Selective serotonin reuptake inhibitors (SSRIs) and heart defects: potential mechanisms for the observed associations.

Several epidemiological investigations have shown an association between congenital heart defects and the selective serotonin reuptake inhibitor (SSRI) class of antidepressants. At first glance this association may not seem to make biological sense, especially since, in many cases, serotonin is thought of as a neurotransmitter involved in signaling between neurons. However, serotonin also acts as a signaling molecule during embryogenesis affecting cell proliferation, migration, death, and differentiation. Serotonin may be particularly important for heart development and evidence suggests that from the time that progenitor heart cells are patterned during the establishment of laterality, to formation of the outflow tract, to myocardial cell differentiation, to septation of the heart chambers, the neurotransmitter may act as an important signaling molecule. Thus, numerous investigations have identified potential target sites where serotonin could regulate key cellular processes in cardiac development, thereby providing biological plausibility for the origin of heart defects caused by SSRIs.

The embryology of body wall closure: relevance to gastroschisis and other ventral body wall defects.

During the 3rd and 4th weeks post-fertilization (5 and 6 weeks from the last normal menstrual period [LNMP]), the human embryo is transformed from a flat disc-shaped organism into the classic shape of an embryo in the "fetal" position. This change is effected by simultaneously rolling the top layer of the disc, the ectoderm, into the neural tube and the bottom layers of the disc, the endoderm and mesoderm, into the gut tube and body wall, respectively. In this manner, the flat disc is transformed into two tubes, one dorsal to the other, surrounded by supporting structures in the body wall. If closure of the neural tube fails, then neural tube defects (NTDs), such as anencephaly and spina bifida, occur; if closure of the ventral body wall fails, then ventral body wall defects, such as ectopia cordis, gastroschisis, and bladder and cloacal exstrophy, occur. Interestingly, no known closure defects have been described for the gut tube. Note, however, that all of the closure defects that do occur have their origins early in gestation during the third and fourth weeks of development.

Embryology of neural tube development.

Neurulation is the process of forming the neural tube, which will become the brain and spinal cord. This article reviews the various cellular processes involved in neurulation and discusses possible roles of folate in this process.

Prevention of fumonisin B1-induced neural tube defects by folic acid.

BACKGROUND: The mycotoxin fumonisin B1 (FB1) inhibits sphingolipid synthesis, blocks folate transport, and has been associated with increased incidences of cancer and neural tube defects. Results from reproductive studies in animal models in vivo and in vitro have demonstrated toxicity in some cases, but no specific terata after fumonisin exposure. No information is available about folic acid's potential to protect against this toxicity. METHODS: Neurulating mouse embryos were exposed to fumonisin or folinic acid in whole embryo culture and assessed for effects on growth and development. RESULTS: Fumonisin exposure inhibited sphingolipid synthesis, reduced growth, and caused cranial neural tube defects in a dose dependent manner. Supplemental folinic acid ameliorated the effects on growth and development, but not inhibition of sphingolipid synthesis. CONCLUSION: Fumonisin has the potential to inhibit embryonic sphingolipid synthesis and to produce embryotoxicity and neural tube defects. Folic acid can reverse some of these effects, supporting results showing that fumonisin disrupts folate receptor function.

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.

Inhibitors of choline uptake and metabolism cause developmental abnormalities in neurulating mouse embryos.

BACKGROUND: Choline is an essential nutrient in methylation, acetylcholine and phospholipid biosynthesis, and in cell signaling. The demand by an embryo or fetus for choline may place a pregnant woman and, subsequently, the developing conceptus at risk for choline deficiency. METHODS: To determine whether a disruption in choline uptake and metabolism results in developmental abnormalities, early somite staged mouse embryos were exposed in vitro to either an inhibitor of choline uptake and metabolism, 2-dimethylaminoethanol (DMAE), or an inhibitor of phosphatidylcholine synthesis, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH(3)). Cell death following inhibitor exposure was investigated with LysoTracker Red and histology. RESULTS: Embryos exposed to 250-750 microM DMAE for 26 hr developed craniofacial hypoplasia and open neural tube defects in the forebrain, midbrain, and hindbrain regions. Embryos exposed to 125-275 microM ET-18-OCH(3) exhibited similar defects or expansion of the brain vesicles. ET-18-OCH(3)-affected embryos also had a distended neural tube at the posterior neuropore. Embryonic growth was reduced in embryos treated with either DMAE (375, 500, and 750 microM) or ET-18-OCH(3) (200 and 275 microM). Whole mount staining with LysoTracker Red and histological sections showed increased areas of cell death in embryos treated with 275 microM ET-18-OCH(3) for 6 hr, but there was no evidence of cell death in DMAE-exposed embryos. CONCLUSIONS: Inhibition of choline uptake and metabolism during neurulation results in growth retardation and developmental defects that affect the neural tube and face.

