Effect of route of administration on phenytoin teratogenicity in A/J mice

Acute administration of the anticonvulsant drug, phenytoin (PHT) has been shown to result in embryotoxicity and teratogenicity in several strains of mice. The A/J strain is reported to be most susceptible to the effects of the drug including an increased incidence of resorptions and orofacial clefts in treated animals. When administered chronically, the drug has been shown to be teratogenic in the absence of maternal toxicity and embryolethality in Swiss Webster mice [Hansen and Billings, 1985]. In this paper, we have compared the embryopathic effects of chronic and acute administrations of PHT to A/J mice. PHT was administered to pregnant females by intraperitoneal (i.p.) injection on day 10 of gestation at a dose of either 60 or 75 mg/kg body weight. Alternatively, PHT was added to ground chow and fed to animals prior to and throughout gestation; animals received a daily dose of either 60 or 75 mg/kg body weight. Pregnant animals were sacrificed on day 18 or 19 of gestation, and fetuses were examined for the presence of orofacial clefts and other anomalies. There was a significant increase in the frequency of cleft lip and palate in animals receiving the drug by i.p. administration, but there was no increase in the incidence of clefts if the drug were added to the diet. The results of this study reiterate the importance of the route of administration of a drug in determining its embryopathic effect.     

Antioxidant and GSH-related enzyme response to a single teratogenic exposure to the anticonvulsant phenytoin: temporospatial evaluation

BACKGROUND: It has been proposed that the anticonvulsant drug phenytoin (PHT) requires bioactivation to reactive intermediate(s) to achieve its recognized teratogenic potential and that embryonal detoxification power may play a fundamental role in the teratogenic response. On this basis, we sought to investigate the potential effects of a teratogenic exposure to PHT on the activities of antioxidant and GSH-related detoxifying enzymes in gestational murine tissues. METHODS: Pregnant Swiss mice were injected intraperitoneally with 0 (vehicle) or 65 mg/kg of PHT on gestation day (GD) 12 (plug day = GD 1). Biochemical determinations, including activities of glutathione transferase, glutathione peroxidase, glutathione reductase, glyoxalase I, glyoxalase II, catalase, and superoxide dismutase, were carried out on maternal and embryonic/fetal livers and in placentas on GD 14 and 19. RESULTS: The major findings of this study show that (1) organogenesis-stage conceptal tissues have detectable levels of all the tested enzymes; (2) most of the embryonic liver and placental enzymes investigated undergo a significant induction within 48 hr (GD 14) after PHT administration; and (3) in the same tissues a down-regulation of enzyme activities is noted near term (GD 19). CONCLUSIONS: Overall, these findings show that teratogenic exposure to PHT is associated with a modulation of reactive-intermediates-scavenging enzyme activities, and provide further support for role of generation of reactive intermediates in PHT-induced teratogenesis.     

Phenytoin-induced cleft palate: evidence for embryonic cardiac bradyarrhythmia due to inhibition of delayed rectifier K+ channels resulting in hypoxia-reoxygenation damage

BACKGROUND: Phenytoin (PHT) teratogenicity has been related to embryonic arrhythmia due to the capacity of PHT to block I(K) channels pharmacologically, resulting in hypoxia-reoxygenation damage. The aim of this study was to further elucidate the proposed mechanism. METHODS: Pregnant CD-1 mice were given PHT (85 mg/kg) or saline intraperitoneally on gestational days 10-11. Embryonic heart rhythm and presence of hemorrhage in orofacial region was recorded on day 12, fetuses were examined for malformations on day 18. Embryonic heart rate was also recorded on individual days after dosing days 9-16. In addition, PHT was given at doses of 10, 25, or 85 mg/kg on day 12 for analysis of plasma concentrations. RESULTS: PTH-induced bradycardia and arrhythmia in approximately 20% of the embryos, 48% showed hemorrhage in the orofacial region; 39% of the fetuses had cleft palate. The region in which hemorrhages were visible in the embryo corresponded with the region where tissue deficiency (cleft palate) was visible in the fetus at term. None of the controls showed hemorrhages, dysrhythmia, or cleft palate. PHT affected embryonic heart rates on days 9-13, but not on days 14-16. Single dose administration on day 12, the most sensitive day, resulted in a dose-dependent decrease in embryonic heart rate (12-34%). Embryonic arrhythmia occurred at 25 and 85, but not at 10 mg/kg or in the controls. Mean maternal free plasma concentrations were 6 and 14 micromol/L in the 10- and 25-mg/kg groups, respectively. CONCLUSIONS: PHT-induced cleft palate was preceded by embryonic dysrhythmia and hemorrhage in the orofacial region. Embryonic heart rhythm was phase specifically affected, as described for selective I(Kr) channel blockers, at clinically relevant concentrations. The results support the idea that PHT teratogenicity is a consequence of pharmacologically induced dysrhythmia and hypoxia-related damage.     

