Elevated nuclear sphingoid base-1-phosphates and decreased histone deacetylase activity after fumonisin B1 treatment in mouse embryonic fibroblasts.

Fumonisin B1 (FB1) is a mycotoxin produced by a common fungal contaminant of corn. Administration of FB1 to pregnant LM/Bc mice induces exencephaly in embryos, and ingestion of FB1-contaminated food during early pregnancy is associated with increased risk for neural tube defects (NTDs) in humans. FB1 inhibits ceramide synthase enzymes in sphingolipid biosynthesis, causing sphinganine (Sa) and bioactive sphinganine-1-phosphate (Sa1P) accumulation in blood, cells, and tissues. Sphingosine kinases (Sphk) phosphorylate Sa to form Sa1P. Upon activation, Sphk1 associates primarily with the plasma membrane, while Sphk2 is found predominantly in the nucleus. In cells over-expressing Sphk2, accumulation of Sa1P in the nuclear compartment inhibits histone deacetylase (HDAC) activity, causing increased acetylation of histone lysine residues. In this study, FB1 treatment in LM/Bc mouse embryonic fibroblasts (MEFs) resulted in significant accumulation of Sa1P in nuclear extracts relative to cytoplasmic extracts. Elevated nuclear Sa1P corresponded to decreased histone deacetylase (HDAC) activity and increased histone acetylation at H2BK12, H3K9, H3K18, and H3K23. Treatment of LM/Bc MEFs with a selective Sphk1 inhibitor, PF-543, or with ABC294640, a selective Sphk2 inhibitor, significantly reduced nuclear Sa1P accumulation after FB1, although Sa1P levels remained significantly increased relative to basal levels. Concurrent treatment with both PF-543 and ABC294640 prevented nuclear accumulation of Sa1P in response to FB1. Other HDAC inhibitors are known to cause NTDs, so these results suggest that FB1-induced disruption of sphingolipid metabolism leading to nuclear Sa1P accumulation, HDAC inhibition, and histone hyperacetylation is a potential mechanism for FB1-induced NTDs.

Elevated Nuclear and Cytoplasmic FTY720-Phosphate in Mouse Embryonic Fibroblasts Suggests the Potential for Multiple Mechanisms in FTY720-Induced Neural Tube Defects.

FTY720 (fingolimod) is a U.S. Food and Drug Administration-approved drug to treat relapsing remitting multiple sclerosis. FTY720 treatment in pregnant inbred LM/Bc mice results in approximately 60% of embryos having a neural tube defect (NTD). Sphingosine kinases (Sphk1, Sphk2) phosphorylate FTY720 in vivo to form the bioactive metabolite FTY720-1-phosphate (FTY720-P). Cytoplasmic FTY720-P is an agonist for 4 of the 5 sphingosine-1-phosphate (S1P) receptors (S1P1, 3-5) and can also act as a functional antagonist of S1P1, whereas FTY720-P generated in the nucleus inhibits histone deacetylases (HDACs), leading to increased histone acetylation. This study demonstrates that treatment of LM/Bc mouse embryonic fibroblasts (MEFs) with FTY720 results in a significant accumulation of FTY720-P in both the cytoplasmic and nuclear compartments. Elevated nuclear FTY720-P is associated with decreased HDAC activity and increased histone acetylation at H3K18 and H3K23 in LM/Bc MEFs. Treatment of LM/Bc MEFs with FTY720 and a selective Sphk2 inhibitor, ABC294640, significantly reduces the amount of FTY720-P that accumulates in the nucleus. The data provide insight into the relative amounts of FTY720-P generated in the nuclear versus cytoplasmic subcellular compartments after FTY720 treatment and the specific Sphk isoforms involved. The results of this study suggest that FTY720-induced NTDs may involve multiple mechanisms, including: (1) sustained and/or altered S1P receptor activation and signaling by FTY720-P produced in the cytoplasm and (2) HDAC inhibition and histone hyperacetylation by FTY720-P generated in the nucleus that could lead to epigenetic changes in gene regulation.

