Janee Gelineau-van Waes

Reproductive and Sphingolipid Metabolic Effects of Fumonisin B1 and its Alkaline Hydrolysis Product in LM/Bc Mice: Hydrolyzed Fumonisin B1 Did Not Cause Neural Tube Defects

Fumonisins are mycotoxins produced by Fusarium verticillioides. They are toxic to animals and exert their effects through mechanisms involving disruption of sphingolipid metabolism. Fumonisins are converted to their hydrolyzed analogs by alkaline cooking (nixtamalization). Both fumonisins and hydrolyzed fumonisins are found in nixtamalized foods such as tortillas, and consumption of tortillas has been implicated as a risk factor for neural tube defects (NTD). Fumonisin B(1) (FB(1)) induced NTD when given (ip) to pregnant LM/Bc mice; however, neither the NTD induction potential of hydrolyzed fumonisin B(1) (HFB(1)) nor its affect on sphingolipid metabolism in pregnant mice have been reported. The teratogenic potential of FB(1) and HFB(1) was therefore compared using the LM/Bc mouse model. Dams were dosed (ip) with 2.5, 5.0, 10, or 20 mg/kg (< or = 49 micromol/kg) body weight (bw) HFB(1) on embryonic day (E)7-E8. Negative and positive control groups were given vehicle or 10 mg/kg (14 micromol/kg) bw FB(1), respectively. The high dose of HFB(1) disrupted sphingolipid metabolism, albeit slightly, but did not cause maternal liver lesions or NTD (n = 8-10 litters per group). In contrast, 10 mg/kg bw FB(1) markedly disrupted maternal sphingolipid metabolism, caused hepatic apoptosis in the dams, increased fetal death rates, and decreased fetal weights. Furthermore, NTD were found in all FB(1)-exposed litters (n = 10), and 66 +/- 24% of the fetuses were affected. The findings indicate that HFB(1) does not cause NTD in the sensitive LM/Bc mouse model and only weakly disrupts sphingolipid metabolism at doses up to sevenfold higher (micromole per kilogram body weight basis) than the previously reported lowest observed adverse effect level for FB(1).

Importance of folate-homocysteine homeostasis during early embryonic development

Abstract Although the beneficial effects of maternal folate supplementation in the periconceptional period have been shown to prevent neural tube defects, congenital heart defects and orofacial clefts, the exact protective mechanism of folates remains unknown. Folates affect DNA synthesis, amino acid metabolism and methylation of genes, proteins and lipids via S-adenosylmethionine-mediated one-carbon transfer reactions. Our laboratory has created several mouse knock out models of folate transport using gene targeting to inactivate folate receptor 1 (Folr1), folate receptor 2 (Folr2) and reduced folate carrier 1 (Slc19a1) genes. Gene ablation of both Folr1 and Slc19a1 leads to lethality, but with maternal folate supplementation, nullizygous embryos for both genes present with neural tube defects (NTDs) and congenital heart defects (CHDs). Folr1 nullizygous mice also exhibit orofacial clefts when the dams are provided with low folate supplementation during pregnancy. Finally, women with NTD-affected pregnancies have been reported to have high autoantibody titers against the folate receptor, potentially inhibiting the transport of folate to the developing embryo. This may be an explanation for some of the folate-responsive NTDs and perhaps other congenital malformations. Herein, we propose how homocysteinylation of the folate receptor may contribute to generation of these autoantibodies against the folate receptor. Clin Chem Lab Med 2007;45:1717–27.

