Biometric Alterations of Mouse Embryonic Eye Structures Due to Short-Term Folic Acid Deficiency.

PURPOSE: Folic acid (FA) is an essential nutrient for normal embryonic development. FA deficiency (FAD) in maternal diet increases the risk of several defects among the progeny, especially, neural tube defects. The eye begins its development from the neural tube; however, the relationship between FAD and ocular development in the offspring has been little explored and it isn't known how the FAD affects the formation of the eye. Our objective was to analyze the effect of maternal FAD on mouse embryos ocular biometry. METHODS: Female mice C57/BL/6J were distributed into three different groups, according to the assigned diet: control group fed a standard FA diet (2 mg FA/kg), FAD group for short term fed (0 mg FA/kg + 1% succinylsulfathiazole) from the day after mating until day 14.5 of gestation, and FAD group for long term fed the same FA-deficient diet for 6 weeks prior mating and continued with this diet during gestation. A total of 57 embryos (19 embryos of each dietary group) at 14.5 gestational days were evaluated. As indicators of changes in ocular biometry, we analyze two parameters: area and circularity of the lens and whole eye, and the area of the retina. The program used in the treatment and selection of the areas of interest was ImageJ. The statistical analysis was performed by IBM SPSS Statistics 19. RESULTS: Regarding the measures of the area, FA-deficient lenses and eyes were smaller than that of controls. We have also observed increase in the size of the neural retina, spatially, in embryos from females fed FAD diet during long term. On the other hand, as regard to circularity measures, we have seen that eyes and lenses were more circular than control. CONCLUSION: Maternal FAD diet for a very short term generates morphological changes in ocular structures to the offspring.

Alteration of lens and retina textures from mice embryos with folic acid deficiency: image processing analysis.

PURPOSE: Folic acid (FA) is an essential vitamin for embryonic development. It plays particularly a critical role in RNA, DNA and protein synthesis. On the other hand, the collagen IV and laminin-1 are important proteins during embryonic development. This study was done to find if FA deficiency at a short and a long term in mothers could alter the tissue texture of retina and lens of the progeny. METHODS: Collagen IV and laminin-1 were localized by immunohistochemistry in the lens and retina of the FA-deficient embryos. To carry out the image processing, texture segmentation was performed through canny edge detection and Fourier transform (FT). We defined a parameter, the grain size, to describe the texture of the lens and retina. A bootstrap method to estimate the distribution and confidence intervals of the mean, standard deviation, skewness and kurtosis of the grain size has been developed. RESULTS: Analysis through image processing using Matlab showed changes in the grain size between control- and FA-deficient groups in both studied molecules. Measures of texture based on FT exhibited changes in the directionality and arrangements of type IV collagen and laminin-1. CONCLUSIONS: Changes introduced by FA deficiency were visible in the short term (2 weeks) and evident in the long term (8 weeks) in both grain size and orientation of fibre structures in the tissues analysed (lens and retina). This is the first work devoted to study the effect of FA deficit in the texture of eye tissues using image processing techniques.

Gene promoter DNA methylation patterns have a limited role in orchestrating transcriptional changes in the fetal liver in response to maternal folate depletion during pregnancy.

SCOPE: Early-life exposures are critical in fetal programming and may influence function and health in later life. Adequate maternal folate consumption during pregnancy is essential for healthy fetal development and long-term offspring health. The mechanisms underlying fetal programming are poorly understood, but are likely to involve gene regulation. Epigenetic marks, including DNA methylation, regulate gene expression and are modifiable by folate supply. We observed transcriptional changes in fetal liver in response to maternal folate depletion and hypothesized that these changes are concomitant with altered gene promoter methylation. METHODS AND RESULTS: Female C57BL/6J mice were fed diets containing 2 or 0.4 mg folic acid/kg for 4 wk before mating and throughout pregnancy. At 17.5-day gestation, genome-wide gene expression and promoter methylation were measured by microarray analysis in male fetal livers. While 989 genes were differentially expressed, 333 promoters had altered methylation (247 hypermethylated, 86 hypomethylated) in response to maternal folate depletion. Only 16 genes had both expression and methylation changes. However, most methylation changes occurred in genomic regions neighboring expression changes. CONCLUSION: In response to maternal folate depletion, altered expression at the mRNA level was not associated with altered promoter methylation of the same gene in fetal liver.