Embryotoxicity of phenytoin in adrenalectomized CD-1 mice.

It has been proposed that the anticonvulsant drug phenytoin (PHT) and glucocorticoids induce orofacial clefting by the same mechanism. Previous work had demonstrated that PHT treatment significantly increased endogenous maternal corticosterone concentrations for approximately 48 hr after dosing in A/J mice. The purpose of the present investigation was to determine whether PHT is embryotoxic in the absence of endogenous maternal glucocorticoids. Maternal adrenal glands were removed on Day 7 of gestation, and the incidence of clefting after PHT treatment was determined. There was a high level of maternal toxicity following adrenalectomy (ADX) and PHT treatment at either 60 or 75 mg/kg. This increased toxicity did not appear to be due to altered maternal drug levels in ADX mice. There was a significant increase in the clefting incidence among offspring of ADX dams treated with PHT at 60 mg/kg. This dose of PHT did not elevate maternal corticosterone levels in ADX dams. These data suggest that PHT is capable of producing clefts in the absence of endogenous maternal corticosterone.     

Quantity and quality of embryos collected from mice passively immunized against estradiol

The objective of this study was to determine the effects of passive immunization against estradiol on the occurrence and timing of estrus, ovulation and fertilization rates and on early embryonic development in mice. Swiss Webster fermale mice were randomly assigned to one of the three treatment groups to be injected with 0.1 ml saline (control; n=15), 0.1 ml anti-estradiol antisera (high dose: n=17) or 0.1 ml antiestradiol antisera diluted 1:10 with saline (low dose; n=17) at seven weeks of age. Immediately after injection mice were placed with males and observed daily for the presence of vaginal plugs for 10 d. Three days after vaginal plugs were observed, mice were terminated and the uteri were removed and flushed to determine the number and quality of unfertilized ova and embryos. No differences were observed in the timing of vaginal plug formation, the proportion of mice with vaginal plugs, or the mean number of unfertilized ova or embryos collected from each treatment group. However, the proportion of excellent or good quality embryos was reduced in the high dosage treatment. It was concluded from this study that passive immunization of mice against estradiol did not increase the number of embryos obtained from mice and that a high dosage of antisera against estradiol reduced embryo quality. These results provide evidence that alterations in levels of estradiol may adversely affect embryonic development.     

Methylenetetrahydrofolate reductase deficiency and low dietary folate increase embryonic delay and placental abnormalities in mice

BACKGROUND: Despite extensive research on mild methylenetetrahydrofolate reductase (MTHFR) deficiency and low dietary folate in different disorders, the association of these metabolic disturbances with a variety of congenital defects and pregnancy complications remains controversial. In this study we investigated the effects of MTHFR and dietary folate deficiency at 10.5 days post coitum (dpc) in our mouse model of mild MTHFR deficiency. METHODS: Mthfr +/+ and +/? female mice were fed a control or folic acid–deficient diet for 6 weeks, then mated with Mthfr +/? males. At 10.5 dpc, embryos were examined and placentae were collected for histologic evaluation. RESULTS: Maternal MTHFR and folate deficiencies resulted in increased developmental delays and smaller embryos. We also observed a low frequency of a variety of embryonic defects in the experimental groups, such as neural tube, heart looping, and turning defects; these results mimic the low incidence and multifactorial nature of these anomalies in humans. Folate‐deficient mice also had increased embryonic losses and severe placental defects, including placental abruption and disturbed patterning of placental layers. Folate‐deficient placentae had decreased ApoA‐I expression, and there was a trend toward a negative correlation between ApoA‐I expression with maternal homocysteine concentrations. CONCLUSIONS: Our study provides biological evidence linking maternal MTHFR and dietary folate deficiencies to adverse pregnancy outcomes in mice. It underscores the importance of folate not only in reducing the incidence of early embryonic defects, but also in the prevention of developmental delays and placental abnormalities that may increase susceptibility to other defects and to reproductive complications. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

