Respiratory changes in 9-methyl PGA teratogenesis

Cellular respiration was accurately measured following dispersion of rat embryos too large for reliable assessments in the intact condition. The technique used in this study for cell dispersion of day 13 embryos effectively eliminated restrictions to oxygen diffusion caused by thickness of the intact animals and permitted valid measurements of oxygen consumption in these embryos. Significantly decreased rates of oxygen consumption were measured by the Warburg technique in such embryonic rat cells following a teratogenic maternal folate deficiency and folate-antagonism using 9-methyl pteroyl-glutamic acid. These results suggest an association between altered embryonic oxidative metabolism and induction of malformations.     

The genetic background of the curly tail strain confers susceptibility to folate‐deficiency‐induced exencephaly

BACKGROUND : Suboptimal maternal folate status is considered a risk factor for neural tube defects (NTDs). However, the relationship between dietary folate status and risk of NTDs appears complex, as experimentally induced folate deficiency is insufficient to cause NTDs in nonmutant mice. In contrast, folate deficiency can exacerbate the effect of an NTD‐causing mutation, as in splotch mice. The purpose of the present study was to determine whether folate deficiency can induce NTDs in mice with a permissive genetic background which do not normally exhibit defects. METHODS : Folate deficiency was induced in curly tail and genetically matched wild‐type mice, and we analyzed the effect on maternal folate status, embryonic growth and development, and frequency of NTDs. RESULTS : Folate‐deficient diets resulted in reduced maternal blood folate, elevated homocysteine, and a diminished embryonic folate content. Folate deficiency had a deleterious effect on reproductive success, resulting in smaller litter sizes and an increased rate of resorption. Notably, folate deficiency caused a similar‐sized, statistically significant increase in the frequency of cranial NTDs among both curly tail (Grhl3 mutant) embryos and background‐matched embryos that are wild type for Grhl3. The latter do not exhibit NTDs under normal dietary conditions. Maternal supplementation with myo‐inositol reduced the incidence of NTDs in the folate‐deficient wild‐type strain. CONCLUSIONS : Dietary folate deficiency can induce cranial NTDs in nonmutant mice with a permissive genetic background, a situation that likely parallels gene‐nutrient interactions in human NTDs. Our findings suggest that inositol supplementation may ameliorate NTDs resulting from insufficient dietary folate. Birth Defects Research (Part A), 2010. © 2009 Wiley‐Liss, Inc.     

Malabsorption of protein bound vitamin B12.

Patients with subnormal serum vitamin B12 concentrations were tested for absorption of protein bound vitamin B12 and compared with controls. Absorption of the protein bound vitamin appeared to decrease with increasing age in healthy subjects. Differences between the result of this test and the result of the Schilling test in patients who had undergone gastric surgery were confirmed; such differences were also seen in some patients who had iron deficiency anaemia, an excessive alcohol intake, or folate deficiency. Defective absorption was also found in six patients with an adequate dietary intake of vitamin B12, normal Schilling test results, low serum vitamin concentrations, and tissue changes responding to treatment with vitamin B12. Malabsorption of the vitamin from protein bound sources, which is not detected by the Schilling test, may produce vitamin B12 deficiency of clinical importance.     

Interactions of cadmium and zinc in cultured rat embryos

Rat embryos were cultured in serum taken from animals dosed with cadmium, or serum with cadmium added $\text{in}$$\text{vitro}$ in the presence or absence of additional zinc. Embryos explanted at day ten and grown in serum taken from animals sooner than 4 h after dosing had a reduced DNA content after 24 h culture. In one-hour serum, the yolk sac had become thick and brittle. Zinc ameliorated the effects but had no stimulatory effect on post eight-hour serum when serum zinc levels were at their lowest. The hypothesis that cadmium induces a maternal zinc deficiency sufficient to cause teratogenic changes could not be sustained. Embryos explanted at nine days were much more susceptible to cadmium added $\text{in}$$\text{vitro}$ than ten-day embryos. The principal anomaly, apart from a reduced DNA content, was a thickening of the yolk sac similar to that seen in embryos grown in serum taken from animals one hour after cadmium dosing. Addition of zinc to the medium prevented both of these effects. The suggestion is made that the cadmium-induced dysgenesis of the yolk sac precludes appropriate embryonic nutrition.     

