ offspring fed FADD developed fewer adenomas than those fed CD. Mthfr+/− genotype of the mother or of the offspring also reduced adenoma numbers in the Apcmin/+ offspring. Adenoma number was inversely correlated with plasma homocysteine (r = −0.49, p<0.005, intestinal dUTP/dTTP ratios (r = −0.42, p = 0.05), and levels of intestinal apoptosis (r = −0.36, p = 0.08). Conclusions: Low dietary folate and Mthfr deficiency reduce adenoma formation in mice predisposed to tumorigenesis, possibly through increased apoptosis consequent to hyperhomocysteinaemia and nucleotide imbalances.

Severe methylenetetrahydrofolate reductase deficiency in mice results in behavioral anomalies with morphological and biochemical changes in hippocampus.

The brain is particularly sensitive to folate metabolic disturbances, since methyl groups are critical for its functions. Methylenetetrahydrofolate reductase (MTHFR) generates the primary circulatory form of folate required for homocysteine remethylation to methionine. Neurological disturbances have been described in homocystinuria caused by severe MTHFR deficiency. The goal of this study was to determine if behavioral anomalies are present in severe Mthfr-deficient (Mthfr(-/-)) mice and to identify neurobiological changes that could contribute to these anomalies. Adult male mice of 3 Mthfr genotypes (+/+, +/-, -/-) were tested on motor, anxiety, exploratory and cognitive tasks. Volumes (whole brain and hippocampus) and morphology, global DNA methylation, apoptosis, expression of choline acetyltransferase (ChAT) and glucocorticoid receptor (GR), and concentrations of choline metabolites were assessed in hippocampus. Mthfr(-/-) mice had impairments in motor function and in short- and long-term memory, increased exploratory behavior and decreased anxiety. They showed decreased whole brain and hippocampal volumes, reduced thickness of the pyramidal cell layer of CA1 and CA3, and increased apoptosis in hippocampus. There was a disturbance in choline metabolism as manifested by differences in acetylcholine, betaine or glycerophosphocholine concentrations, and by increased ChAT levels. Mthfr(-/-) mice also had increased GR mRNA and protein. Our study has revealed significant anomalies in affective behavior and impairments in memory of Mthfr(-/-) mice. We identified structural changes, increased apoptosis, altered choline metabolism and GR dysregulation in hippocampus. These findings, as well as some similar observations in cerebellum, could contribute to the behavioral changes and suggest that choline is a critical metabolite in homocystinuria.

Methionine synthase reductase deficiency results in adverse reproductive outcomes and congenital heart defects in mice

Low dietary folate and polymorphisms in genes of folate metabolism can influence risk for pregnancy complications and birth defects. Methionine synthase reductase (MTRR) is required for activation of methionine synthase, a folate- and vitamin B(12)-dependent enzyme. A polymorphism in MTRR (p.I22M), present in the homozygous state in 25% of many populations, may increase risk for neural tube defects. To examine the impact of MTRR deficiency on early development and congenital heart defects, we used mice harboring a gene-trapped (gt) allele in Mtrr. Female mice (Mtrr(+/+), Mtrr(+/gt), and Mtrr(gt/gt)) were mated with male Mtrr(+/g) mice. Reproductive outcomes and cardiac phenotype (presence of defects and myocardial thickness) were assessed at E14.5. Mtrr-deficient mothers had more resorptions and more delayed embryos per litter (resorptions per litter: 0.29+/-0.13; 1.21+/-0.41; 1.87+/-0.38 and delayed embryos per litter: 0.07+/-0.07; 0.14+/-0.14; 0.60+/-0.24 in Mtrr(+/+), Mtrr(+/gt), and Mtrr(gt/gt) mothers respectively). Placentae of Mtrr(gt/gt) mothers were smaller and their embryos were smaller, with myocardial hypoplasia and a higher incidence of ventricular septal defects (VSD) per litter (0; 0.57+/-0.30; 1.57+/-0.67 in Mtrr(+/+), Mtrr(+/gt), and Mtrr(gt/gt) groups respectively). Embryonic Mtrr(gt/gt) genotype was associated with reduced embryonic length, reduced embryonic and placental weight, and higher incidence of VSD, but did not affect myocardial thickness or embryonic delay. We conclude that Mtrr deficiency adversely impacts reproductive outcomes and cardiac development in mice. These findings may have implications for nutritional prevention of heart defects, particularly in women with the common MTRR polymorphism.