The genetic background of the curly tail strain confers susceptibility to folate‐deficiency‐induced exencephaly

BACKGROUND : Suboptimal maternal folate status is considered a risk factor for neural tube defects (NTDs). However, the relationship between dietary folate status and risk of NTDs appears complex, as experimentally induced folate deficiency is insufficient to cause NTDs in nonmutant mice. In contrast, folate deficiency can exacerbate the effect of an NTD‐causing mutation, as in splotch mice. The purpose of the present study was to determine whether folate deficiency can induce NTDs in mice with a permissive genetic background which do not normally exhibit defects. METHODS : Folate deficiency was induced in curly tail and genetically matched wild‐type mice, and we analyzed the effect on maternal folate status, embryonic growth and development, and frequency of NTDs. RESULTS : Folate‐deficient diets resulted in reduced maternal blood folate, elevated homocysteine, and a diminished embryonic folate content. Folate deficiency had a deleterious effect on reproductive success, resulting in smaller litter sizes and an increased rate of resorption. Notably, folate deficiency caused a similar‐sized, statistically significant increase in the frequency of cranial NTDs among both curly tail (Grhl3 mutant) embryos and background‐matched embryos that are wild type for Grhl3. The latter do not exhibit NTDs under normal dietary conditions. Maternal supplementation with myo‐inositol reduced the incidence of NTDs in the folate‐deficient wild‐type strain. CONCLUSIONS : Dietary folate deficiency can induce cranial NTDs in nonmutant mice with a permissive genetic background, a situation that likely parallels gene‐nutrient interactions in human NTDs. Our findings suggest that inositol supplementation may ameliorate NTDs resulting from insufficient dietary folate. Birth Defects Research (Part A), 2010. © 2009 Wiley‐Liss, Inc.     

Late Maternal Folate Supplementation Rescues from Methyl Donor Deficiency-Associated Brain Defects by Restoring Let-7 and miR-34 Pathways

The micronutrients folate and vitamin B12 are essential for the proper development of the central nervous system, and their deficiency during pregnancy has been associated with a wide range of disorders. They act as methyl donors in the one-carbon metabolism which critically influences epigenetic mechanisms. In order to depict further underlying mechanisms, we investigated the role of let-7 and miR-34, two microRNAs regulated by methylation, on a rat model of maternal deficiency. In several countries, public health policies recommend periconceptional supplementation with folic acid. However, the question about the duration and periodicity of supplementation remains. We therefore tested maternal supply (3 mg/kg/day) during the last third of gestation from embryonic days (E) 13 to 20. Methyl donor deficiency-related developmental disorders at E20, including cerebellar and interhemispheric suture defects and atrophy of selective cerebral layers, were associated with increased brain expression (by 2.5-fold) of let-7a and miR-34a, with subsequent downregulation of their regulatory targets such as Trim71 and Notch signaling partners, respectively. These processes could be reversed by siRNA strategy in differentiating neuroprogenitors lacking folate, with improvement of their morphological characteristics. While folic acid supplementation helped restoring the levels of let-7a and miR-34a and their downstream targets, it led to a reduction of structural and functional defects taking place during the perinatal period. Our data outline the potential role of let-7 and miR-34 and their related signaling pathways in the developmental defects following gestational methyl donor deficiency and support the likely usefulness of late folate supplementation in at risk women.     