Embryonic development in the reduced folate carrier knockout mouse is modulated by maternal folate supplementation

BACKGROUND The reduced folate carrier (RFC1) is a ubiquitously expressed integral membrane protein that mediates delivery of 5-methyltetrahydrofolate into mammalian cells. In this study, embryonic/fetal development is characterized in an RFC1 knockout mouse model in which pregnant dams receive different levels of folate supplementation. METHODS RFC1(+/-) males were mated to RFC1(+/-) females, and pregnant dams were treated with vehicle (control) or folic acid (25 or 50 mg/kg) by daily subcutaneous injection (0.1 mL/10 g bwt), beginning on E0.5 and continuing throughout gestation until the time of sacrifice. RESULTS Without maternal folate supplementation, RFC1 nullizygous embryos die shortly postimplantation. Supplementation of pregnant dams with 25 mg/kg/day folic acid prolongs survival of mutant embryos until E9.5-E10.5, but they are developmentally delayed relative to wild-type littermates, display a marked absence of erythropoiesis, severe neural tube and limb bud defects, and failure of chorioallantoic fusion. Fgfr2 protein levels are significantly reduced or absent in the extraembryonic membranes of RFC1 nullizygous embryos. Maternal folate supplementation with 50 mg/kg/day results in survival of 22% of RFC1 mutants to E18.5, but they develop with multiple malformations of the eyelids, lungs, heart, and skin. CONCLUSIONS High doses of daily maternal folate supplementation during embryonic/fetal development are necessary for early postimplantation embryonic viability of RFC1 nullizygous embryos, and play a critical role in chorioallantoic fusion, erythropoiesis, and proper development of the neural tube, limbs, lungs, heart, and skin.

Genetic basis of susceptibility to environmentally induced neural tube defects.

Abstract: Neural tube defects (NTDs) are among the most common of all human congenital defects, with multifactorial etiologies comprising both environmental and genetic components. Several murine model systems have been developed in an effort to elucidate genetic factors regulating expression of NTDs. Strain‐dependent differences in susceptibility to teratogenic insults and altered patterns of gene expression observed within the neuroepithelium of affected embryos support the hypothesis that subtle genetic changes can result in NTDs. Since several affected genes are folate‐regulated, transgenic knockout mice lacking a functional folate receptor were developed. Nullizygous embryos died in utero with significant morphological defects, supporting the critical role of folic acid in early embryogenesis. While epidemiological studies have not established an association between polymorphisms in the human folate receptor gene and NTDs, it is known that folate supplementation reduces infant NTD risk. Continued efforts are therefore necessary to reveal the mechanism by which folate works and the nature of the gene(s) responsible for human NTDs.

Phenytoin-induced alterations in craniofacial gene expression

In utero exposure to the anticonvulsant drug phenytoin has been shown to alter normal embryonic development, leading to a pattern of dysmorphogenesis known as the Fetal Hydantoin Syndrome. This embryopathy is characterized by growth retardation, microcephaly, mental deficiency, and craniofacial malformations, although the precise mechanism(s) by which phenytoin alters normal developmental pathways remains unknown. To better understand the molecular events involved in the pathogenesis of phenytoin-induced congenital defects, alterations in gene expression were examined during critical periods of craniofacial development. Pregnant SWV mice were administered phenytoin (60 mg/kg/day) from gestational day 6.5 until they were sacrificed at selected developmental time points. Tissue from the craniofacial region of control and exposed embryos was isolated, and samples were subjected to in situ transcription, antisense RNA amplification, and hybridization on reverse Northern blots to quantitatively assess expression of 36 candidate genes. Chronic phenytoin exposure significantly altered expression of several genes at distinct times during morphogenesis. Results of these studies show that expression of the retinoic acid receptors (RAR) alpha, beta, and gamma were significantly increased by phenytoin exposure. Elevations in gene expression of laminin beta 1, and the growth factors IGF-2, TGF alpha, and TGF beta 1, were also demonstrated in the craniofacial region of phenytoin-exposed embryos. As several of these genes are transcriptionally regulated by retinoic-acid-responsive elements in their promoter regions, phenytoin-induced alterations in expression of the RAR isoforms may have severe downstream consequences in the regulation of events necessary for normal craniofacial development. Such alterations occurring coordinately at critical times during craniofacial development may account for the dysmorphogenesis often associated with phenytoin exposure.