Improved development of rat embryos in culture during the period of craniofacial morphogenesis

To study the mammalian craniofacial development, the culture conditions of rat whole embryo during the period of major craniofacial morphogenesis were examined. The improved rotating apparatus which is gassed continuously was used. Rat embryos explanted at 11.5 days (plug day 0) developed in vitro for up to 72 hr, that is, throughout the period of major craniofacial morphogenesis, and cultured embryos showed normal facial formation. The medium was equilibrated with a gas mixture of 95% 02, 5% CO2. The 100% rat serum improved the protein content of embryos cultured for 48 hr compared with the medium consisting of 50% rat serum and 50% Tyrode solution, although somite number was not altered. Furthermore, 100% rat serum containing 2 mg/ml glucose was the best medium for supporting growth of embryos when it was measured by protein content. Thus, the best culture medium was pure rat serum containing 50 units/ml penicillin, 50 micrograms/ml streptomycin, and 2 mg/ml glucose. Protein content, body weight, craniofacial formation, and somite number of embryos cultured for 48 hr with continuous gassing were much better than those cultured with noncontinuous gassing.     

Phenytoin teratogenicity and effects on embryonic and maternal folate metabolism [published errtum appears in Teratology 1986 Dec;34(3):487

It has been postulated that the mechanism of teratogenicity of the anticonvulsant drug phenytoin (PHT), is via a deficiency of folic acid. To test this hypothesis, Swiss Webster mice were administered PHT in the diet prior to and throughout gestation. Animals received a daily dose of approximately 75 mg/kg body weight. The maternal plasma PHT levels were within the therapeutic range for this drug. This dose increased the incidence of malformations, primarily cleft palate, in the absence of embryolethality. There was a decrease in maternal plasma folate levels on day 12 of gestation but no effect on days 10 and 18. Even in the presence of a maternal folate deficiency on day 12, PHT had no effect on total embryonic folate levels on days 10, 12, and 14. Previous experiments have demonstrated that PHT decreases activity of the enzyme 5,10-methylenetetrahydrofolate reductase in the liver of nonpregnant Swiss Webster mice. Data from the current study indicate that this enzyme activity is also decreased in hepatic tissue of pregnant mice, but it is not altered in embryos on the days examined. These data show that a teratogenic dose of PHT affects maternal folate metabolism. However embryonic folate metabolism, when measured in total embryos, was not affected.     

Development of rat embryos in culture media containing different concentrations of normal and diabetic rat serum.

In vitro culture of rodent embryos has been extensively used in the search for teratologic agents, with possible relevance to diabetic pregnancy. However, the high concentrations of rat serum added to the culture medium (approximately 75%) have raised concern that the teratogenic effects of some compounds may be attenuated or masked in this culture system and thereby forced the addition of pharmacological concentrations of the compounds (e.g., D-glucose and beta-hydroxybutyrate) to the medium. This issue has been examined in the present study where the effects of different concentrations of rat serum on growth and differentiation of rat embryos were recorded in cultures supplemented with increased concentrations of D-glucose and beta-hydroxybutyrate. The embryonic development was also evaluated after culture in medium supplied with serum from diabetic rats. Compared with normal rat serum, the diabetic serum had an elevated glucose concentration as well as markedly increased levels of triglycerides and branched amino acids, indicating a potentially rich supply of major nutrients for the cultured embryos. Lowering the serum concentration in the culture medium from 80% to 50% yielded progressively retarded embryonic growth but no increased rate of other morphological malformations. At 40% serum concentration, however, there was a sharp rise in the incidence of somatic malformations, in addition to the prevailing growth retardation. When the embryonic growth and development were compared at 50% and 80% serum concentrations, increased D-glucose or beta-hydroxybutyrate concentrations caused similar degrees of embryonic dysmorphogenesis. Also, the uptake of each compound by the embryos exposed to elevated levels of the two agents were similar in 50% and 80% serum cultures. There was, therefore, no protection against the teratogenic and growth-retarding effects of increased D-glucose or beta-hydroxybutyrate offered by high serum concentrations in the culture medium (i.e., 80% vs. 50%). Embryos cultured in 50% or 80% diabetic rat serum at 30 mmol/L or 50 mmol/L D-glucose concentration showed similar rates of somatic malformations as did embryos exposed to the same proportion of normal rat serum at similar glucose concentrations. By contrast, the diabetic rat serum amplified the general retarding effects of high D-glucose levels, yielding lower protein levels and somite numbers in embryos from diabetic serum culture than in embryos cultured in normal rat serum.(ABSTRACT TRUNCATED AT 400 WORDS)     

