Induction Of Neural Tube Defects Research Articles

Alcohol induces neural tube defects by reducing retinoic acid signaling and promoting neural plate expansion.

Introduction: Neural tube defects (NTDs) are among the most debilitating and common developmental defects in humans. The induction of NTDs has been attributed to abnormal folic acid (vitamin B9) metabolism, Wnt and BMP signaling, excess retinoic acid (RA), dietary components, environmental factors, and many others. In the present study we show that reduced RA signaling, including alcohol exposure, induces NTDs. Methods: Xenopus embryos were exposed to pharmacological RA biosynthesis inhibitors to study the induction of NTDs. Embryos were treated with DEAB, citral, or ethanol, all of which inhibit the biosynthesis of RA, or injected to overexpress Cyp26a1 to reduce RA. NTD induction was studied using neural plate and notochord markers together with morphological analysis. Expression of the neuroectodermal regulatory network and cell proliferation were analyzed to understand the morphological malformations of the neural plate. Results: Reducing RA signaling levels using retinaldehyde dehydrogenase inhibitors (ethanol, DEAB, and citral) or Cyp26a1-driven degradation efficiently induce NTDs. These NTDs can be rescued by providing precursors of RA. We mapped this RA requirement to early gastrula stages during the induction of neural plate precursors. This reduced RA signaling results in abnormal expression of neural network genes, including the neural plate stem cell maintenance genes, geminin, and foxd4l1.1. This abnormal expression of neural network genes results in increased proliferation of neural precursors giving rise to an expanded neural plate. Conclusion: We show that RA signaling is required for neural tube closure during embryogenesis. RA signaling plays a very early role in the regulation of proliferation and differentiation of the neural plate soon after the induction of neural progenitors during gastrulation. RA signaling disruption leads to the induction of NTDs through the mis regulation of the early neuroectodermal network, leading to increased proliferation resulting in the expansion of the neural plate. Ethanol exposure induces NTDs through this mechanism involving reduced RA levels.

High glucose-induced oxidative stress represses sirtuin deacetylase expression and increases histone acetylation leading to neural tube defects.

Aberrant epigenetic modifications are implicated in maternal diabetes-induced neural tube defects (NTDs). Because cellular stress plays a causal role in diabetic embryopathy, we investigated the possible role of the stress-resistant sirtuin (SIRT) family histone deacetylases. Among the seven sirtuins (SIRT1-7), pre-gestational maternal diabetes in vivo or high glucose in vitro significantly reduced the expression of SIRT 2 and SIRT6 in the embryo or neural stem cells, respectively. The down-regulation of SIRT2 and SIRT6 was reversed by superoxide dismutase 1 (SOD1) over-expression in the in vivo mouse model of diabetic embryopathy and the SOD mimetic, tempol and cell permeable SOD, PEGSOD in neural stem cell cultures. 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a superoxide generating agent, mimicked high glucose-suppressed SIRT2 and SIRT6 expression. The acetylation of histone 3 at lysine residues 56 (H3K56), H3K14, H3K9, and H3K27, putative substrates of SIRT2 and SIRT6, was increased by maternal diabetes in vivo or high glucose in vitro, and these increases were blocked by SOD1 over-expression or tempol treatment. SIRT2 or SIRT6 over-expression abrogated high glucose-suppressed SIRT2 or SIRT6 expression, and prevented the increase in acetylation of their histone substrates. The potent sirtuin activator (SRT1720) blocked high glucose-increased histone acetylation and NTD formation, whereas the combination of a pharmacological SIRT2 inhibitor and a pan SIRT inhibitor mimicked the effect of high glucose on increased histone acetylation and NTD induction. Thus, diabetes in vivo or high glucose in vitro suppresses SIRT2 and SIRT6 expression through oxidative stress, and sirtuin down-regulation-induced histone acetylation may be involved in diabetes-induced NTDs. The mechanism underlying pre-gestational diabetes-induced neural tube defects (NTDs) is still elusive. Our study unravels a new epigenetic mechanism in which maternal diabetes-induced oxidative stress represses sirtuin deacetylase 2 (SIRT2) and 6 (SIRT6) expression leading to histone acetylation and gene expression. SIRT down-regulation mediates the teratogenicity of diabetes leading to (NTD) formation. The study provides a mechanistic basis for the development of natural antioxidants and SIRT activators as therapeutics for diabetic embryopathy.

Enantioselective pharmacodynamic and pharmacokinetic analysis of two chiral CNS-active carbamate derivatives of valproic acid.

