In May of 2006, the Hrubec group suddenly began to observe neural tube defects (NTDs) in embryos of untreated control mice. Unintentional exposure to a teratogenic agent in tap water was identified as the cause. We aimed to identify the contaminant, but first we demonstrated that the NTDs were pathological being present on both gestational day 9 and 10. We also found that a second species, rats, developed NTDs when exposed to tap waters. Disinfection by-products (DBPs) arise when natural organic matter in municipal water sources reacts with disinfectants used in the water treatment process. Purge and trap gas chromatography-mass spectrometry (PT GC-MS) and animal exposure studies were used to determine if the teratogenic contaminant was a DBP. Since the distribution pattern of DBPs did not match the distribution pattern of NTDs, we concluded that a DBP was not likely to be responsible for the observed malformations. Pharmaceuticals and personal care products have emerged as ubiquitous contaminants of ground and surface waters, and have been detected in drinking water. In order to analyze for these compounds, we submitted different water samples to a commercial water analysis lab (AXYS Analytical Services, Sidney, BC, Canada). Several pharmaceuticals were identified in a number of samples, including a known teratogenic drug used to treat mood disorders and seizures: carbamazepine. Further analysis for carbamazepine was conducted in-house. Carbamazepine was found in several ground, surface, and tap waters, at various concentrations. To establish whether or not carbamazepine was responsible for NTDs in our mice, we conducted 2 dosing studies. Carbamazepine was provided to mice at concentrations detected in tap water, as well as approximately 2 x and 1000 x that concentration. Both studies found no significant differences in NTD rates among the dose groups. As no dose effect was observed, we concluded that CBZ was not directly responsible for the malformations. The identity of the teratogenic contaminant is not known at this time, but is unlikely to be a DBP or low concentrations of the pharmaceutical carbamazepine. / Master of Science

Neural tube defects (NTDs) are a world health issue, affecting approximately 1 in every 1000 live births. These congenital defects arise from the improper closure of the neural tube during development, resulting in significant, life-threatening malformations of the central nervous system. Although it has been observed that supplementing women of child-bearing age with folates greatly decreases the chances of having an NTD affected baby, unfortunately these defects still occur. It is accepted that these complex disorders arise from a combination of genetic, environmental, and dietary influences. One such dietary influence is the one-carbon metabolism metabolite, homocysteine. Homocysteine is a byproduct of methylation reactions in the cell that exists in an inverse homeostasis with folate. Homocysteine can also undergo a transformation that allows it to then react with exposed lysine or cysteine residues on proteins, in a process known as N-homocysteinylation or S-homocysteinylation respectively. High levels of homocysteine have been long correlated with many disease states, including NTDs. One potential mechanism by which homocysteine confers its negative effects is through protein N-homocysteinylation. Here, a novel and high-throughput assay for N-homocysteinylation determination is described. This assay is shown to be accurate with mass spectrometry then shown to be biologically relevant using known hyperhomocysteinemia mouse models. This assay was then applied to a cohort of neural tube closure staged mouse embryos with two different genetic mutations that have previously been shown to predispose mice to NTDs. The genotypes explored here are mutations to the LRP6 gene and the Folr1 gene, both of which have been described as folate-responsive NTD mouse models. It was seen that maternal diet and embryonic genotype had the largest influence on the developmental outcome of these embryos; however, the inverse relationship between folate and homocysteine seemed to be established at this early time point, emphasizing the importance of the balance in one-carbon metabolism. One of these genes, LRP6, was then explored in a human cohort of spina bifida cases. Four novel mutations to the LRP6 gene were found and compared to the mouse model used in the previous study. One of the mutations found in the human population was seen to mimic that of the LRP6 mouse model, therefore expanding the potential of this NTD model. / text