叶酸缺乏致胎鼠宫内发育迟缓及肝脏胰岛素生长因子系统表达变化

目的：叶酸是一种水溶性B族维生素,在体内氨基酸与核苷酸代谢中起重要作用,是胎儿生长发育所必须的营养素。本文通过建立叶酸缺乏的孕鼠模型,探讨叶酸缺乏对胎鼠宫内发育的影响,并研究胎鼠肝脏组织中胰岛素生长因子（IGF）系统的表达变化。方法：雌性C57BL／6J小鼠叶酸缺乏组6只、正常对照组6只,分别饲以不舍叶酸和含2mg叶酸／kg的纯合饲料。四周后与雄鼠交配,于怀孕第13．5天（13．5dpc）对孕鼠剖腹取胎,观察和评价胎鼠发育指标,并对宫内发育迟缓（IUGR）比率进行统计。用Real-timePCR法检测胎鼠肝脏组织中胰岛素生长因子I（IGFI）、胰岛素生长因子I受体（IGFIR）、胰岛素生长因子II（IGFII）、胰岛素生长因子II受体（IGFIIR）、胰岛素生长因子结合蛋白1（IGFBP-1）和胰岛素生长因子结合蛋白3（IGFBP-3）mRNA的相对表达水平。结果：叶酸缺乏组雌鼠合笼前每日体重增长量降低,13．5dpc胎鼠吸收胎和死胎比率升高,胎重下降,IUGR比率显著升高,差异有统计学意义（P〈0．05）;叶酸缺乏组胎鼠肝脏组织中IGFII和IGFIIRmRNA的相对表达水平均低于正常对照组（P〈0．05）,IGFI、IGFIR、IGFBP-1和IGFBP-3mRNA的相对表达水平两组间没有差异（P〉0．05）。结论：叶酸缺乏会导致小鼠孕中期胎鼠IUGR比率升高及胎肝IGFII和IGFIIRmRNA的表达水平降低,提示叶酸缺乏对IGF系统基因的调控,可能与胎鼠I-UGR发生机制有关。     

Methylenetetrahydrofolate reductase deficiency and low dietary folate increase embryonic delay and placental abnormalities in mice

BACKGROUND: Despite extensive research on mild methylenetetrahydrofolate reductase (MTHFR) deficiency and low dietary folate in different disorders, the association of these metabolic disturbances with a variety of congenital defects and pregnancy complications remains controversial. In this study we investigated the effects of MTHFR and dietary folate deficiency at 10.5 days post coitum (dpc) in our mouse model of mild MTHFR deficiency. METHODS: Mthfr +/+ and +/? female mice were fed a control or folic acid–deficient diet for 6 weeks, then mated with Mthfr +/? males. At 10.5 dpc, embryos were examined and placentae were collected for histologic evaluation. RESULTS: Maternal MTHFR and folate deficiencies resulted in increased developmental delays and smaller embryos. We also observed a low frequency of a variety of embryonic defects in the experimental groups, such as neural tube, heart looping, and turning defects; these results mimic the low incidence and multifactorial nature of these anomalies in humans. Folate‐deficient mice also had increased embryonic losses and severe placental defects, including placental abruption and disturbed patterning of placental layers. Folate‐deficient placentae had decreased ApoA‐I expression, and there was a trend toward a negative correlation between ApoA‐I expression with maternal homocysteine concentrations. CONCLUSIONS: Our study provides biological evidence linking maternal MTHFR and dietary folate deficiencies to adverse pregnancy outcomes in mice. It underscores the importance of folate not only in reducing the incidence of early embryonic defects, but also in the prevention of developmental delays and placental abnormalities that may increase susceptibility to other defects and to reproductive complications. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