2-Ethyl-3-methylbutyl-carbamate (EMC) and 2-isopropylpentyl-carbamate (IPC) are among the most potent anticonvulsant carbamate derivatives of valproic acid. EMC and IPC are chiral compounds. Consequently, the aim of the current study was to comparatively evaluate the pharmacokinetic (PK) and pharmacodynamic (PD anticonvulsant activity) profile of EMC and IPC individual enantiomers. The anticonvulsant activity of EMC and IPC individual enantiomers was evaluated in several anticonvulsant rodent models including maximal electroshock (MES), 6 Hz psychomotor, subcutaneous (pentylenetetrazole) (scMet), and the pilocarpine-induced and soman-induced status epilepticus (SE). The PK-PD relationship of EMC and IPC individual enantiomers was evaluated following intraperitoneal administration (50 mg/kg) to rats. Induction of neural tube defects (NTDs) was evaluated in a mouse strain that was highly susceptible to teratogen-induced NTDs. In mice and rats, (2S)-EMC exhibited anticonvulsant activity similar to that of racemic EMC in the MES and scMet tests, whereas in the 6 Hz test, racemic EMC was more potent than its two individual enantiomers. Racemic EMC exhibited a potent activity in the soman-induced SE model when administered 5 and 20 min after seizure onset with median effective dose (ED50 ) values of 33 and 48 mg/kg, respectively. (2R)-IPC and (2S)-IPC exhibited ED50 values similar to those of racemic IPC in the mouse and rat MES and scMet models. (2R)-IPC had similar ED50 values on the 6 Hz tests. Racemic IPC had an ED50 value of 107 mg/kg in the pilocarpine-induced SE model when given 30 min after seizure onset. Racemic EMC and IPC and their enantiomers had similar clearance (3.8-5.5 L/h/kg) and short half-life (<1 h). EMC and its enantiomers did not cause NTDs at doses 3-10 times higher than their anticonvulsant ED50 values. EMC and IPC did not exhibit enantioselective PK, a fact that may contribute to their nonenantioselective activity in any of the anticonvulsant models. The nonsignificant difference between racemic EMC and racemic IPC and their enantiomers, suggests that their wide spectrum of anticonvulsant activity is likely to be caused by multiple mechanisms of action.

Risk Factors Associated with Lipomyelomeningocele: A Case-Control Study

Background: In general, it seems that both genetic and environmental factors play important roles in the induction of neural tube defects. Lipomyelomeningocele (LipoMMC) is a rather common type of closed neural tube defect, but only limited studies have investigated the potential risk factors of this anomaly. Therefore, the purpose of this case-control study was to investigate the risk factors involved in LipoMMC formation. Material and Method: Various risk factors were evaluated in 35 children between 1 month and 10 years of age with LipoMMC in a hospital-based case-control study. The 2 control arms consisted of 35 children with myelomeningocele (MMC group) and 35 children with congenital anomalies other than central nervous system problems (control group). All groups were matched for age and visited the same hospital. A structured questionnaire was used for the collection of all data, including the mothers' weight and height during pregnancy, education, reproductive history, previous abortions, and socioeconomic status, as well as the parents' consanguinity and family history of the same anomalies. Results: Univariate analysis of the children with LipoMMC compared to the control group showed that the use of periconceptional folic acid supplementation was significantly lower in the MMC and LipoMMC groups compared to the control group. In addition, comparison of the MMC and control groups revealed statistically significant differences regarding the use of folic acid and maternal obesity. In multivariate analysis, use of folic acid in the periconceptional period and during the first trimester was an independent risk factor for LipoMMC and MMC. Furthermore, maternal obesity was a significantly positive risk factor for MMC. Conclusion: The probable risk factors for LipoMMC were investigated in this case-control study. Consumption of folic acid in the periconceptional period and during the first trimester is an independent protective factor against LipoMMC. It seems that larger studies are needed to examine other possible risk factors.

Fumonisin B1induced neural tube defects were not increased in LM/Bc mice fed folate-deficient diet

Fumonisin B1 (FB1 ) is found in corn-based foods and is a possible risk factor for neural tube defects (NTDs). The mechanism(s) underlying NTD induction by FB1 in LM/Bc mice is not understood; however, evidence suggests disrupted folate transport is involved. Female LM/Bc mice were fed folate-sufficient (control) or folate-deficient diet beginning 5 wk before mating, treated with 0 (vehicle), 2.5 or 10 mg/kg FB1 by intraperitoneal injection on embryonic days 7 (E7) and E8, and their fetuses examined on E16. Dose-dependent NTD induction was found in groups fed the control diet: 3 of 13 low-dose and 10 of 11 high-dose litters were affected. Among groups fed folate-deficient diet, NTDs were found only in 4 of 11 high-dose litters. In another trial, consumption of folate-deficient diet also resulted in fewer NTDs at a dose of 10 mg/kg FB1 and reduced maternal red blood cell folate levels by 80%. In utero death did not fully account for the differences in NTD rates. Folate deficiency does not exacerbate NTD induction by FB1 in LM/Bc mice. Interactions between folate, other nutritional factors, and FB1 in this mouse model for NTDs are complex and require further investigation.