Role of methotrexate exposure in apoptosis and proliferation during early neurulation.

Apoptosis and proliferation play important roles in embryonic development and are required for neural tube closure. The antifolate drug methotrexate (MTX) induces folate dysmetabolism by inhibition of dihydrofolate reductase and causes abnormal apoptosis and proliferation. In this study, we established an animal model of neural tube defects (NTDs) using MTX to investigate the role of apoptosis and proliferation in NTDs caused by folate deficiency. Differential gene expressions were studied by microarray and reverse transcription-polymerase chain reaction in the NTD animal model. Results showed that 30.8% of NTDs were caused by using MTX in treatment regimens. Microarray indicated that 166 genes were significantly different between the control and NTD mice, including four apoptosis-related genes (Endog, Trp53, Casp3, Bax) and three proliferation-related genes (Ptch1, Pla2g4a, Foxg1). Levels of Endog, Trp53, Casp3, Bax (fold change>1.5) were upregulated but Ptch1, Pla2g4a, Foxg1 (fold change<0.67) were downregulated (P<0.05). These results were confirmed by reverse transcription-polymerase chain reaction. TUNEL, immunohistochemical assays and Western blot were further used to detect apoptosis and proliferation in the NTD animal model. It was found that apoptosis in neuroepithelial cells was increased as determined by TUNEL (P<0.05). Expressions of caspase-3 were significantly enhanced (P<0.05) but expressions of phosphohistone H3 were greatly decreased (P<0.05). These results concluded that MTX caused a folate and folate-associated dysmetabolism, and further induced abnormal apoptosis and proliferation, which may play a critical role in the occurrence of NTDs caused by folate deficiency.

The heart-placenta axis in the first month of pregnancy: induction and prevention of cardiovascular birth defects.

Extrapolating from animal studies to human pregnancy, our studies showed that folate (FA) deficiency as well as one-time exposure to environmental factors in the first two to three weeks of human gestation can result in severe congenital heart defects (CHDs). Considering that approximately 49% of pregnancies are unplanned, this period of pregnancy can be considered high-risk for cardiac, as well as for neural, birth defects, as the woman usually is not aware of her pregnancy and may not yet be taking precautionary actions to protect the developing embryo. Using avian and mouse vertebrate models, we demonstrated that FA supplementation prevents CHD induced by alcohol, lithium, or elevation of the metabolite homocysteine, a marker for FA deficiency. All three factors affected the important Wnt signaling pathway by suppressing Wnt-mediated gene expression in the heart fields, resulting in a delay of cardiomyocyte migration, cardiomyogenesis, and CHD. Optimal protection of cardiogenesis was observed to occur with FA supplementation provided upon morning after conception and at higher doses than the presently available in prenatal vitamin supplementation. Our studies demonstrate pathways and cell processes that are involved with protection of one-carbon metabolism during heart development.

Fumonisin FB1 treatment acts synergistically with methyl donor deficiency during rat pregnancy to produce alterations of H3- and H4-histone methylation patterns in fetuses.

SCOPE: Prenatal folate and methyl donor malnutrition lead to epigenetic alterations that could enhance susceptibility to disease. Methyl-deficient diet (MDD) and fumonisin FB1 are risk factors for neural tube defects and cancers. Evidence indicates that FB1 impairs folate metabolism. METHODS AND RESULTS: Folate receptors and four heterochromatin markers were investigated in rat fetuses liver derived from dams exposed to MDD and/or FB1 administered at a dose twice higher than the provisional maximum tolerable daily intake (PMTDI = 2 µg/kg/day). Even though folate receptors transcription seemed up-regulated by methyl depletion regardless of FB1 treatment, combined MDD/FB1 exposure might reverse this up-regulation since folate receptors transcripts were lower in the MDD/FB1 versus MDD group. Methyl depletion decreased H4K20me3. Combined MDD/FB1 decreased H4K20me3 even more and increased H3K9me3. The elevated H3K9me3 can be viewed as a defense mechanism inciting the cell to resist heterochromatin disorganization. H3R2me2 and H4K16Ac varied according to this mechanism even though statistical significance was not consistent. CONCLUSION: Considering that humans are exposed to FB1 levels above the PMTDI, this study is relevant because it suggests that low doses of FB1 interact with MDD thus contributing to disrupt the epigenetic landscape.