Evidence for fumonisin inhibition of ceramide synthase in humans consuming maize-based foods and living in high exposure communities in Guatemala.

SCOPE: Fumonisin (FB) occurs in maize and is an inhibitor of ceramide synthase (CerS). We determined the urinary FB1 (UFB1 ) and sphingoid base 1-phosphate levels in blood from women consuming maize in high and low FB exposure communities in Guatemala. METHODS AND RESULTS: FB1 intake was estimated using the UFB1 . Sphinganine 1-phosphate (Sa 1-P), sphingosine 1-phosphate (So 1-P), and the Sa 1-P/So 1-P ratio were determined in blood spots collected on absorbent paper at the same time as urine collection. In the first study, blood spots and urine were collected every 3 months (March 2011 to February 2012) from women living in low (Chimaltenango and Escuintla) and high (Jutiapa) FB exposure communities (1240 total recruits). The UFB1 , Sa 1-P/So 1-P ratio, and Sa 1-P/mL in blood spots were significantly higher in the high FB1 intake community compared to the low FB1 intake communities. The results were confirmed in a follow-up study (February 2013) involving 299 women living in low (Sacatepéquez) and high (Santa Rosa and Chiquimula) FB exposure communities. CONCLUSIONS: High levels of FB1 intake are correlated with changes in Sa 1-P and the Sa 1-P/So 1-P ratio in human blood in a manner consistent with FB1 inhibition of CerS.

A blood spot method for detecting fumonisin-induced changes in putative sphingolipid biomarkers in LM/Bc mice and humans.

Fumonisins (FB) are mycotoxins found in maize. They are hypothesised risk factors for neural tube defects (NTDs) in humans living where maize is a dietary staple. In LM/Bc mice, FB1-treatment of pregnant dams induces NTDs and results in increased levels of sphingoid base 1-phosphates in blood and tissues. The increased level of sphingoid base 1-phosphates in blood is a putative biomarker for FB1 inhibition of ceramide synthase in humans. Collection of blood spots on paper from finger sticks is a relatively non-invasive way to obtain blood for biomarker analysis. The objective of this study was to develop and validate in an animal model, and ultimately in humans, a method to estimate the volume of blood collected as blood spots on absorbent paper so as to allow quantification of the molar concentration of sphingoid base 1-phosphates in blood. To accomplish this objective, blood was collected from unexposed male LM/Bc and FB1-exposed pregnant LM/Bc mice and humans and applied to two types of absorbent paper. The sphingoid base 1-phosphates, absorbance at 270 nm (A270), and total protein content (Bradford) were determined in the acetonitrile:water 5% formic acid extracts from the dried blood spots. The results show that in both mouse and human the A270, total protein, and blood volume were closely correlated and the volume of blood spotted was accurately estimated using only the A270 of the extracts. In mouse blood spots, as in tissues and embryos, the FB1-induced changes in sphingolipids were correlated with urinary FB1. The half-life of FB1 in the urine was short (<24 h) and the elevation in sphingoid base 1-phosphates in blood was also short, although more persistent than the urinary FB1.

Urinary fumonisin B1 and estimated fumonisin intake in women from high- and low-exposure communities in Guatemala.

SCOPE: Fumonisin (FB) intake can be high when maize is a dietary staple. We determined (i) urinary FB (UFB) in women consuming maize in high- and low-exposure communities in Guatemala, (ii) the FB levels in maize, (iii) the relationship between UFB and FB intake, and (iv) the relative excretion of UFB1 , UFB2 , and UFB3 . METHODS AND RESULTS: Urine and maize were analyzed for FB for 1 year in three departments. Maize consumption was estimated by an interview questionnaire. Fumonisin B1 , B2 , and B3 (FB1 , FB2 and FB3 ), were detected in 100% of maize samples. FB1 in maize and urine was significantly higher in Jutiapa compared to Chimaltenango or Escuintla. The FB intake paralleled UFB1 in a dose-dependent manner but UFB1 was present in much higher levels than UFB2 or UFB3 compared to maize. CONCLUSION: In Jutiapa, agroecological conditions favored FB production. UFB1 mirrored the estimated FB intake. UFB1 > 0.1 ng/mL resulted in a dose-dependent increase in the risk of exceeding FB intake of 2 µg/kg b.w./day compared to women with no detectable UFB1 . More than 50% exceeded 2 µg/kg b.w./day when UFB1 was >0.5 ng/mL. UFB2 and UFB3 were rarely detected confirming that FB1 is either absorbed better or preferentially excreted in urine.