Stereoselective anticonvulsant and pharmacokinetic analysis of valnoctamide, a CNS-active derivative of valproic acid with low teratogenic potential.

Valnoctamide (VCD), a central nervous system (CNS)-active chiral constitutional isomer of valpromide, the corresponding amide of valproic acid (VPA), is currently undergoing phase IIb clinical trials in acute mania. VCD exhibits stereoselective pharmacokinetics (PK) in animals and humans. The current study comparatively evaluated the pharmacodynamics (PD; anticonvulsant activity and teratogenicity) and PK of the four individual stereoisomers of VCD. The anticonvulsant activity of VCD individual stereoisomers was evaluated in several rodent anticonvulsant models including maximal electroshock, 6Hz psychomotor, subcutaneous metrazol, and the pilocarpine-induced and soman-induced status epilepticus (SE). The PK-PD (anticonvulsant activity) relationship of VCD stereoisomers was evaluated following intraperitoneal administration (70mg/kg) to rats. Induction of neural tube defects (NTDs) by VCD stereoisomers was evaluated in a mouse strain that was highly susceptible to teratogen-induced NTDs. VCD had a stereoselective PK, with (2S,3S)-VCD exhibiting the lowest clearance, and consequently a twice-higher plasma exposure than all other stereoisomers. Nervertheless, there was less stereoselectivity in VCD anticonvulsant activity and each stereoisomer had similar median effective dose (ED)50 values in most models. VCD stereoisomers (258 or 389mg/kg) did not cause NTDs. These doses are 3-12 times higher than VCD anticonvulsant ED50 values. VCD displayed stereoselective PK that did not lead to significant stereoselective activity in various anticonvulsant rodent models. If VCD exerted its broad-spectrum anticonvulsant activity using a single mechanism of action (MOA), it is likely that it would exhibit a stereoselective PD. The fact that there was no significant difference between racemic VCD and its individual stereoisomers suggests that VCD's anticonvulsant activity is due to multiple MOAs.

C-Jun NH2-Terminal Kinase 1/2 and Endoplasmic Reticulum Stress as Interdependent and Reciprocal Causation in Diabetic Embryopathy

Embryos exposed to high glucose exhibit aberrant maturational and cytoarchitectural cellular changes, implicating cellular organelle stress in diabetic embryopathy. c-Jun-N-terminal kinase 1/2 (JNK1/2) activation is a causal event in maternal diabetes–induced neural tube defects (NTD). However, the relationship between JNK1/2 activation and endoplasmic reticulum (ER) stress in diabetic embryopathy has never been explored. We found that maternal diabetes significantly increased ER stress markers and induced swollen/enlarged ER lumens in embryonic neuroepithelial cells during neurulation. Deletion of either jnk1 or jnk2 gene diminished hyperglycemia-increased ER stress markers and ER chaperone gene expression. In embryos cultured under high-glucose conditions (20 mmol/L), the use of 4-phenylbutyric acid (4-PBA), an ER chemical chaperone, diminished ER stress markers and abolished the activation of JNK1/2 and its downstream transcription factors, caspase 3 and caspase 8, and Sox1 neural progenitor apoptosis. Consequently, both 1 and 2 mmol/L 4-PBA significantly ameliorated high glucose–induced NTD. We conclude that hyperglycemia induces ER stress, which is responsible for the proapoptotic JNK1/2 pathway activation, apoptosis, and NTD induction. Suppressing JNK1/2 activation by either jnk1 or jnk2 gene deletion prevents ER stress. Thus, our study reveals a reciprocal causation of ER stress and JNK1/2 in mediating the teratogenicity of maternal diabetes.