Maternal folate supply and sex influence gene-specific DNA methylation in the fetal gut.

SCOPE: Epidemiological evidence supports the developmental origins of health and disease hypothesis that developmental under/over-nutrition increases adulthood disease risk. Epigenetic markings are one potential mechanism mediating these effects. Altered folate supply may influence methyl group availability for DNA methylation. We reported low folate supply in utero was associated with reduced global DNA methylation in the murine small intestine of adult offspring. We hypothesised that aberrant methylation would be observed during early development. METHODS AND RESULTS: Female C57BL/6J mice were fed diets containing 2 mg folic acid/kg or 0.4 mg folic acid/kg 4 wk before mating and during pregnancy. At 17.5 day gestation, gene methylation in fetal gut was analysed by Pyrosequencing(®) . Low folate reduced overall methylation of Slc394a by 3.4% (p=0.038) but did not affect Esr1 or Igf2 differentially methylated region (DMR) 1. There were sex-specific differences in Slc394a and Esr1 methylation (2.4% higher in females (p=0.002); 4% higher in males (p=0.0014), respectively). CONCLUSION: This is the first study reporting causal effects of maternal folate depletion on gene-specific methylation in fetal gut. These observations support reports that altered methyl donor intake during development affects DNA methylation in the offspring. The consequences of epigenetic changes for health throughout the life course remain to be investigated.

Perinatal folate supply: relevance in health outcome parameters.

The importance of physiological supply of folate is well recognized in human health; the crucial roles of folate in one-carbon metabolism for physiological DNA synthesis and cell division, as well as in the conversion of homocysteine (Hcy) to methionine, and subsequently, to S-adenosylmethionine, have been convincingly demonstrated. Improved folate status may reduce the risk of macrocytic anaemia, cardiovascular diseases, neuropsychiatric disorders and adverse pregnancy outcomes. Inadequate folate status results in a decrease in the methylation cycle and in increased blood levels of the neurotoxic Hcy. The aim of this review is to provide insight into the influence of folate status on pregnancy health outcomes, and to consider increasing evidence of a link between the extent of genome/epigenome damage and elevated risk for adverse obstetrical endpoints. Pregnant women are at risk for folate insufficiency because of the increased need for folate for rapid fetal growth, placental development and enlargement of the uterus. Inadequate folate status may cause fetal malformations, impaired fetal growth, pre-term delivery and maternal anaemia. Even some diseases of the placenta may arise from folate deficiencies. Fetal growth seems to be vulnerable to maternal folate status during the periconception period, because it has the potential to affect both the closure of the neural tube and several epigenetic mechanisms within the placenta and the fetus. Mainly on the basis of the well recognized link between maternal folate status and fetal neural tube defects, women are advised to receive folic acid supplement during the periconceptional period. Because an adequate folate supply seems to play an important role in the implantation and development of the placenta and in improving endothelial function, folic acid supplementation in the late first trimester or early second trimester might also be beneficial.

Sacarose como veículo de suplementação dietética de ácido fólico em camundongos prenhes / Saccharose as a vehicle for the supplementation of folic acid in pregnant mice

INTRODUÇÃO: A ingestão adequada de folato é essencial durante a embriogênese, e sua deficiência está associada à ocorrência de defeitos no fechamento do tubo neural. OBJETIVO: Determinar se a sacarose é um bom veículo para a suplementação de folato em camundongos. MATERIAL E MÉTODOS: Quarenta camundongos Swiss fêmeas foram divididos nos grupos: C: ração comercial + água ad libitum; DS: ração balanceada isenta de folato + folato adicionado à sacarose diluída na água por 14 dias; D/DS: ração balanceada isenta de folato + água com sacarose sem folato por 14 dias seguida de ração balanceada isenta de folato + folato adicionado à sacarose diluída na água por mais 14 dias; D: ração balanceada isenta de folato + água com sacarose sem folato por 14 dias. Os animais de todos os grupos experimentais receberam ração balanceada isenta de folato + folato adicionado à sacarose diluída na água durante os três dias do acasalamento e nos 15 dias restantes até o sacrifício. RESULTADOS: Os animais dos grupos D e D/DS apresentaram alopecia, palidez ocular e adinamia enquanto consumiam água com sacarose sem folato, sinais que foram revertidos quando receberam folato adicionado à sacarose diluída na água. Não houve diferença entre os grupos em relação a prenhez, implantes, fetos vivos, reabsorção, morte fetal tardia, nível sérico de folato e contagem de hemácias ao final do experimento, não tendo sido observadas anomalias congênitas em nenhum dos grupos. CONCLUSÃO: A sacarose é um meio adequado para a suplementação de folato na dieta.