Susceptible periods during embryogenesis of the heart and endocrine glands.

One of the original principles of teratology states that, "Susceptibility to teratogenesis varies with the developmental stage at the time of exposure to an adverse influence" [Wilson JG. Environment and Birth Defects. New York:Academic Press, 1973]. The time of greatest sensitivity encompasses the period of organ formation during weeks 3-8 following fertilization in human gestation. At this time, stem cell populations for each organ's morphogenesis are established and inductive events for the initiation of differentiation occur. Structural defects of the heart and endocrine system are no exception to this axiom and have their origins during this time frame. Although the function and maturation of these organs may be affected at later stages, structural defects and loss of cell types usually occur during these early phases of development. Thus, to determine critical windows for studying mechanisms of teratogenesis, it is essential to understand the developmental processes that establish these organs.

Workshop to identify critical windows of exposure for children's health: cardiovascular and endocrine work group summary.

The work group on cardiovascular and endocrine effects was asked to review the current state of knowledge about children's windows of vulnerability to developmental toxicants and to recommend how that information may be used to improve risk assessment and public health. We considered differences between structural defects, where periods of vulnerability are rather well defined, and functional defects, where periods of vulnerability are quite elusive.

Embryology of the sternum.

Development of the sternum during the growth of the embryo is described. Anterior body wall defects in the thoracic region may be severe, leading to ectopia cordis, or mild, as in skin-covered sternal clefts. The embryologic basis for other sternal abnormalities, such as pectus excavatum and pectus carniatum, is not clear; however, abnormalities of rib morphogenesis and growth are the most likely causes.

Disruption of Msx-1 and Msx-2 reveals roles for these genes in craniofacial, eye, and axial development.

In mouse embryos, the muscle segment homeobox genes, Msx-1 and Msx-2 are expressed during critical stages of neural tube, neural crest, and craniofacial development, suggesting that these genes play important roles in organogenesis and cell differentiation. Although the patterns of expression are intriguing, little is known about the function of these genes in vertebrate embryonic development. Therefore, the expression of both genes, separately and together, was disrupted using antisense oligodeoxynucleotides and whole embryo culture techniques. Antisense attenuation of Msx-1 during early stages of neurulation produced hypoplasia of the maxillary, mandibular, and frontonasal prominences, eye anomalies, and somite and neural tube abnormalities. Eye defects consisted of enlarged optic vesicles, which may ultimately result in micropthalmia similar to that observed in Small eye mice homozygous for mutations in the Pax-6 gene. Histological sections and SEM analysis revealed a thinning of the neuroepithelium in the diencephalon and optic vesicle and mesenchymal deficiencies in the craniofacial region. Injections of Msx-2 antisense oligodeoxynucleotides produced similar malformations as those targeting Msx-1, with the exception that there was an increase in number and severity of neural tube and somite defects. Embryos injected with the combination of Msx-1 + Msx-2 antisense oligodeoxynucleotides showed no novel abnormalities, suggesting that the genes do not operate in a redundant manner.

Mouse embryos in culture: models for understanding diabetes-induced embryopathies and gene function.

Both the metabolic studies on diabetes and the genetic studies using antisense oligodeoxynucleotides clearly demonstrate the importance and usefulness of rodent whole embryo culture. Without this technique, these studies would be impossible and, consequently, our knowledge of both normal and abnormal development would not be as advanced as it is today. The culture system fills a unique niche in studies in the fields of developmental biology and teratology and these sciences would have been less well served without Dr. New's contribution.

NIEHS/EPA Workshops. Growth and differentiation factors.