Comparative pharmacodynamic and pharmacokinetic analysis of two anticonvulsant halo derivatives of 2,2,3,3‐tetramethylcyclopropanecarboxamide, an amide of a cyclic analog of valproic acid

α-Fluoro-2,2,3,3-tetramethylcyclopropanecarboxamide (α-F-TMCD) and α-Cl-TMCD, are α-halo derivatives of TMCD, the corresponding amide of a cyclopropane analog of valproic acid (VPA). This study aimed to comparatively evaluate the pharmacodynamics and pharmacokinetics of α-F-TMCD and α-Cl-TMCD in rodent models of epilepsy and for antiepileptic drug (AED)-induced teratogenicity. The potential of α-F-TMCD as an antiallodynic and antinociceptive compound was also evaluated. α-F-TMCD and α-Cl-TMCD were synthesized. α-Cl-TMCD anticonvulsant activity was evaluated in comparison to VPA in the mouse maximal-electroshock-seizure (MES), Metrazol (scMet), and 6-Hz psychomotor-seizure tests. Neurotoxicity was assessed by the Rotorod-ataxia test. Induction of neural tube defects (NTDs) by α-Cl-TMCD and α-F-TMCD was evaluated after intraperitoneal administration to a mouse strain highly susceptible to VPA-induced teratogenicity. The ability of α-F-TMCD to reduce pain was evaluated in the rat spinal nerve ligation (SNL) model for neuropathic pain and in the formalin test. α-F-TMCD and α-Cl-TMCD pharmacokinetics was evaluated following intraperitoneal (40 mg/kg) and oral (60 mg/kg) administration to rats. α-F-TMCD and α-Cl-TMCD had similar potencies in the 6-Hz test and were more potent than VPA in this model and in the scMet test. Neither induced NTDs, and both exhibited wide safety margins. α-F-TMCD was active in the two pain models, and was found to be equipotent to gabapentin in the SNL model (ED(50) = 37 and 32 mg/kg, respectively). Comparative pharmacokinetic analysis showed that α-Cl-TMCD is less susceptible to liver first-pass effect than α-F-TMCD because of lower total (metabolic) clearance and liver extraction ratio. Based on their potent anticonvulsant activity and lack of teratogenicity, α-F-TMCD and α-Cl-TMCD have the potential for development as new antiepileptics and central nervous system (CNS) drugs.

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).

Evaluation of stereoselective anticonvulsant, teratogenic, and pharmacokinetic profile of valnoctylurea (capuride): A chiral stereoisomer of valproic acid urea derivative

The purpose of this study was to evaluate the stereoselective anticonvulsant activity, neurotoxicity, pharmacokinetics, and teratogenic potential of two stereoisomers of valnoctylurea (VCU), a central nervous system (CNS)-active urea derivative of valnoctic acid, which is a constitutional isomer of valproic acid (VPA). VCU stereoisomers (2S,3S)-VCU and (2R,3S)-VCU were synthesized. Their anticonvulsant activity was determined and compared to VPA and racemic-VCU in rats utilizing the maximal electroshock seizure (MES) and the subcutaneous pentylenetetrazole (scMet) tests. Neurotoxicity was determined in rats using the positional sense test, muscle tone test, and gait and stance test. The induction of neural tube defects (NTDs) by VCU stereoisomers was evaluated in a mouse strain highly susceptible to VPA-induced teratogenicity. The pharmacokinetics of VCU was studied in a stereoselective manner following intraperitoneal (i.p.) administration to rats. (2S,3S)-VCU and (2R,3S)-VCU median effective dose ED(50) values were 29 mg/kg [95% confidence interval (CI) = 8-60 mg/kg] and 42 mg/kg (95% CI = 36-51 mg/kg) (MES) and 22 mg/kg (95% CI = 13-51 mg/kg) and 12 mg/kg (95%CI = 7-21 mg/kg) (scMet), respectively. (2S,3S)-VCU was more potent and had a wider safety margin (p < 0.05), defined as the protective index (PI = TD(50)/ED(50)), at both the MES (PI > 17) and scMet (PI > 23) tests than racemic-VCU or (R,S)-VCU (PI = 2.8 and 9.9, respectively). VCU stereoisomers caused NTDs at doses >4 times that of their anticonvulsant ED(50) values. At a dose of 112 mg/kg, (2R,3S)-VCU was nonteratogenic and less embryotoxic than its stereoisomer (2S,3S)-VCU. No stereoselective pharmacokinetics was observed following intraperitoneal dosing of racemic-VCU to rats. VCU was mainly eliminated by metabolism with a half-life of 2 h. VCU anticonvulsant activity and wide PI values make it a potential candidate for development as a new, potent antiepileptic drug (AED).