Adequate folate intake is essential during embryogenesis and its deficiency is associated with neural tube defects. OBJECTIVE: To investigate if saccharose is a good vehicle for the supplementation of folate in mice. MATERIAL AND METHODS: 40 Swiss female mice were allocated into the following groups: C (commercial mouse food + ad libitum water); DS (folate-free balanced diet + saccharose with folate diluted in water for 14 days); D/DS (folate-free balanced diet + folate-free saccharose diluted in water for 14 days, followed by folate-free balanced diet + saccharose with folate diluted in water for 14 days); D (folate-free balanced diet + folate-free saccharose diluted in water for 14 days). Mice from all experimental groups received folate-free balanced diet + saccharose with folate diluted in water during their three-day mating period and thereafter 15 days until animals were put down. RESULTS: Mice from groups D and D/DS showed alopecia, pale eyes and adynamia while on folate-free saccharose water regimen. These symptoms disappeared after the introduction of saccharose with folate diluted in water. No statistical difference was noted among groups as to pregnancy, number of implants, live fetuses, reabsorption, late fetal death, serum folate levels and red blood cells count and no congenital abnormalities were identified in any groups by the end of the experiment. CONCLUSION: Saccharose is a suitable vehicle for the dietary supplementation of folate.

Gene-environment interactions in the causation of neural tube defects: folate deficiency increases susceptibility conferred by loss of Pax3 function.

Risk of neural tube defects (NTDs) is determined by genetic and environmental factors, among which folate status appears to play a key role. However, the precise nature of the link between low folate status and NTDs is poorly understood, and it remains unclear how folic acid prevents NTDs. We investigated the effect of folate level on risk of NTDs in splotch (Sp(2)(H)) mice, which carry a mutation in Pax3. Dietary folate restriction results in reduced maternal blood folate, elevated plasma homocysteine and reduced embryonic folate content. Folate deficiency does not cause NTDs in wild-type mice, but causes a significant increase in cranial NTDs among Sp(2)(H) embryos, demonstrating a gene-environment interaction. Control treatments, in which intermediate levels of folate are supplied, suggest that NTD risk is related to embryonic folate concentration, not maternal blood folate concentration. Notably, the effect of folate deficiency appears more deleterious in female embryos than males, since defects are not prevented by exogenous folic acid. Folate-deficient embryos exhibit developmental delay and growth retardation. However, folate content normalized to protein content is appropriate for developmental stage, suggesting that folate availability places a tight limit on growth and development. Folate-deficient embryos also exhibit a reduced ratio of s-adenosylmethionine (SAM) to s-adenosylhomocysteine (SAH). This could indicate inhibition of the methylation cycle, but we did not detect any diminution in global DNA methylation, in contrast to embryos in which the methylation cycle was specifically inhibited. Hence, folate deficiency increases the risk of NTDs in genetically predisposed splotch embryos, probably via embryonic growth retardation.

Developmental consequences of in utero sodium arsenate exposure in mice with folate transport deficiencies.