Microarray analysis of E9.5 reduced folate carrier (RFC1; Slc19a1) knockout embryos reveals altered expression of genes in the cubilin-megalin multiligand endocytic receptor complex

BackgroundThe reduced folate carrier (RFC1) is an integral membrane protein and facilitative anion exchanger that mediates delivery of 5-methyltetrahydrofolate into mammalian cells. Adequate maternal-fetal transport of folate is necessary for normal embryogenesis. Targeted inactivation of the murine RFC1 gene results in post-implantation embryolethality, but daily folic acid supplementation of pregnant dams prolongs survival of homozygous embryos until mid-gestation. At E10.5 RFC1-/- embryos are developmentally delayed relative to wildtype littermates, have multiple malformations, including neural tube defects, and die due to failure of chorioallantoic fusion. The mesoderm is sparse and disorganized, and there is a marked absence of erythrocytes in yolk sac blood islands. The identification of alterations in gene expression and signaling pathways involved in the observed dysmorphology following inactivation of RFC1-mediated folate transport are the focus of this investigation.ResultsAffymetrix microarray analysis of the relative gene expression profiles in whole E9.5 RFC1-/- vs. RFC1+/+ embryos identified 200 known genes that were differentially expressed. Major ontology groups included transcription factors (13.04%), and genes involved in transport functions (ion, lipid, carbohydrate) (11.37%). Genes that code for receptors, ligands and interacting proteins in the cubilin-megalin multiligand endocytic receptor complex accounted for 9.36% of the total, followed closely by several genes involved in hematopoiesis (8.03%). The most highly significant gene network identified by Ingenuity™ Pathway analysis included 12 genes in the cubilin-megalin multiligand endocytic receptor complex. Altered expression of these genes was validated by quantitative RT-PCR, and immunohistochemical analysis demonstrated that megalin protein expression disappeared from the visceral yolk sac of RFC1-/- embryos, while cubilin protein was widely misexpressed.ConclusionInactivation of RFC1 impacts the expression of several ligands and interacting proteins in the cubilin-amnionless-megalin complex that are involved in the maternal-fetal transport of folate and other nutrients, lipids and morphogens such as sonic hedgehog (Shh) and retinoids that play critical roles in normal embryogenesis.

Folic acid protects SWV/Fnn embryo fibroblasts against arsenic toxicity

It has been proposed that arsenic exerts its toxic effects, in part, by perturbing cellular methyl metabolism. Based on the hypothesis that folic acid treatment will attenuate the cytotoxic and growth inhibitory effects of arsenic, SWV/Fnn embryo fibroblasts were cultured in media supplemented with various concentrations of folic acid during treatment with sodium arsenite or dimethylarsinic acid (DMA). It was found that folic acid protects SWV/Fnn embryo fibroblasts from sodium arsenite and DMA cytotoxicity in a dose-dependent manner. In contrast, folic acid supplementation has no effect on toxicity resulting from treatment with ethanol or staurosporine, suggesting that folic acid is not generally protective against necrosis and apoptosis. Although folic acid protects against acute arsenic toxicity, this agent shows a modest and delayed ability to attenuate the growth inhibitory effect of arsenic on these cells. These results support a model in which perturbations of methyl metabolism contribute to the acute cytotoxicity of arsenic.

Reduction of Fumonisin Toxicity by Extrusion and Nixtamalization (Alkaline Cooking)

Fumonisins are mycotoxins found in corn. They are toxic to animals and cause cancer in rodents and neural tube defects in LM/Bc mice. Reducing their concentrations in corn-based foods is therefore desirable. Chemical analysis or in vitro bioassays of food extracts might not detect toxic fumonisin reaction products that are unknown or unextractable from food matrices, thus potentially underestimating in vivo toxicity. The effectiveness of two common cooking methods, extrusion and nixtamalization (alkaline cooking), to reduce the toxicity of fumonisin-contaminated corn grits (extrusion) and whole kernel corn (nixtamalization) was shown by means of rat feeding bioassays using fumonisin-specific kidney effects as indicators of potential toxicity. A third bioassay showed that in contrast to fumonisin B1 (FB1), hydrolyzed fumonisin B1 (HFB1; formed from FB1 during nixtamalization) did not cause neural tube defects in LM/Bc mice. The findings indicate that extrusion and nixtamalization reduce the potential toxicity of FB1-contaminated corn.