The work group identified a number of research areas where they felt there were significant data gaps where additional research was critical to better under standing of the origins of birth defects and to developing ways for their prevention. These included: 1. Studies designed to determine the role of growth and differentiation factors during pre- and postimplantation stages of development. These investigations could include descriptive studies involving the localization and timing of genes expressed and their products, but must also emphasize and include studies involving the function of these molecules and their interactions in normal and abnormal development. 2. Studies designed to develop and utilize models for investigating normal and abnormal development. These approaches could include in vivo and in vitro techniques, such as creation of genetically defined systems and cell, organ, and whole embryo cultures. These technologies should emphasize ways to study the functions of growth and differentiation factors and the effects of environmental factors. 3. Studies designed to identify environmental agents and their targets in embryonic, extraembryonic, and maternal tissues that may play a role in producing developmental abnormalities through peturbations of growth and differentiation factors. 4. Studies designed to determine cellular and molecular mechanisms for protection and recovery from environmental insults.

Regenerating (reg) and insulin genes are expressed in prepancreatic mouse embryos.

The pancreatic regenerating (reg) gene is proposed to be involved in pancreatic beta-cell growth. Up- or down-regulation of reg gene expression has been shown to parallel variations in beta-cell mass and function in the adult pancreas. In several species at least two nonallelic reg genes have been identified. In this study we investigated the expression of each individual reg gene (reg-I and reg-II) during embryogenesis in the mouse. Single mouse embryos were harvested at 8.5, 9, 10, and 12 days of development, homogenized and subjected individually to reverse transcription (RT)-PCR, with a single primer pair to amplify both reg-I and -II mRNAs. Southern blot analysis of the RT-PCR products revealed the presence of reg mRNA at day 9 of embryogenesis, just before the beginning of pancreatic organogenesis. Slot-blot analysis with internal oligonucleotide probes that specifically recognize reg-I or -II sequences demonstrated that only reg-I mRNA was present in day 9 and day 10 prepancreatic embryos. Reg-II mRNA was not detected until day 12, a stage corresponding to late organogenesis. RT-PCR for insulin mRNA from the same samples used for the amplification of reg mRNA showed that the earliest insulin expression occurred at day 8.5, and coincided with the onset of reg-I expression. Hybridization with gene-specific oligonucleotide probes revealed that only insulin-II mRNA was detectable at this time. Insulin-I mRNA was not detectable until day 12 and coincided with early reg-II expression. These results suggest that the two nonallelic reg genes and the two insulin genes are expressed differentially during early embryogenesis. Differential expression of reg-I and -II suggests that they may be induced by different and independent stimuli and have distinct functions.

Mouse embryonic cardiac metabolism under euglycemic and hypoglycemic conditions.

Children of mothers with insulin-dependent diabetic mothers (IDDM) have a 2-4 times higher incidence of congenital birth defects as compared to the general population, including cardiac abnormalities, of unknown etiology. Using rodent embryos to explore potential teratogenic factors of the altered IDDM metabolism, it has been shown that exposure to hypoglycemia in vitro results in a variety of defects, including cardiac malformations. Since pregnant diabetics experience frequent episodes of low blood glucose, it was hypothesized that hypoglycemia may play a role in the generation of heart abnormalities seen in children born to IDDM mothers. Several studies have indicated that during embryogenesis the heart is dependent on glucose for energy production such that under hypoglycemic conditions, insufficient amounts of ATP may be produced resulting in abnormalities. To test this hypothesis, cardiac ATP content was monitored in D10-D12 (plug day = D1) hearts. In addition, the contribution of glycolysis and the Krebs cycle to ATP production was monitored. D10 hearts exposed to euglycemic control conditions were found to be primarily dependent on glycolysis for ATP production from glucose before switching to the Krebs cycle and oxidative phosphorylation for energy production from this substrate on D11. Exposure to hypoglycemia did not alter the timing of this maturation process or deplete cardiac ATP content. However, cardiac lactate levels increased approximately twofold in the presence of hypoglycemia on d10. Since increased concentrations of lactate are harmful to many tissues and have been shown to be detrimental to the adult rat heart, lactic acidosis may explain the origin of cardiac defects produced by hypoglycemia, and not a deficiency of ATP.

Effects of cocaine and cocaine metabolites on mouse developmentin vitro.