The development and role of international biological reference materials in the diagnosis of anaemia

Anaemia is a major global health problem. Although the main cause is iron deficiency, anaemia also results from other nutritional deficiencies (folate and vitamin B₁₂), haemolytic disorders including haemoglobinopathies, and bone marrow disorders. Accurate diagnosis of anaemia is dependent on reliable diagnostic tests and reference ranges, which in turn are dependent on effective standardisation. Standardisation is achieved through the availability of reference materials and reference measurement procedures. International biological reference materials have therefore been developed to standardise and control diagnostic tests for anaemia for a diverse range of analytes including total haemoglobin and haemoglobin types, ferritin, the serum transferrin receptor, serum vitamin B₁₂ and folate, whole blood folate, and alloantibodies which mediate immune haemolytic anaemia.     

Iron in bone marrow

In the bone-marrow, non-haemoglobin iron can predominantly be found in the reticulum. Slight granules containing iron can also be observed in parts of erythroblasts by means of the Berlin blue reaction. These cells are called sideroblasts. In chemical respect, non-haemoglobin iron consists of ferritin soluble in water and haemosiderin insoluble in water. Erythroblasts will only take their iron from plasma transferrin. For the most part, this iron uptake is being regulated by erythropoietin adapting erythropoiesis to the oxygen requirements of the tissue. The iron contained in erythroblasts is predominantly utilized for haemoglobin synthesis in these cells. A slight part is being taken up by ferritin. The bone-marrow reticulum will phagocytise aged erythrocytes and store liberated iron as ferritin and haemosiderin. Part of the iron is being delivered again to plasma transferrin. With constant serum iron level the liberation of iron from the reticulo-endothelial tissue must correspond to the iron uptake by erythropoiesis. The absence of iron capable of being coloured in the bone-marrow reticulum is considered to be a reliable parameter of iron deficiency. It enables the diagnosis of iron deficiency anaemia to be made even in those patients with serum iron level and a total iron binding capacity lying within the normal range and no hypochromia of erythrocytes being present. It enables iron deficiency anaemia to be separated from sideropenic anaemia with reticulo-endothelial siderosis in differential-diagnostic manner. Even in patients with sideroblastic anaemia, iron colouring of bone-marrow smears is required for ensuring the diagnosis. Recently, a separation has also been made for idiopathic anaemia with abnormal sideroblasts. In these patients there is an increased risk for acute leukemia to develop.     

Pigmentation in Megaloblastic Anaemia Associated with Pregnancy and Lactation

Generalized skin pigmentation in five African women with megaloblastic anaemia in the postnatal period was associated with low serum folate levels, as distinct from vitamin B₁₂ deficiency. It is suggested that the occurrence of pigmentation in both folate and vitamin B₁₂ deficiency may reflect a common abnormality of metabolism.     

Polymorphism C1420T of Serine hydroxymethyltransferase gene on maternal risk for Down syndrome

Recent researches have investigated the factors that determine the maternal risk for Down syndrome (DS) in young woman. In this context, some studies have demonstrated the association between polymorphisms in genes involved on folate metabolism and the maternal risk for DS. These polymorphisms may result in abnormal folate metabolism and methyl deficiency, which is associated with aberrant chromosome segregation leading to trisomy 21. In this study, we analyzed the influence of the polymorphism C1420T in Serine hydroxymethyltransferase (SHMT) gene on maternal risk for DS and on metabolites concentrations of the folate pathway (serum folate and plasma homocysteine and methylmalonic acid). The study group was composed by 105 mothers with DS children (case group) and 185 mothers who had no children with DS (control group). The genotype distribution did not show significant statistical difference between case and control mothers (P?=?0.24) however a protective effect between genotypes CC (P?=?0.0002) and CT (P?<?0.0001) and maternal risk for DS was observed. Furthermore, the SHMT C1420T polymorphism (rs1979277) does not affect the concentration of metabolites of folate pathway in our DS mothers. In conclusion, our data showed a protective role for the genotypes SHMT CC and CT on maternal risk for DS. The concentrations of metabolites of folate pathway did not differ significantly between the genotypes SHMT.     