Fumonisin Inhibition of Ceramide Synthase: A Possible Risk Factor for Human Neural Tube Defects

Fumonisins are carcinogenic mycotoxins that cause farm animal diseases. They commonly contaminate maize and are suspected, but not proven, to cause human disease. Their mode of action involves inhibition of the enzyme (ceramide synthase) that controls the formation of sphingolipids, important regulators of pathways involved in cell death and survival. Sphingolipids are needed for the proper function of receptors associated with lipid rafts (for example, the high affinity folate-binding protein). Fumonisin disruption of folate transport interferes with neural tube closure in animal models in vitro, and this effect is reduced by folate supplementation. In vivo studies in the LMBc mouse strain have shown that maternal fumonisin administration during pregnancy causes a dose-related increase in the frequency of neural tube defects (NTDs) in the embryos. Co-exposing the dams to folate or ganglioside GM1 is protective, suggesting that fumonisin alters sphingolipid-dependent lipid raft function. In addition, altered expression of cytokines, inducible nitric oxide synthase, elevated levels of sphinganine and sphinganine 1-phosphate and several genes involved in redox homeostasis are observed in affected embryos. NTDs are the second most common birth defect in humans. The etiology of human NTDs is complex. Increased risk has been associated with genetic predisposition, dietary exposure to environmental contaminants, and reduced intake of folate and other vitamins/nutrients. Human clinical and epidemiological studies show folate supplementation reduces the risk for NTDs. While there is no direct evidence for fumonisin as a cause of NTD in humans, the incidence of NTD is higher where maize consumption is high, and both fumonisin exposure and folate deficient diets are likely. Thus, it has been hypothesized that fumonisin inhibition of ceramide synthase is a risk factor for NTDs in humans with folate deficient diets who consume large quantities of low quality maize.

Nutrient effects upon embryogenesis: folate, vitamin A and iodine.

The period of human ‘embryogenesis’, the foundation of this chapter, is generally taken to include the initial 8-week period of human development, from fertilization through organogenesis. Knowledge of the effects of nutrients upon the normal development of the embryo during this period typically has been acquired by observation of the effects that accompany some perturbation of the delivery of a given nutrient; therefore this chapter will focus upon the results of ‘perturbed’ delivery of folic acid, vitamin A, and iodine. While a developmental defect may occur at virtually any time during gestation, only perturbations that occur during embryogenesis can produce major anatomical malformations of organs that develop from the neural tube and the neural crest. Defects of the neural tube and neural crest are the most common and the most devastating in terms of mortality and morbidity, stillbirths, and spontaneous abortions. These include neural tube closure defects such as spina bifida, orofacial defects, and conotruncal heart defects [1–3]. Based upon these data, it may be argued that the most important nutrient effects during embryogenesis are those that impact upon the development of the neural tube and the neural crest; therefore, these effects will be the principal topic of this chapter. Cells of the neural tube and neural crest essentially are identical during embryogenesis [4], therefore a nutrient that is essential for neural tube development is essential for neural crest development as well. This relationship is demonstrated by both folic acid and vitamin A: a deficiency of either during embryogenesis is related to an increase in the occurrence of Hornstra G, Uauy R, Yang X (eds): The Impact of Maternal Nutrition on the Offspring. Nestlé Nutrition Workshop Series Pediatric Program, vol 55, pp 29–47, Nestec Ltd., Vevey/S. Karger AG, Basel,