Folate Deficiency after Anticonvulsant Drugs: An Effect of Hepatic Enzyme Induction?

Serum and red cell folate levels were reduced in 59% and 58% respectively of 75 children with epilepsy attending a residential school. The degree of folate deficiency was significantly related to increased hepatic microsomal enzyme activity, assessed from increased urinary excretion of D-glucaric acid and also correlated with the daily dose of anticonvulsant taken. Anticonvulsant drugs are known to have inducing properties, and since folate is required as a cofactor in drug hydroxylations it is suggested that folate depletion results from increased demand for the cofactor after induction of drug-metabolizing enzymes. As folate deficiency may ultimately limit drug metabolism this hypothesis would explain why blood phenytoin levels decrease and fit control may worsen after correction of folate deficiency in epileptic patients.     

Phenytoin teratogenicity: hypoxia marker and effects on embryonic heart rhythm suggest an hERG-related mechanism

BACKGROUND: The antiepileptic drug phenytoin (PHT) is a human and animal teratogen. The teratogenicity has been linked to PHT-induced embryonic cardiac arrhythmia and hypoxic damage during a period when regulation of embryonic heart rhythm is highly dependent on a specific K(+) ion current (I(Kr)). PHT has been shown to inhibit I(Kr). The aims of this study were to investigate whether teratogenic doses cause embryonic hypoxia during and after the I(Kr) susceptible period and to further characterize PHT effects on embryonic heart rhythm. METHODS: Pregnant C57BL mice were administered the hypoxia marker pimonidazole followed by PHT or saline (controls) on GD 10 or GD 15. The embryos were fixed and sectioned, and the immunostained sections were analyzed with a computer assisted image analysis. Effects of PHT (0-250 microM) on heart rhythm in GD 10 embryos cultured in vitro were videotaped and then analyzed by using a digitalization technique. RESULTS: PHT dose-dependently increased the hypoxia staining (6- and 11-fold after maternal dosing of 100 and 150 mg/kg, respectively) during the period I(Kr) is expressed and functional (GD 10). In contrast, there were no differences between the PHT doses in hypoxia staining, and much less pronounced hypoxia after this period (GD 15). With increasing PHT concentrations, increased length of the interval (bradycardia) and large variations in length between individual heartbeats (arrhythmia) were recorded. CONCLUSIONS: PHT induced bradycardia/arrhythmia and severe embryonic hypoxia during the I(Kr) susceptible period, supporting the idea of an I(Kr)-arrhythmia-hypoxia-related teratogenic mechanism.     

Respiratory changes in 9-methyl PGA teratogenesis

Cellular respiration was accurately measured following dispersion of rat embryos too large for reliable assessments in the intact condition. The technique used in this study for cell dispersion of day 13 embryos effectively eliminated restrictions to oxygen diffusion caused by thickness of the intact animals and permitted valid measurements of oxygen consumption in these embryos. Significantly decreased rates of oxygen consumption were measured by the Warburg technique in such embryonic rat cells following a teratogenic maternal folate deficiency and folate-antagonism using 9-methyl pteroyl-glutamic acid. These results suggest an association between altered embryonic oxidative metabolism and induction of malformations.     

Maternal fumonisin exposure and risk for neural tube defects: mechanisms in an in vivo mouse model