Anticonvulsant profile and teratogenicity of 3,3‐dimethylbutanoylurea: A potential for a second generation drug to valproic acid

The purpose of this study was to evaluate the anticonvulsant activity and teratogenic potential of branched aliphatic acylureas represented by isovaleroylurea (IVU), pivaloylurea (PVU) and 3,3-dimethylbutanoylurea (DBU), as potential second-generation drugs to valproic acid (VPA). The anticonvulsant activity of IVU, PVU, and DBU was determined in mice and rats utilizing the maximal electroshock seizure (MES) and the pentylenetetrazole (scMet) tests. The ability of DBU to block electrical-, or chemical-induced seizures was further examined in three acute seizure models: the psychomotor 6 Hz model, the bicuculline and picrotoxin models and one model of chronic epilepsy (i.e., the hippocampal kindled rat model). The induction of neural tube defects (NTDs) by IVU, PVU, and DBU was evaluated after i.p. administration at day 8.5 of gestation to a mouse strain highly susceptible to VPA-induced teratogenicity. The pharmacokinetics of DBU was studied following i.v. administration to rats. DBU emerged as the most potent compound having an MES-ED(50)of 186 mg/kg (mice) and 64 mg/kg (rats) and an scMet-ED(50)of 66 mg/kg (mice) and 26 mg/kg (rats). DBU underwent further evaluation in the hippocampal kindled rat (ED(50)= 35 mg/kg), the psychomotor 6 Hz mouse model (ED(50)= 80 mg/kg at 32 mA and ED(50)= 133 mg/kg at 44 mA), the bicuculline- and picrotoxin-induced seizure mouse model (ED(50)= 205 mg/kg and 167 mg/kg, respectively). In contrast to VPA, DBU, IVU, and PVU did not induce a significant increase in NTDs as compared to control. DBU was eliminated by metabolism with a half-life of 4.5 h. DBU's broad spectrum and potent anticonvulsant activity, along with its high safety margin and favorable pharmacokinetic profile, make it an attractive candidate to become a new, potent, and safe AED.

Hydroxamic acid and fluorinated derivatives of valproic acid: Anticonvulsant activity, neurotoxicity and teratogenicity

Purpose Fluorinated and non-fluorinated valproic acid (VPA) analogues with hydroxamic acid moieties were tested for their teratogenic, anticonvulsant and neurotoxic potencies in mice. Methods Compounds were synthesized from their corresponding acids. The induction of neural tube defects (exencephaly) of the resulting hydroxamates (applied on day 8.25 of gestation) was tested in the offspring of pregnant animals (Han:NMRI mice). The anticonvulsant activity was evaluated in the subcutaneous pentylenetetrazole (PTZ) seizure threshold test and neurotoxicity in the rotorod neurotoxicity test. Results All tested hydroxamates showed no or greatly reduced teratogenic potency in mice compared to the free acids. Furthermore all compounds exhibited anticonvulsant activity with ED 50 doses ranging from 0.16 mmol/kg to 0.59 mmol/kg (VPA 0.57 mmol/kg). Neurotoxicity of the hydroxamates was increased compared to VPA. TD 50 doses range from 0.70 mmol/kg to 1.42 mmol/kg (VPA 1.83 mmol/kg). Conclusion Hydroxamic acid derivatives of VPA with improved protective index and little or undetectable teratogenic potency compared to the free acids are described. α-fluorination of VPA also resulted in loss of teratogenic activity. Such fluorination of the hydroxamic acids also led to compounds with an improved anticonvulsant profile compared to non-fluorinated hydroxamates. The non-chiral 2-Fluoro-VPA-hydroxamic acid was the most promising compound with a protective index (ratio of TD 50 to ED 50) of 4.4 compared to 3.2 for VPA. This compound combines an improved ratio of anticonvulsant potency/neurotoxicity with the advantage of not being teratogenic in the mouse neural tube defect model used.

Prevention of ochratoxin A‐induced neural tube defects by folic acid in the genetic polydactyly/arhinencephaly mouse,Pdn/Pdn

The gene responsible for the polydactyly/arhinencephaly (Pdn/Pdn) mouse, which exhibits polysyndactyly and arhinencephaly and has a 13.2% risk of neural tube defects (NTD), has been identified as Gli3. Ochratoxin A (OTA) is a teratogen causing NTD in mice. When Pdn/Pdn embryos were exposed to 2 mg/kg of OTA on day 7.5, the incidence of NTD in Pdn/Pdn fetuses increased to 51.6%. Pre-treatment with folinic acid (FA), metabolically the most active form of folic acid, before OTA-treatment decreased the incidence of NTD to 20.8%. We investigated the effect of OTA and FA on gene expression in day 9 embryos using whole-mount in situ hybridization and real-time PCR. Over-expression of Fgf8 was observed at the anterior neural ridge (ANR) in the non-treated Pdn/Pdn. Over-expression at the ANR expanded in the OTA-treated Pdn/Pdn, and it was ameliorated by pretreatment with FA. Emx2 signal was observed in the dorsal forebrain in the non-treated +/+, but disappeared in the OTA-treated +/+, and was recovered by FA. The Emx2 signal was pale and the expression amount was depressed in the non-treated and OTA-treated Pdn/Pdn embryos. It was suggested that down-regulation of Gli3 induced the over-expression of Fgf8 at the ANR, that OTA treatment accelerated the over-expression, and that pretreatment with FA ameliorated the OTA-induced over-expression of Fgf8 in the Pdn/Pdn. It was also suggested that down-regulation of Gli3 induced the down-regulation of Emx2 in the Pdn/Pdn. It was further speculated that the over-expression of Fgf8 at the ANR and down-regulation of Emx2 in the dorsal forebrain may contribute to NTD induction.