Previous studies have demonstrated that mice lacking a functional folate binding protein 2 gene (Folbp2-/-) were significantly more sensitive to in utero arsenic exposure than were the wild-type mice similarly exposed. When these mice were fed a folate-deficient diet, the embryotoxic effect of arsenate was further exacerbated. Contrary to expectations, studies on 24-h urinary speciation of sodium arsenate did not demonstrate any significant difference in arsenic biotransformation between Folbp2-/- and Folbp2+/+ mice. To better understand the influence of folate pathway genes on arsenic embryotoxicity, the present investigation utilized transgenic mice with disrupted folate binding protein 1 (Folbp1) and reduced folate carrier (RFC) genes. Because complete inactivation of Folbp1 and RFC genes results in embryonic lethality, we used heterozygous animals. Overall, no RFC genotype-related differences in embryonic susceptibility to arsenic exposure were observed. Embryonic lethality and neural tube defect (NTD) frequency in Folbp1 mice was dose-dependent and differed from the RFC mice; however, no genotype-related differences were observed. The RFC heterozygotes tended to have higher plasma levels of S-adenosylhomocysteine (SAH) than did the wild-type controls, although this effect was not robust. It is concluded that genetic modifications at the Folbp1 and RFC loci confers no particular sensitivity to arsenic toxicity compared to wild-type controls, thus disproving the working hypothesis that decreased methylating capacity of the genetically modified mice would put them at increased risk for arsenic-induced reproductive toxicity.

Investigation of the effects of folate deficiency on embryonic development through the establishment of a folate deficient mouse model.

BACKGROUND: Folic acid (FA) has been shown to reduce the incidence of neural tube, craniofacial, and cardiovascular defects and low birth weight. The mechanism(s) by which the vitamin is effective, however, has not been determined. Therefore, a folic acid deficient mouse model was developed. METHODS: To create a folic acid deficiency, ICR female mice were placed on a diet containing no FA and including 1% succinyl sulfathiazole (SS) for 4 weeks before mating. Control mice were fed diets with either: 1) FA and 1% SS [+SS only diet]; 2) FA [normal diet]; or 3) a breeding diet. Dams and fetuses were examined during various days of gestation. RESULTS: Blood analysis showed that by gestational day 18, plasma folate concentrations in the -FA+SS fed dams decreased to 1.13 ng/ml, a concentration approximately 3% of that in breeding diet fed dams (33.24 ng/ml) and 8% of that in +SS only/normal fed dams (13.59 ng/ml). RBC folate levels showed a similar decrease, whereas homocysteine concentrations increased. Reproductive outcome in the -FA+SS fed dams was poor with increased fetal deaths, decreased fetal weight, and delays in palate and heart development. CONCLUSIONS: Female mice fed a folic acid deficient diet and 1% succinyl sulfathiazole exhibited many of the characteristics common to human folic acid deficiency, including decreased plasma and RBC folate, increased plasma homocysteine, and poor reproductive outcomes. Thus, an excellent model has been created to investigate the mechanism(s) underlying the origin of birth defects related to folic acid deficiency.

Effects of folate deficiency on embryonic development.

While there is strong evidence that folate deficiency including the use of anti-folate drugs in early pregnancy is teratogenic and may lead to a range of serious abnormalities of the developing fetus including intrauterine death, the mechanism(s) for these effects have not yet been delineated. In neural tube defects, there is increasing evidence that marginal folate status exacerbates the effect of an underlying genetic defect in the mother, the fetus, or both. An abnormal relationship between the ingestion of folate and the folate levels in red blood cells has been found in women who have given birth to infants with neural tube defects. Periconceptional folate supplementation has been shown to give effective protection against the development of neural tube defects. The mechanism of the prevention is as yet unknown. However, folic acid will not prevent all cases of neural tube defect. Moreover, neither determinations of periconceptional vitamin profiles (Mooij et al, 1993) nor determinations of methylmalonic acid and homocysteine levels will detect all women at risk. Anencephaly and spina bifida can be identified prenatally by detection of excessive levels of alpha-fetoprotein in amniotic fluid and maternal serum and by ultrasonographic scanning (Wilson and Rudd, 1993). Both genetic counselling and prenatal diagnosis should be offered to women who are recognized to be at high risk. Research into the underlying biology of neural tube defects is of major importance. The identification of underlying genetic defects would allow for genetic testing and better counselling of families at risk for the occurrence of a neural tube defect.

Whole-body autoradiographic disposition and plasma pharmacokinetics of 5,10-dideazatetrahydrofolic acid in mice fed folic acid-deficient or regular diets.