BACKGROUND: Fumonisin B1 (FB1) is a mycotoxin produced by the fungus Fusarium verticillioides, a common contaminant of corn worldwide. FB1 disrupts sphingolipid biosynthesis by inhibiting the enzyme ceramide synthase, resulting in an elevation of free sphingoid bases and depletion of downstream glycosphingolipids. A relationship between maternal ingestion of FB1-contaminated corn during early pregnancy and increased risk for neural tube defects (NTDs) has recently been proposed in human populations around the world where corn is a dietary staple. The current studies provide an in vivo mouse model of FB1 teratogenicity. METHODS: Pregnant LM/Bc mice were injected with increasing doses of FB1 on GD 7.5 and 8.5, and exposed fetuses were examined for malformations. Sphingolipid profiles and (3)H-folate concentrations were measured in maternal and fetal tissues. Immunohistochemical expression of the GPI-anchored folate receptor (Folbp1) and its association with the lipid raft component, ganglioside GM1, were characterized. Rescue experiments were performed with maternal folate supplementation or administration of gangliosides. RESULTS: Maternal FB1 administration (20 mg/kg of body weight) during early gestation resulted in 79% NTDs in exposed fetuses. Sphingolipid profiles were significantly altered in maternal and embryonic tissues following exposure, and (3)H-folate levels and immunohistochemical expression of Folbp1 were reduced. Maternal folate supplementation partially rescued the NTD phenotype, whereas GM1 significantly restored folate concentrations and afforded almost complete protection against FB1-induced NTDs. CONCLUSIONS: Maternal FB1 exposure altered sphingolipid metabolism and folate concentrations in LM/Bc mice, resulting in a dose-dependent increase in NTDs that could be prevented when adequate folate levels were maintained.     

Growth of 9.5-day rat embryos in folic-acid-deficient serum

Rat embryos (9.5-day-old) were cultured for 48 hours in serum from diet-induced folic-acid-deficient rats. Resultant embryos were frequently abnormal; many were growth retarded and exhibited a defect in the turning mechanism that inverts the embryo from ventrally to dorsally convex. Affected embryos displayed abnormal twisting or kinking of the neural tube. Gross anaemia was also frequently observed, and the protein content of the embryos was markedly less than that of embryos grown in normal rat serum. Supplementation of the deficient serum with folic acid improved growth and greatly reduced the occurrence of deformities. It virtually eliminated the incidence of gross anaemia but only partially restored the protein content of the embryos to the level observed in those grown in normal rat serum. The effects of the folate deficiency could be eliminated by supplementation with normal rat serum. The data indicate that embryos have a requirement for adequate folate in order for normal growth and differentiation to take place; they also suggest that some of the embryopathic effects of maternal folate deficiency are mediated by secondary effects on maternal metabolism. This may take the form of a disturbance in the production of maternally synthesised growth factors necessary for normal embryonic development.     

Reduction of caffeine teratogenicity in mice by inducing maternal drug metabolism with beta-naphthoflavone

The effect of stimulating maternal drug metabolism on caffeine teratogenicity was investigated in C57BL/6J (cytochrome P1-450 inducible) and AKR/J (cytochrome P1-450 noninducible) mice. The inducing agent, beta-naphthoflavone (beta-NF) in corn oil, was administered intraperitoneally (IP) to dams at 20 or 80 mg/kg/d on days 9 and 10 of gestation. Teratogenic injections of 175 mg/kg/d caffeine in deionized water were administered IP on days 11 and 12 of gestation. All dams were sacrificed on day 18 of gestation, and fetuses were fixed for razor blade sectioning and skeletal examination. Caffeine, without maternal metabolism stimulation, caused similar types and rates of malformations in both strains of mice. Inducing drug metabolism during pregnancy with beta-NF protected the embryos from the congenital toxicities of large injections of caffeine. Reductions in embryolethality, limb malformations, and hematoma formation were evident in the inducible strain but not in the strain incapable of being induced. A dosage of eighty mg/kg/d was more effective than 20 mg/kg/d beta-NF in decreasing malformations, suggesting that stimulation of metabolism and caffeine-induced teratogenicity are inversely related. Rapid elimination of caffeine resulting from increasing drug metabolism with the concomitant decrease in toxicity would indicate that caffeine, and not a metabolite, is the toxicant.     