Teratogenic effect of the cholesterol synthesis inhibitor AY 9944 on rat embryos in vitro.

AY 9944 [trans-1,4-bis(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride] is an amphiphilic cationic molecule. This chemical is an established inhibitor of cholesterol synthesis and is teratogenic in rats. The mechanisms of this teratogenicity remain to be clarified. This study used cultured rat whole embryos to ascertain whether AY 9944 had a direct effect on embryos, or whether its action was indirect, via the maternal cholesterol metabolism. Four experimental conditions were investigated: (A) controls; (B) 10 day untreated embryos were cultured in serum of treated rats; (C) 10 day untreated embryos were cultured in serum containing added AY 9944 (0-1,000 micrograms/ml); and (D) 10 day embryos from females treated on day 4 of gestation were cultured in normal serum. In group B there was no growth retardation; some slight nonspecific abnormalities were not significant. In group C, direct addition of AY 9944 to culture medium retarded growth and differentiation in a dose-dependent manner. No malformation was observed, but histological examinations showed numerous areas of cell necrosis, especially in the CNS. In group D, not only was growth retardation observed, but also characteristic malformations of AY 9944 teratogenesis, including pituitary agenesis. These results show that AY 9944 teratogenicity is initiated prior to day 10.     

Tissue-specific distribution of aberrant DNA methylation associated with maternal low-folate status in human neural tube defects

This study compares the density and tissue-specific distribution of 5-methyl cytosine (5mC) in genomic DNA from human fetuses with or without neural tube defects (NTD) and examines whether low maternal serum folate is a possible correlate and/or risk factor for NTD. The results demonstrate significant hypomethylation of brain genomic DNA in NTD fetuses relative to controls (P<.01), as well as relative hypermethylation of skin and heart in NTD fetuses. In normal fetuses, the level of 5mC in liver genomic DNA decreased from fetal week 18 to 28 and increased over the same developmental period in kidney genomic DNA, but these trends were absent in genomic DNA from NTD fetuses. Mean maternal serum folate was significantly lower in NTD fetuses than in controls (P<.01), and maternal serum folate correlated with density of 5mC in genomic brain DNA from NTD fetuses (r=0.610). The results indicate that aberrant DNA methylation in NTD may be due to maternal folate deficiency and may be involved in the pathogenesis of NTD in humans.     

Endogenous folate of normal fibroblasts using high-performance liquid chromatography and modified extraction procedure

The endogenous levels of the various folate monoglutamate compounds in cultured human fibroblasts were determined using high-performance liquid chromatography for the separation of folate monoglutamate. Endogenous folates were converted to monoglutamate forms using conjugase enzyme present in rat serum and incubation was carried out at pH 6.5. This minimized folate coenzyme interconversion during processing. Using methanol for precipitation of protein instead of heat minimized degradation of labile folates. Recovery of all folates except 10-formyltetrahydrofolic acid (10-CHO H4PteGlu) using this procedure was more than 90%. Disruption of cells by boiling appeared to cause less postextraction changes of cell folates than did freezing and thawing or sonication. When heat to release endogenous folate, conjugase treatment with rat serum at pH 6.5, and precipitation of protein with methanol were used, more than half of the intracellular folate of normal fibroblasts in confluent growth was 5-methyltetrahydrofolic acid (5-CH3 H4PteGlu), and 10-CHO H4PteGlu and tetrahydrofolic acid (H4PteGlu) comprised 29 and 6%, respectively.     