The effect of folic acid depletion on the tissue distribution and plasma pharmacokinetics of the oncolytic agent 5,10-dideazatetrahydrofolic acid (DDATHF) was evaluated in mice fed either folic acid-deficient or regular diets. Mice were maintained on diets for 2 weeks prior to receiving a single i.v. 30 mg/kg dose of [14C]DDATHF (tissue distribution) or DDATHF (plasma pharmacokinetics). Whole-body autoradiographic evaluation and plasma analysis for DDATHF were conducted in mice at 5 min and 6, 24, 48, 96, 120, and 168 h postdose. Radiocarbon associated with [14C]DDATHF was readily distributed to all tissues in both diet groups at the early time points and was rapidly cleared from most tissues at 24 h postdose. At the later time points, substantial amounts of radioactivity remained in liver from mice fed either diet. However, levels of radiocarbon in liver from mice fed the folic acid-deficient diet were approximately 2.5-4.2-fold the radiocarbon levels in liver from mice fed the regular diet. Similarly, plasma pharmacokinetics indicated that mice fed the folic acid-deficient diet had sustained plasma concentrations of DDATHF compared to plasma concentrations in mice fed the regular diet. These data indicated that a deficiency in dietary folic acid in mice caused increased hepatic retention of radioactivity and sustained plasma concentrations of DDATHF which are probably responsible for the observed toxicity of DDATHF in mice.

Normal and abnormal development of the lip and palate.

Largely because fusion of the lip and the palate are developmental weak points, common facial clefts may arise in a great many ways, both experimentally and in man. However, we believe that the vast majority of human clefts have similar origins with minor variations. One must always question the appropriateness of animal models. Apparently appropriate animal models are giving us considerable insight into the manner in which genetic and environmental factors alter development and how they interact with one another in the developing embryo. These studies help us understand the nature of the multifactorial threshold concept as it applies to cleft lip and cleft palate, and they emphasize the potential importance of even "minor" environmental factors in determining on which side of the threshold for clefting an individual embryo may fall.

Folate deficiency and in vitro palatogenesis II: Effects of methotrexate on rabbit palate fusion, folate pools, and dihydrofolate reductase activity.

Folate deficiency during pregnancy induced by dietary deprivation or folate antagonists is teratogenic. Methotrexate is a commonly used antifolate drug that produces congenital defects, with the craniofacial complex being especially vulnerable. The molecular action of this compound, therefore, was examined in vitro using fetal rabbit palates in an organ culture system. Dihydrofolate reductase activity was measured in these palates on the 4 days preceding fusion. There was no significant fluctuation in activity with gestational age. Methotrexate treatment of the palates in vitro resulted in significant reduction of enzyme activity and also decreased total folate concentration. However, the in vitro fusion ability of these palates was not altered by exposure to methotrexate. These results suggest that since methotrexate is a potent inhibitor of DNA synthesis and has its maximal effect on rapidly dividing systems, the peak in cellular proliferative activity in the fetal rabbit palate occurs very early in its development.

Folate deficiency and in vitro palatogenesis I: effects of a folate-deficient culture on rabbit palate fusion and folate pools.

Folate compounds are essential for nucleic acid metabolism and data from in vitro experiments have shown that folatee deficiency during pregnancy induced by dietary deprivation or folate antagonists is teratogenic. The craniofacial complex is a main target of this teratogen, with the lip and palate being especially vulnerable. The direct effect of folate deficiency on a target teratogenic tissue was, therefore, examined in vitro using fetal rabbit palates in an organ culture system. Folate pool sizes in the palates on the 4 days preceding fusion were determined by competitive binding assay. There was a sharp decrease in folate levels from 2.63 +/- 0.86 ng/mg wet wt on Day 15 of gestation to 1.23 +/- 0.35 ng/mg on Day 16 and 0.59 +/- 0.12 ng/mg on Day 17. The folat pool size on Day 14 was found to be quite variable with a mean of 1.93 ng/mg wet wt. Comparable results were obtained when the folate pool sizes were expressed as nanograms of folate per milligram of protein. The effect of a folate-deficiency culture medium on in vitro fusion and folate pool sizes was examined. The results of incubating palatal shelves excised on Days 14, 15, 16, and 17 of gestation for 5, 4, 3, and 2 days, respectively, show that a folate-depleted medium neither prevented nor delayed fusion. An examination of the palatal pool size after incubation in folate-deficient media revealed that while there was some decrease in folate levels, the palates apparently retained sufficient folate to support cellular activity.