Anticonvulsant activity, neural tube defect induction, mutagenicity and pharmacokinetics of a new potent antiepileptic drug, N-methoxy-2,2,3,3-tetramethylcyclopropane carboxamide

N-methoxy-2,2,3,3-tetramethylcyclopropane carboxamide (OM-TMCD) is a methoxyamide derivative of a cyclopropyl analogue of valproic acid (VPA). The structural considerations used in the design of OM-TMCD were aimed to enhance OM-TMCD anticonvulsant potency (compared to VPA) and to prevent VPA's two life-threatening side effects, i.e., induction of neural tube defects (NTDs) and hepatotoxicity. Following i.p. administration to rats OM-TMCD demonstrated a broad spectrum of anticonvulsant activity and showed better potency than VPA in the maximal electroshock seizure and subcutaneous pentylenetetrazole tests as well as in the hippocampal kindling model. OM-TMCD was inactive in the mouse 6-Hz test at 100 mg/kg dose. Teratogenicity studies performed in a SWV/Fnn-mouse model for VPA-induced-exencephaly showed that on the equimolar basis OM-TMCD possesses the same fetal toxicity and ability to induce NTDs as VPA, but since OM-TMCD is a much more potent anticonvulsant its activity/exencephaly formation ratio appears to be much more beneficial than that of VPA. OM-TMCD was found to be non-mutagenic and non-pro-mutagenic in the Ames test. It showed a beneficial pharmacokinetic profile in rats, having a high oral bioavailability of 75% and satisfactory values of clearance and volume of distribution. These results support further studies to fully characterize the therapeutic potential of OM-TMCD.

Fumonisins: current research trends in developmental toxicology

Fumonisin B1 (FB1) is a mycotoxin produced byFusarium verticillioides that is found in maize and maize-based foods. Reproductive studies in CD1 mice, rats and rabbits initially found no evidence that fumonisins are teratogenic. However, more recent findings suggest that they might increase the risk of neural tube defects (NTDs) in populations consuming large amounts of fumonisin-contaminated corn. When ≥15 mg/kg body weight fumonisin B1 (FB1) was given to pregnant LM/Bc mice by intraperitoneal (ip) injection, all litters were positive for NTDs. To determine if NTD induction is unique to the inbred LM/Bc mouse strain, NTD induction in LM/Bc and CD1 mice was compared: (a) in a study in whichF. verticillioides culture material providing ≤150 ppm FB1 was fed to female mice before and during gestation, and (b) in a study in which FB1 was given by ip injection to CD1 dams on gestation days 7 and 8, the critical time for NTD development. In the feeding study, one of five LM/Bc litters from dams fed the 150 ppm FB1 diet was positive for NTDs whereas no NTDs were found in the CD1 litters. In the ip injection study, 40% of the litters at the highest dose tested, 45 mg/kg body weight, were positive for NTDs and one of nine low-dose (15 mg/kg body weight) litters was also positive. Thus, FB1 induced NTDs in both LM/Bc and CD1 mice although the latter strain appears less sensitive. Comparative investigations using these strains will be useful for elucidating the mechanisms underlying fumonisin-induced NTDs in mice and determining the suitability of mouse models for studying the relationships between fumonisins and NTDs in humans.

S-2-PENTYL-4-PENTYNOIC HYDROXAMIC ACID AND ITS METABOLITE S-2-PENTYL-4-PENTYNOIC ACID IN THE NMRI-EXENCEPHALY-MOUSE MODEL: PHARMACOKINETIC PROFILES, TERATOGENIC EFFECTS, AND HISTONE DEACETYLASE INHIBITION ABILITIES OF FURTHER VALPROIC ACID HYDROXAMATES AND AMIDES