Increased sphingoid base-1-phosphates and failure of neural tube closure after exposure to fumonisin or FTY720.

BACKGROUND: Fumonisin B(1) (FB(1)) is a mycotoxin produced by a common fungal contaminant of corn. Ingestion of FB(1)-contaminated food is associated with increased risk for neural tube defects (NTDs). FB(1) induces NTDs in inbred LM/Bc mice. FB(1) inhibits ceramide synthase in de novo sphingolipid biosynthesis, resulting in accumulation of sphinganine and sphinganine-1-phosphate (Sa1P). Sa1P functions as a ligand for a family of G protein-coupled S1P receptors. METHODS: Pregnant SWV and LM/Bc mice were treated with FB(1) (20 mg/kg/day intraperitoneally on embryonic day (ED) 7.5-8.5) or the known S1P receptor agonist FTY720 (10 mg/kg/day oral gavage on ED 6.5-8.5). LC/MS was used to detect sphingoid base-1-phosphates in maternal blood spots, plasma, and embryonic tissue. Strain-specific SWV and LM/Bc mouse embryonic fibroblasts (MEFs) and serum free mouse embryo (SFME) neural progenitor cells were treated with FB(1) (40 µM for 24 hr) and LC/MS was used to detect sphingoid base-1-phosphates. RESULTS: FTY720 induced NTDs in both the SWV and the LM/Bc strains of mice. Sphinganine-1-P (Sa1P) and FTY720-P were elevated in the blood spots and plasma of mice treated with FB(1) or FTY720, respectively. FTY720-P was elevated in ED 9.5 exencephalic embryos. Sa1P was elevated in SFME and MEF cells treated with FB(1), and Sa1P was higher in MEFs generated from the FB(1)-NTD-susceptible LM/Bc strain. CONCLUSIONS: Elevated sphingoid base-1-P after FB(1) or FTY720 suggest a potential role for these bioactive lipid ligands and activation of S1P receptor signaling pathways in the failure of neural tube closure after FB(1) or FTY720. Sa1P may represent a biomarker for FB(1)-NTD risk assessment.

The kinetics of urinary fumonisin B1 excretion in humans consuming maize-based diets.

SCOPE: Fumonisins (FB) are mycotoxins found in maize. The purpose of this study was to (i) determine the relationship between FB(1) , FB(2) , and FB(3) intake and urinary excretion in humans, (ii) validate a method to isolate urinary FB on C(18) -SPE cartridges for international shipment, and (iii) test the method using samples from Guatemala. METHODS AND RESULTS: Volunteers (n = 10) consumed 206 grams/day of tortillas and biscuits prepared from masa flour and a product containing maize flour. Volunteers estimated their daily urine output and samples were analyzed for FB(1) , FB(2) , and FB(3) and hydrolyzed FB(1) . Only FB(1) was detected in urine suggesting lower absorption of FB(2) and FB(3) . Excretion was highly variable peaking soon after consumption began and decreasing rapidly after consumption stopped. Within 5 days after consumption ended, FB(1) was not detected in urine. In a study with eight volunteers, the average total urinary FB(1) was 0.5% of the intake. FB(1) was detected in 61% (107/177) of the samples collected in Guatemala. CONCLUSION: The results support the use of urinary FB(1) to assess ongoing exposure in population-based studies. However, relating the FB(1) concentration in urine to dietary intake of FB by individual subjects will be complicated due to interindividual variability and the rapidity of clearance.

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.

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.

BACKGROUND: The 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. RESULTS: Affymetrix 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 Ingenuitytrade mark 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. CONCLUSION: Inactivation 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.