Yeung Sau-Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 139-172). / Abstracts in English and Chinese. / Title page --- p.i / Acknowledgements --- p.ii / Table of Content --- p.iv / List of Figures --- p.viii / List of Graphs --- p.x / List of Tables --- p.xi / Abbreviations --- p.xiv / Abstract --- p.xv / Chinese Abstract --- p.xvii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Diabetes Mellitus --- p.2 / Chapter 1.1.1 --- Type 1 diabetes mellitus --- p.3 / Chapter 1.1.2 --- Type 2 diabetes mellitus --- p.5 / Chapter 1.1.3 --- Maturity onset diabetes of the young (MODY) --- p.6 / Chapter 1.1.4 --- Gestational diabetes --- p.7 / Chapter 1.2 --- Effect of Diabetes on Pregnancy --- p.9 / Chapter 1.3 --- Suggested Causes of Diabetic Embryopathy --- p.10 / Chapter 1.3.1 --- Glucose --- p.10 / Chapter 1.3.2 --- Ketone bodies --- p.11 / Chapter 1.3.3 --- Somatomedin inhibitors --- p.12 / Chapter 1.3.4 --- TNF-α --- p.12 / Chapter 1.3.5 --- Oxidative stress --- p.13 / Chapter 1.4 --- Animal Model of Diabetes --- p.15 / Chapter 1.4.1 --- Chemically-induced --- p.15 / Chapter 1.4.2 --- Mutants --- p.17 / Chapter 1.5 --- Gene-teratogen Interaction under Diabetic Pregnancy --- p.19 / Chapter 1.6 --- Strategy of the Thesis --- p.21 / Chapter Chapter 2 --- General Materials and Methods --- p.24 / Chapter 2.1 --- Mouse Maintenance and Mating Method --- p.25 / Chapter 2.2 --- Induction of Diabetes --- p.25 / Chapter 2.3 --- Preparation of All-trans Retinoic Acid --- p.26 / Chapter 2.4 --- Dissection of Embryos --- p.26 / Chapter 2.5 --- DNA Extraction from Yolk Sac for Genotyping --- p.27 / Chapter 2.6 --- Genotyping of Embryos --- p.28 / Chapter 2.7 --- Preparation of RNA Probes for In Situ Hybridization --- p.29 / Chapter 2.7.1 --- Mini-scale preparation of plasmid DNA --- p.29 / Chapter 2.7.2 --- Linearization of plasmid DNA --- p.30 / Chapter 2.7.3 --- In vitro transcription --- p.31 / Chapter 2.8 --- Whole Mount In Situ Hybridization --- p.33 / Chapter 2.8.1 --- Fixation and dehydration of embryos --- p.33 / Chapter 2.8.2 --- Hybridization --- p.33 / Chapter 2.8.3 --- Post-hybridization wash --- p.34 / Chapter 2.8.4 --- Antibody wash and color development --- p.35 / Chapter 2.8.5 --- Embryo powder preparation --- p.36 / Chapter 2.8.6 --- Pre-absorption of antibody --- p.35 / Chapter 2.9 --- Whole Mount TUNEL Staining --- p.36 / Chapter Chapter 3 --- "Maternal Diabetes, Sp2H and RA Interaction" --- p.39 / Chapter 3.1 --- Introduction --- p.40 / Chapter 3.1.1 --- Neural tube defects --- p.41 / Chapter 3.1.2 --- Retinoic acid as environmental factor --- p.41 / Chapter 3.1.3 --- Sp2H as genetic factor --- p.44 / Chapter 3.1.4 --- Experimental design of this chapter --- p.46 / Chapter 3.2 --- Material and Methods --- p.47 / Chapter 3.2.1 --- Sp2H mice --- p.47 / Chapter 3.2.2 --- Mating and RA injection protocol --- p.47 / Chapter 3.2.3 --- Dissection of fetuses and analysis of neural tube development --- p.48 / Chapter 3.3 --- Results --- p.49 / Chapter 3.3.1 --- Maternal diabetes alone --- p.50 / Chapter 3.3.2 --- Sp2H mutation alone --- p.51 / Chapter 3.3.3 --- RA alone --- p.52 / Chapter 3.3.4 --- Maternal diabetes and RA interaction --- p.53 / Chapter 3.3.5 --- Sp2H mutation and RA interaction --- p.55 / Chapter 3.3.6 --- Sp2H mutation and maternal diabetes interaction --- p.57 / Chapter 3.3.7 --- "Maternal diabetes, Sp2H mutation and RA interaction" --- p.59 / Chapter 3.4 --- Discussion --- p.62 / Chapter 3.4.1 --- Maternal diabetes alone does not cause neural tube defects --- p.62 / Chapter 3.4.2 --- RA induces neural tube defects --- p.63 / Chapter 3.4.3 --- Interaction of maternal diabetes with RA in increasing the susceptibility to neural tube defects --- p.64 / Chapter 3.4.4 --- Embryos with Sp2H allele show increased susceptibility to neural tube defects when triggered by maternal diabetes and RA --- p.67 / Chapter Chapter 4 --- Molecular and Cellular Bases of Interaction --- p.71 / Chapter 4.1 --- Introduction --- p.72 / Chapter 4.1.1 --- Mechanism of diabetic embryopathy --- p.72 / Chapter 4.1.2 --- Mechanism of Sp2H mutation in development of neural tube defects --- p.74 / Chapter 4.1.3 --- Mechanism of RA teratogenicity --- p.75 / Chapter 4.1.4 --- "Possible common pathways shared by maternal diabetes, RA and Sp2H mutation" --- p.76 / Chapter 4.1.5 --- Experimental design of this chapter --- p.78 / Chapter 4.2 --- Materials and Methods --- p.80 / Chapter 4.2.1 --- Sample collection for studying Pax3 expression in Sp2H/+ And +/+ embryos in response to maternal diabetes or RA by whole mount in situ hybridization --- p.80 / Chapter 4.2.2 --- "Sample collection for studying the level of apoptosis in response to the interaction of maternal diabetes, Sp2H mutation and RA by whole mount TUNEL staining" --- p.82 / Chapter 4.3 --- Results --- p.86 / Chapter 4.3.1 --- Expression levels of Pax3 mRNA detected by whole mount in situ hybridization / Chapter 4.3.1.1 --- Expression of Pax3 in Sp2H/+/- and +/+ embryos --- p.86 / Chapter 4.3.1.2 --- Effect of maternal diabetes on Pax3 expression in Sp2H/+ and +/+ embryos --- p.87 / Chapter 4.3.1.3 --- Effect of RA on Pax3 expression in Sp2H /+ and +/+ embryos --- p.88 / Chapter 4.3.2 --- Level of apoptosis detected by whole mount TUNEL --- p.89 / Chapter 4.3.2.1 --- Effect of Sp2H allele on apoptosis --- p.94 / Chapter 4.3.2.2 --- Effect of maternal diabetes on apoptosis in Sp2H/+ and +/+ embryos --- p.95 / Chapter 4.3.2.3 --- Effect of RA on apoptosis in Sp2H/+ and +/+ embryos --- p.96 / Chapter 4.3.2.4 --- Effect of maternal diabetes and RA on apoptosis in Sp2H/+ and +/+ embryos --- p.97 / Chapter 4.4 --- Discussion --- p.99 / Chapter 4.4.1 --- Underexpression of Pax3 and increases in apoptosis under maternal diabetes --- p.99 / Chapter 4.4.2 --- "RA does not down regulate Pαx3, but increases apoptosis" --- p.102 / Chapter 4.4.3 --- Interaction of maternal diabetes and RA in increasing apoptosis --- p.104 / Chapter Chapter 5 --- "Maternal Diabetes, NOD and RA Interaction" --- p.108 / Chapter 5.1 --- Introduction --- p.109 / Chapter 5.1.1 --- Diabetic embryopathy in NOD mice --- p.109 / Chapter 5.1.2 --- Experimental design of this chapter --- p.110 / Chapter 5.2 --- Materials and Methods --- p.112 / Chapter 5.2.1 --- NOD mice --- p.112 / Chapter 5.2.2 --- Mating and RA Injection Protocol --- p.112 / Chapter 5.2.3 --- Sample Collection for the Study of Pax3 Expression --- p.113 / Chapter 5.3 --- Results --- p.115 / Chapter 5.3.1 --- Maternal diabetic alone --- p.116 / Chapter 5.3.2 --- NOD mutation alone --- p.117 / Chapter 5.3.3 --- RA alone --- p.118 / Chapter 5.3.4 --- Maternal diabetes and RA interaction --- p.119 / Chapter 5.3.5 --- NOD mutation and RA interaction --- p.121 / Chapter 5.3.6 --- NOD mutation and maternal diabetes interaction --- p.123 / Chapter 5.3.7 --- "Maternal diabetes, NOD mutation and RA interaction" --- p.125 / Chapter 5.3.8 --- Expression of Pax3 in embryos with different copies of NOD alleles --- p.128 / Chapter 5.4 --- Discussion --- p.130 / Chapter 5.4.1 --- Maternal diabetes interacts with NOD mutation to increase susceptibility to neural tube defects --- p.130 / Chapter 5.4.2 --- Interaction of maternal diabetes with NOD mutation is greatly exacerbated when exposed to RA --- p.131 / Chapter 5.4.3 --- Pax3 is not involved in the interaction --- p.133 / Chapter Chapter 6 --- Conclusion and Future Perspectives --- p.134 / References --- p.139 / Figures / Graphs