Structure-activity relationship studies of valproic acid (VPA) derivatives have revealed a quantitative correlation between histone deacetylase (HDAC) inhibition and induction of neural tube defects (NTDs) in the NMRI-exencephaly-mouse model, but this correlation has been, so far, limited to congeners with a carboxylic acid function. Whereas the classical HDAC inhibitor trichostatin A is active only as a hydroxamate but not as a carboxylic acid, we found that neither VPA amides nor hydroxamates inhibit HDACs, but can cause NTDs; e.g., 2-pentyl-4-pentynoic hydroxamic acid with its S-enantiomer being the potent teratogen. We therefore investigated the hypothesis that hydroxamic acid derivatives of VPA might be metabolized in vivo and may possibly be pro-teratogenic, as had been shown for valpromide but not valproic hydroxamic acid. We developed two stereoselective quantification methods based on chiral derivatization of VPA hydroxamates with (1R,2S,5R)-(-)-menthylchloroformate and carboxylic acid derivatives with (S)-(-)-1-naphthylethylamine, followed by gas chromatography-nitrogen phosphor detector analysis of biological samples. We then determined the pharmacokinetic profiles of S-2-pentyl-4-pentynoic hydroxamic acid and of S-2-pentyl-4-pentynoic acid in mice. S-2-Pentyl-4-pentynoic hydroxamic acid was found to be extensively metabolized to the corresponding carboxylic acid without affecting the stereochemistry at position C2. Furthermore, the metabolite S-2-pentyl-4-pentynoic acid was found to be very stable in vivo, with an extended half-life of 4.2 h compared with that of VPA, 1.4 h. Comparison of the individual HDAC inhibition abilities of additional VPA amides and hydroxamates, as measured by cellular and enzymatic assays, led us to the conclusion that both classes of VPA derivatives can be pro-teratogenic.

Fetotoxicity and neural tube defects in CD1 mice exposed to the mycotoxin fumonisin B1

Fumonisins are produced by Fusarium verticillioides and occur in maize and maize-based foods. Their effects on human health are unclear, however, epidemiological and experimental evidence suggest that they increase the risk of neural tube defects (NTDs) in populations routinely consuming foods prepared from contaminated maize. When given orally to CD1 mice and other laboratory species, fumonisin B1 (FB1) was fetotoxic, but not teratogenic. However, intraperitoneal (ip) injection of FB1 to inbred LM/Bc mice at the critical time for neural tube closure elicited NTDs in a dose-dependent manner. To determine if CD1 mice are susceptible to induction of NTDs by fumonisins, 10 to 100 mg/kg body weight (BWt) FB1 was given ip to females on gestation days 7 and 8. FB1 was fetotoxic at maternal doses≥45 mg/kg BW. The number of litters with one or more NTD-affected fetuses increased in a dose-dependent manner, reaching a maximum of 55 % (six of 11 litters) at 100 mg/kg BW. A no observed adverse affect level was not established as 10 % of litters at the lowest dose, 10 mg/kg BW FB1, were NTD positive. These results demonstrated that NTD induction by FB1 in mice is not unique to the LM/Bc strain. They further showed that CD1 mice are less sensitive to NTD induction by FB 1 than LMBc mice. Comparative investigations using these and other strains will be useful for determining the mechanisms of NTD induction in fumonisin-treated mice and the relevance of these animal models to humans.

Peptide-Mediated Protection from Ethanol-Induced Neural Tube Defects

Ethanol inhibition of L1-mediated cell adhesion may contribute to the spectrum of neurological, behavioral and morphological abnormalities associated with prenatal ethanol exposure. We showed previously that the neuroprotective peptides NAPVSIPQ (NAP) and SALLRSIPA (SAL) antagonize ethanol inhibition of L1 adhesion and prevent ethanol-induced growth retardation in mouse whole embryo culture. Here we ask whether NAP and SAL also prevent ethanol-induced major malformations of the nervous system. Gestational day 8.0 (3–5 somites) C57BL/6J mouse embryos were grown for 6 h in control medium, 100 mM ethanol and 10^–10M peptides and then maintained for an additional 20 h in control medium. At the end of the culture period, only embryos having 18–19 somite pairs were examined and compared for the degree of neural tube closure. Ethanol exposure resulted in neural tube defects (NTDs) consistent with total dysraphia and anencephaly. Co-incubation with ethanol and L-NAP (all L-amino acids), D-NAP (all D-amino acids) or SAL significantly increased the percentage of embryos that had begun to close their neural folds at the level of the forebrain/midbrain junction or that had progressed beyond this stage of closure. P7A-NAP (NAPVSIAQ), which lacks neuroprotective activity, but retains activity as an antagonist of ethanol inhibition of L1 adhesion, was effective in preventing ethanol-induced NTDs. In contrast, I6A-NAP (NAPVSAPQ), which shows reduced efficacy as an ethanol antagonist but retains its neuroprotective efficacy, did not significantly diminish the induction of NTDs by ethanol. These findings demonstrate the ability of NAP and SAL to prevent ethanol-induced NTDs and support the hypothesis that ethanol teratogenesis is caused in part by ethanol inhibition of L1-mediated cell adhesion.