ApcMin/+ mouse model of colon cancer: Gene expression profiling in tumors

The ApcMin/+ mouse is a popular animal model for studies of human colon cancer, but the molecular changes associated with neoplasia in this system have only been partially characterized. Our aim was to identify novel genes involved in tumorigenesis in this model. RNA from intestinal adenomas and from pre‐neoplastic small intestine were prepared from six ApcMin/+ mice. The tumor transcriptomes were analyzed with high‐density oligonucleotide microarrays representing ∼12,000 probe sets; we compared their profiles with those of matched pre‐neoplastic intestine. Stringent analysis revealed reproducible changes for 98 probe sets representing 90 genes, including novel observations regarding 50 genes whose involvement in this mouse model has never been reported. In addition to the expected changes in growth regulatory genes, the altered gene products could be assigned to four functional groupings that should enhance tumorigenesis: metabolic changes that would result in a high rate of glycolysis, alterations in enzymes involved in reactive oxygen species or carcinogen metabolism, cytoskeletal elements, and proteins involved in tumor invasion or angiogenesis. A fifth group consisted of expression changes that might restrict tumor progression, suggesting that the adenomatous state reflects a balance of pro‐ and anti‐tumorigenic factors. Since many of the altered genes had not previously been reported to be involved in any tumorigenic processes, our observations provide a host of new candidates for potential modulation to prevent or treat intestinal neoplasia. Supplementary material for this article can be found at http://www.mrw.interscience.wiley.com/suppmat/0730‐2312/suppmat/v93.html.

MTHFR deficiency or reduced intake of folate or choline in pregnant mice results in impaired short-term memory and increased apoptosis in the hippocampus of wild-type offspring

Genetic or nutritional disturbances in one-carbon metabolism, with associated hyperhomocysteinemia, can result in complex disorders including pregnancy complications and neuropsychiatric diseases. In earlier work, we showed that mice with a complete deficiency of methylenetetrahydrofolate reductase (MTHFR), a critical enzyme in folate and homocysteine metabolism, had cognitive impairment with disturbances in choline metabolism. Maternal demands for folate and choline are increased during pregnancy and deficiencies of these nutrients result in several negative outcomes including increased resorption and delayed development. The goal of this study was to investigate the behavioral and neurobiological impact of a maternal genetic deficiency in MTHFR or maternal nutritional deficiency of folate or choline during pregnancy on 3-week-old Mthfr(+/+) offspring. Mthfr(+/+) and Mthfr(+/-) females were placed on control diets (CD); and Mthfr(+/+) females were placed on folate-deficient diets (FD) or choline-deficient diets (ChDD) throughout pregnancy and lactation until their offspring were 3weeks of age. Short-term memory was assessed in offspring, and hippocampal tissue was evaluated for morphological changes, apoptosis, proliferation and choline metabolism. Maternal MTHFR deficiency resulted in short-term memory impairment in offspring. These dams had elevated levels of plasma homocysteine when compared with wild-type dams. There were no differences in plasma homocysteine in offspring. Increased apoptosis and proliferation was observed in the hippocampus of offspring from Mthfr(+/-) mothers. In the maternal FD and ChDD study, offspring also showed short-term memory impairment with increased apoptosis in the hippocampus; increased neurogenesis was observed in ChDD offspring. Choline acetyltransferase protein was increased in the offspring hippocampus of both dietary groups and betaine was decreased in the hippocampus of FD offspring. Our results reveal short-term memory deficits in the offspring of dams with MTHFR deficiency or dietary deficiencies of critical methyl donors. We suggest that deficiencies in maternal one-carbon metabolism during pregnancy can contribute to hippocampal dysfunction in offspring through apoptosis or altered choline metabolism.