Obesity in pregnancy has been associated with an elevated risk for neural tube defects, though it is unknown if this is linked to a lower folate status in obese women. Studies have identified a reduced folate status among obese women even after controlling for folate intake. Thus, it is possible that folic acid pharmacokinetics are altered in the obese body. In this study, we compared the pharmacokinetics of folic acid in obese and non-obese women of childbearing age, following administration of a weight-adjusted dose. Area under the concentration-time curve was found to be significantly higher in the obese group, with the dose per kilogram lean body weight most strongly predicting systemic exposure. Estimation of the daily dose required to achieve protective blood concentrations did not identify a need to change supplementation recommendations for obese women. Accordingly, current guidelines appear to suggest adequate doses for obese women of childbearing age.

Obesity in pregnancy has been associated with an elevated risk for neural tube defects, though it is unknown if this is linked to a lower folate status in obese women. Studies have identified a reduced folate status among obese women even after controlling for folate intake. Thus, it is possible that folic acid pharmacokinetics are altered in the obese body. In this study, we compared the pharmacokinetics of folic acid in obese and non-obese women of childbearing age, following administration of a weight-adjusted dose. Area under the concentration-time curve was found to be significantly higher in the obese group, with the dose per kilogram lean body weight most strongly predicting systemic exposure. Estimation of the daily dose required to achieve protective blood concentrations did not identify a need to change supplementation recommendations for obese women. Accordingly, current guidelines appear to suggest adequate doses for obese women of childbearing age.

Neural Tube Defects (NTDs) are a group of common congenital malformations that result from incomplete neural tube closure leading to abnormalities of the brain and/or spinal cord. Unfortunately, their etiology remains unknown, probably due to complex multifactorial interactions. The protective effect of dietary folates in preventing NTDs is well known, but this beneficial effect is limited to the 60 to 70% of cases; leaving 30% of the population without any known option for improving pregnancy outcomes. The mechanism by which folates rescue NTD-affected embryos is poorly understood, but the ability of folate supplementation to overcome a significant percentage of NTDs and the critical role of 5-methyltetrahydrofolate in the remethylation of homocysteine (Hcy) to methionine in the placenta suggests that folate binding and/or transport might play a critical role during development. We hypothesized that maternal autoantibodies (AB) targeting placental folate receptor alpha (FRα) are blocking the receptor and limiting the ability of the FRα to bind folates, reducing intraembryonic folate levels. Furthermore, we hypothesized that AB binding to other relevant proteins required for trophoblastic growth and placentation can be involved in activating pathologic inflammatory pathways that can result in suboptimal uptake of nutrients and contribute to an abnormal closure of the neural tube. We developed a high throughput ELISA to evaluate whether mothers experiencing pregnancies complicated with NTDs are more likely to have placental AB to FRα than are mothers experiencing normal pregnancies. We optimized and simplified a protocol for AB elution from placental tissues and determined whether these antibodies were blocking the FRα from binding with available folates. Although anti-FRα IgG antibodies were not associated to the blocking activity in this study, we found that the blocking activity was higher in the placentas from NTD-affected pregnancies compared to controls, that FRα IgM antibodies are most likely the type of antibody produced during gestation that is most relevant to the blocking activity and that it is unlikely that autoimmunity against other developmental proteins associated with NTDs is generating the NTDs. / text

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [145]-157).

Source: Dissertation Abstracts International, Volume: 70-03, Section: B, page: 1623. Adviser: Ross Shegog. Includes bibliographical references.