Anticonvulsant profile and teratogenicity of N-methyl-tetramethylcyclopropyl carboxamide: a new antiepileptic drug.

The studies presented here represent our efforts to investigate the anticonvulsant activity of N-methyl-tetramethylcyclopropyl carboxamide (M-TMCD) and its metabolite tetramethylcyclopropyl carboxamide (TMCD) in various animal (rodent) models of human epilepsy, and to evaluate their ability to induce neural tube defects (NTDs) and neurotoxicity. The anticonvulsant activity of M-TMCD and TMCD was determined after intraperitoneal (i.p.) administration to CF#1 mice, and either oral or i.p. administration to Sprague-Dawley rats. The ability of M-TMCD and TMCD to block electrical-, chemical-, or sensory-induced seizures was examined in eight animal models of epilepsy. The plasma and brain concentrations of M-TMCD and TMCD were determined in the CF#1 mice after i.p. administration. The induction of NTDs by M-TMCD and TMCD was evaluated after a single i.p. administration at day 8.5 of gestation in a highly inbred mouse strain (SWV) that is susceptible to valproic acid-induced neural tube defects. In mice, M-TMCD afforded protection against maximal electroshock (MES)-induced, pentylenetetrazol (Metrazol)-induced, and bicuculline-induced seizures, as well as against 6-Hz "psychomotor" seizures and sound-induced seizures with ED50 values of 99, 39, 81, 51, and 10 mg/kg, respectively. In rats, M-TMCD effectively prevented MES- and Metrazol-induced seizures and secondarily generalized seizures in hippocampal kindled rats (ED50 values of 82, 45, and 39 mg/kg, respectively). Unlike M-TMCD, TMCD was active only against Metrazol-induced seizures in mice and rats (ED50 values of 57 and 52 mg/kg, respectively). Neither M-TMCD nor TMCD was found to induce NTDs in SWV mice. The results obtained in this study show that M-TMCD is a broad-spectrum anticonvulsant drug that does not induce NTDs and support additional studies to evaluate its full therapeutic potential.

Antisense oligonucleotide down-regulation of E-cadherin in the yolk sac and cranial neural tube malformations.

The cadherins are a family of calcium-dependent cell adhesion molecules that are regulated both spatially and temporally during development. Epithelial cadherin (E-cadherin) is present in epithelial cells in both the embryo and yolk sac during organogenesis. The consequences of disrupting the expression of E-cadherin at this stage of development are poorly understood. We report here our studies on the effects of antisense oligonucleotides on E-cadherin in the rat whole embryo culture system. Four 18-base single strand phosphorothioate oligodeoxynucleotides (AS-oligos), complementary to various regions of the mouse E-cadherin cDNA sequence, were dissolved in saline and injected into the amniotic cavities of 5-7 somite rat embryos; a sense (S-oligo) to oligo-1, an 18-base random sequence oligo (C-oligo), and PBS were used as controls. Embryos were cultured for up to 45 h; embryo morphology and the relative concentrations of E-cadherin protein were examined. All six oligonucleotides (AS-oligos and control oligos) induced malformations when amounts ranging from 25 to 50 pmol of oligonucleotide were injected per embryo. The malformations induced by all the oligos included craniofacial hypoplasia, an enlarged pericardium, twisted spinal cord, swelling of the rhombencephalon, and underdeveloped forelimb. Injection of AS-oligo-1, a sequence starting at the tenth base downstream from the translation initiation codon (ATG), resulted in malformed embryos with a high incidence of cranial neural tube malformations. The effects of AS-oligo-1 on the relative abundance of E- and neural (N)-cadherin proteins were examined by Western blot analysis. In the AS-oligo-1-exposed malformed embryos, the relative abundance of E- and N-cadherin proteins was not altered up to 24 h after injection; E- and N-cadherin concentrations in the embryo were decreased at 45 h postinjection. In contrast, the relative abundance of the E-cadherin protein in the yolk sac was reduced at 1-2 h after injection of AS oligo-1 and returned to control levels by 4 h. S-oligo-1 did not induce any change in the relative abundance of E- or N-cadherins. Thus, there was a tissue-specific and temporary "knockdown" of E-cadherin expression in the yolk sac of embryos exposed to antisense (AS-oligo-1); the down-regulation of yolk sac E-cadherin appears to lead to the induction of neural tube defects in the embryo. The exposure of whole embryos in culture to antisense oligonucleotides provides a model system in which the roles of developmentally important molecules and their spatial and temporal contributions to embryogenesis can be elucidated.