Decreased TGF-beta1 and IGF-1 protein expression in rat embryo skull bone in folic acid-restricted diet

Folic acid deficiency during conception up to the end of the third month of gestation is believed to play the most important factor in neural tube defects (NTDs). However, the exact molecular mechanism remains to be elucidated. It has been suggested that transforming growth factor-beta (TGF-beta1) and insulin-like growth factor-1 (IGF-1) play a critical role in supporting bone formation. Therefore, folic acid deficiency may contribute to NTD occurrence via decreased TGF-beta1 and IGF-1 expression. This study aimed to determine the correlation between folic acid deficiency and the expression of TGF-beta1 and IGF-1 in rat skull bone. Thirty female Sprague-Dawley rats were divided into three groups. Purified diet containing 5 (restricted), 15 (low) and 30 microg (normal) of folic acid was given to the first, second and third groups, respectively. At 16 weeks of a given diet, blood samples were taken to examine folic acid (folate immunoassay method), TGF-beta1 and IGF-1 (enzyme-linked immunosorbent assay method) levels. After forced mating, on the 18th-19th day of gestation (E18-19), the pregnant rats were subjected to hysterectomy. The skull bone samples of E18-19 rats were taken to examine the TGF-beta1 and IGF-1 protein expression by immunohistochemistry. The folic acid-restricted diet (5 microg) resulted in decreased serum TGF-beta1 and IGF-1 levels. Furthermore, protein expression of TGF-beta1 and IGF-1 in E18-19 rat skull bones was also significantly lower in the folic acid-restricted diet than in the normal diet. Folic acid deficiency could result in reduction of TGF-beta1 and IGF-1 protein levels and might contribute to formation of defects in the skull bone as observed in mengingocele patients.     

Cardiovascular abnormalities in Folr1 knockout mice and folate rescue

BACKGROUND: Periconceptional folic acid supplementation is widely believed to aid in the prevention of neural tube defects (NTDs), orofacial clefts, and congenital heart defects. Folate-binding proteins or receptors serve to bind folic acid and 5-methyltetrahydrofolate, representing one of the two major mechanisms of cellular folate uptake. METHODS: We herein describe abnormal cardiovascular development in mouse fetuses lacking a functional folate-binding protein gene (Folr1). We also performed a dose-response study with folinic acid and determined the impact of maternal folate supplementation on Folr1 nullizygous cardiac development. RESULTS: Partially rescued preterm Folr1(-/-) (formerly referred to as Folbp1) fetuses were found to have outflow tract defects, aortic arch artery abnormalities, and isolated dextracardia. Maternal supplementation with folinic acid rescued the embryonic lethality and the observed cardiovascular phenotypes in a dose-dependant manner. Maternal genotype exhibited significant impact on the rescue efficiency, suggesting an important role of in utero folate status in embryonic development. Abnormal heart looping was observed during early development of Folr1(-/-) embryos partially rescued by maternal folinic acid supplementation. Migration pattern of cardiac neural crest cells, genetic signals in pharyngeal arches, and the secondary heart field were also found to be affected in the mutant embryos. CONCLUSIONS: Our observations suggest that the beneficial effect of folic acid for congenital heart defects might be mediated via its impact on neural crest cells and by gene regulation of signaling pathways involved in the development of the pharyngeal arches and the secondary heart field.     

Zinc-deficient rat embryos have increased caspase 3-like activity and apoptosis

Caspase activity is a hallmark of apoptosis. Given that maternal zinc (Zn) deficiency results in apoptosis in the rat embryo, we assessed caspase activity in Zn-deficient embryos. Mid-gestation rat embryos were collected from dams fed either a Zn-deficient (0.5 Zn/g) diet ad libitum, or a Zn-adequate (25 microg Zn/g) diet ad libitum or pair fed to dams fed the Zn-deficient diet. Embryos from dams fed the Zn-adequate diet had a normal level of cell death, while embryos from the dams fed the Zn-deficient diet had either increased or normal levels of cell death. Zn-deficient embryos displaying increased cell death had increased caspase activity. Embryos with normal levels of cell death, regardless of maternal diet, had similar caspase activities. Thus, Zn-deficiency-induced apoptosis in vivo is associated with increased caspase activity.