The use of cocaine use has been associated with adverse developmental effects in humans, and cocaine administration produces developmental toxicity in animal models. However, whether the adverse effects produced during organogenesis are due directly to the effects of cocaine or its metabolites remains to be established. This study was therefore undertaken to compare the morphological effects of cocaine and its metabolites, ecgonine, benzoylecgonine (BE) and ecgonine methyl ester (EME) in whole embryo culture (WEC) using early somite stage ICR mice. Cocaine produced a concentration-dependent induction of defects including effects on craniofacial development such as neural tube closure defects (NTDs). Concentrations of cocaine of 51.4 mum or more produced dysmorphogenesis and 100% of the embryos exhibited NTDs at 441 mum. EME also induced defects at concentrations of 400 mum or above. Neither ecgonine nor BE altered embryogenesis at concentrations of 2000 mum or less. The incidence of cocaine-induced NTDs was dependent on the length of exposure to cocaine. At 294 mum, exposures of 3 hr or more were required to alter development when evaluated at the end of a 24-hr culture period. Lower cocaine concentrations required longer exposure periods (6 or 12 hr) to produce dysmorphogenesis. The incidence of NTDs appears to follow the area under the concentration time curve and is not solely dependent on the peak cocaine concentration in the medium. Exposure of conceptuses to a combination of cocaine and EME produced a high incidence of NTDs. These results suggest that the concentration of cocaine or EME required to induce NTDsin vitro is higher than the teratogenic concentrationin vivo. Additionally, the time required for high concentrations of cocaine to induce NTDs is longer than the serum half-life of cocaine reportedin vivo following a single administration. Thus, NTDs produced by cocaine administration appear not to be due solely to the effect of cocaine or its metabolites on the conceptus but may involve effects on extraembryonic and/or maternal tissues as well.

Effects of cocaine administration during early organogenesis on prenatal development and postnatal growth in mice.

Cocaine use has been associated with adverse developmental effects in humans. However, clinical reports both confirm and deny an association between cocaine use and malformations. Similarly, differences in species and strain, as well as route and timing of cocaine administration, have added to the difficulties in determining the teratogenicity of cocaine in animal models. This study was undertaken to compare the effects of dose, route, and timing of cocaine administration in ICR mice during early organogenesis. A single intraperitoneal (ip) administration of cocaine ( > or = 60 mg/kg) on Day 9 of gestation (plug day = 1) produced maternal lethality. The predominant developmental effect of cocaine administration was an increase in the percentage of litters exhibiting an enlarged renal pelvis. Despite a high incidence of affected pups at these doses, the enlargement was not severe. These results, in agreement with previous reports, provide further evidence that the developing urogenital system is sensitive to cocaine administration. When cocaine was administered using a subcutaneous route, pup weights were greater and the incidence of enlarged renal pelvis was lower than when an ip route was used. To better mimic human binge cocaine abuse, the toxicity of a "split dose" was determined. A 60 mg/kg dose was administered using one administration of 60 mg/kg, two treatments of 30 mg/kg, or three administrations of 20 mg/kg with 1 hr separating the treatments. The incidence of enlarged renal pelvis was similar when cocaine was administered as one or two but was decreased when cocaine was administered as three treatments. Both the route and split-dose studies suggest that high-peak serum concentrations are required to perturb development. There were no differences in the incidence or severity of enlarged renal pelvis when cocaine was administered on Day 8, 9, or 10 or on all 3 days of gestation. This suggested that the increase in enlarged renal pelvis may not be a specific teratogenic effect of cocaine administration but may be a delay of normal development induced by cocaine exposure during this early period of organogenesis. To address this hypothesis, cocaine was administered on Day 9 using an ip route and the pups were allowed to be naturally born. In pups whose mothers received cocaine there was an increase in postnatal deaths and a trend toward a reduction in pup body weight/litter at Postnatal Day 21. However, when renal morphology was assessed on Postnatal Day 21 no abnormal kidneys were seen. This supports the hypothesis that enlarged renal pelvis produced by cocaine administration during early organogenesis represents a developmental delay and not a persistent teratogenic defect. These studies suggest that high peak cocaine concentrations are required to delay normal kidney morphogenesis in mice.

Antisense targeting of engrailed-1 causes abnormal axis formation in mouse embryos.

Antisense oligonucleotide targeting of engrailed-1 (En-1) in early somite mouse embryos resulted in reduced En protein levels and produced abnormalities of the brain, face, and heart and shortening of the embryonic axis (caudal dysgenesis). Defects of the brain and limbs were consistent with abnormalities observed in null mutant mice described by other investigators. Abnormalities of the face and heart may be related to alterations in neural crest cells. Caudal dysgenesis suggested a role for En-1 in axis formation and this hypothesis was supported by results showing that En-1 protein and mRNA are present in the primitive streak. Thus, in addition to participating in the signaling pathway for brain and limb development, En-1 appears to play a role in patterning the embryonic axis.