The transcription factor DEAF-1 is the mammalian homologue of a critical Drosophila developmental gene and is essential for neonatal survival in mice. Haploinsufficiency of Deaf-1 in the testis of adult mice was initially thought to cause loss of spermatogenesis and disrupted morphology of the seminiferous tubules, and this heterozygosity was thought to be sufficient to disrupt epigenetic programming in the developing sperm to produce inheritance of testicular defects in both heterozygous and genotypically normal offspring. Although Deaf-1 knockout mice do display disrupted testis structure, infertility at advanced age, hyperproliferation of early germ cells, and abnormal staging of seminiferous tubules, this phenotype was also observed in normal mouse strains that were born in the SIUC vivarium. Mice ordered from a vendor and raised at SIUC did not show testicular defects. This suggests an environmental factor at the SIUC vivarium may act as an endocrine disruptor during embryonic testicular development. Deaf-1-/- mice die soon after birth, often as the result of exencephaly, a gross neural tube defect (NTD). Unlike many mouse models, exencephalic Deaf-1-/- mice do not display a higher incidence of NTDs in females as compared to their male littermates. DEAF-1 promotes Bax-mediated apoptosis; studies using terminal UTP nick-end labeling (TUNEL) suggest a global increase in apoptosis in both exencephalic and normal Deaf-1-/- fetuses during neurulation as compared to their Deaf-1+/+ littermates. This indicates that Deaf-1 is crucial for correct apoptotic patterning during development, which, in turn, is essential for neural tube closure. Finally, cDNA microarray comparison of e14.5 Deaf-1 knockout and wildtype fetuses reveals expression of translation initiation factor 4g3 (Eif4g3) to be downregulated in Deaf-1-/- fetuses. Electrophoretic mobility shift assay using recombinant DEAF-1, and chromatin immunoprecipitation assay of a human cell line confirmed DEAF-1 could bind the eIF4G3 promoter both in vitro and in vivo. Additionally, transcription of the Deaf-1 Antisense Transcript (Das) was found to be significantly downregulated in both e14.5 fetuses and e18.5 fetal brains from Deaf-1-/- mice, suggesting that either lack of Deaf-1 protein or lack of exons 2 through 5 in Deaf-1 knockout mice causes changes in levels of the noncoding RNA that shares Deaf-1's promoter in the mouse.

With a birth prevalence of 1 in 1000, neural tube defects (NTD)s contribute considerably to morbidity and healthcare costs. Known genetic and environmental (non-inherited) risk factors for NTDs account for a small portion of risk, suggesting unidentified risk factors. In animal studies, maternal alcohol and pesticide exposures, independently, led to excess neural cell death, resulting in too few cells for neural tube closure. Human studies report no association between alcohol exposure and NTDs, but small to moderate positive associations for pesticide exposure. Such human etiologic studies of NTDs require a large base population, but frequently include only live births. Exclusion of cases by pregnancy outcomes may create ascertainment and response bias, complicating interpretation of findings. Using data from the National Birth Defects Prevention Study (NBDPS) and the Iowa Registry for Congenital and Inherited Disorders (IRCID), the independent effects of maternal periconceptional (1 month prior through 2 months postconception) alcohol and occupational pesticide exposure on the development of NTDs were examined, and differences in Iowa NTD cases were characterized by pregnancy outcome. Maternal reports of alcohol exposure were obtained for 1223 NTD case infants and 6807 control infants. Adjusted odds ratios, estimated using multivariate logistic regression, were near unity for NTDs by any maternal alcohol exposure, binge episode(s), and type(s) of alcohol consumed. Occupational pesticide exposure was assigned by industrial hygienists for mothers of 502 case and 2950 control infants. Adjusted odds ratios for any exposure and cumulative exposure to any pesticide, insecticides only, and insecticides + herbicides + fungicides were near unity for NTDs. Insecticide + herbicide exposure was positively associated with spina bifida. Among the 279 Iowa NTD case infants ascertained by the IRCID, 167 live births and 112 were other pregnancy outcomes (fetal deaths and elective terminations), which increased in proportion over time. Selected infant and maternal characteristics of live births and other pregnancy outcomes were similar. NBDPS eligibility varied significantly by pregnancy outcome, but participation rates did not. NTD case mothers were similar to Iowa NBDPS control mothers. Efforts were made to improve upon prior etiologic studies of these exposures and NTDs, including increased sample size and improved exposure specificity. Some exposure strata (e.g., herbicides only) and outcome strata (e.g., other rare subtypes) were limited by small numbers. All results may have been affected by response and ascertainment bias. Future studies should aim to use similarly detailed exposure classification methods, increase sample size in less prevalent NTD subtypes, and improve ascertainment of fetal deaths.