碱基切除修复基因Lig3对小鼠胚胎神经发育的影响

摘要 目的：DNA连接酶III（DNA ligase III, Lig3）基因是碱基切除修复通路中的关键基因,在胚胎发育过程中发挥重要作用,通过研究Lig3基因在叶酸代谢障碍状态下的表达情况,探讨其对小鼠胚胎神经发育的影响。方法：采用无特定病原体（specific pathogen free, SPF）级C57BL/6J成年小鼠（8-9周,18-20 g）,雌雄1:1合笼,孕鼠随机分为实验组和对照组,孕7.5天实验组腹腔注射4.5 mg/kg体重甲氨蝶呤（Methotrexate, MTX,二氢叶酸还原酶抑制剂）诱导产生叶酸代谢障碍的小鼠神经管畸形（neural tube defects, NTDs）模型,对照组腹腔注射等体积的生理盐水。孕10.5天体视显微镜下观察胎鼠的发育情况。同时利用200 nM的MTX建立叶酸代谢障碍的小鼠神经干细胞模型。在模型建立成功的基础上,应用实时荧光定量聚合酶链反应(Real time quantitative PCR,RT-qPCR)及免疫印迹（Western blot）等方法研究碱基切除修复通路相关基因Lig3的表达水平。结果：4.5 mg/kg 体重MTX处理孕鼠后胎鼠NTDs的发生率为31.1%（19/61）,而正常对照组未见胎鼠NTDs的发生。在体视显微镜下可见NTDs胎鼠神经管未闭合,而正常胎鼠发育完好。RT-qPCR检测发现叶酸代谢障碍小鼠NTDs 胚胎神经组织中Lig3 mRNA的表达水平明显低于对照组（P<0.05）。Western blot检测发现,与对照组相比,叶酸代谢障碍NTDs胎鼠神经组织中Lig3蛋白水平明显降低（P<0.05）。同时,在MTX处理的神经干细胞中,Lig3的表达水平明显低于对照组（P<0.05）。对凋亡相关蛋白Cleaved caspase-3进行检测发现MTX处理后的NTDs胎鼠神经组织及细胞模型中其表达均明显增加,表明细胞凋亡增加。结论：在叶酸代谢障碍前提下,Lig3表达降低,DNA修复功能减弱,细胞凋亡增加,导致NTDs的发生,为NTDs及出生缺陷的防控提供新思路。     

Teratogenic effects of maternal biotin deficiency on mouse embryos examined at midgestation

Pregnant mice were fed a basal diet that not only did not contain biotin, but also contained the spray-dried egg white including avidin that caused the biotin deficiency. The effects of maternal biotin deficiency on craniofacial and limb development in embryos were examined at two stages of midgestation. On day 12.6 of gestation, male and female embryos weighted less and digit development was retarded in the biotin-deficient group. On day 15.6 of gestation (dg), the embryos also weighted less and external malformations, such as micrognathia (94.8%), micromelia (41.4%), and exencephaly (11.4%), were observed. The inhibition of palatal and digit formation by biotin deficiency at midgestation is responsible for later formation of cleft palate and micromelia. On dg 12.6 the liver biotin level of biotin-deficient dams was reduced to 20% of control values. Interestingly, the biotin content of the whole embryonic body was about ninefold greater than liver biotin levels in their dams.     

Marked accumulation of valproic acid in embryonic neuroepithelium of the mouse during early organogenesis

Valproic acid, an antiepileptic drug, causes neural tube defects in mice and man. 14C-labeled valproic acid (sodium-salt) was administered to pregnant mice on days 8 and 9 of gestation (period of high sensitivity in regard to formation of neural tube defects in this species). Two dose levels of valproic acid (1 and 400 mg/kg) were used; in each case the total radioactivity administered was the same: 400 microCi/kg or 14.7 MBq/kg. Autoradiography combined with computerized densitometry revealed that in low-dose animals most of the radioactivity was confined to maternal liver and kidney, while at high doses more activity was observed in soft tissues and fluids, including amniotic fluid. In the embryo, the neuroepithelium showed the highest concentration, irrespective of dose and survival interval (30 min, 3 h, and 6 h). Upon administration of the high dose, up to five times more radioactivity (approximately 2,000 times more valproic acid) was recovered in embryonic tissues than after the low dose. It is concluded that high doses of VPA saturate the capacities of metabolism, excretion, and protein binding in the maternal organism, resulting in a higher proportion of the dose reaching the embryo, allowing more of the drug to be accumulated by the target organ, the neuroepithelium.     

Teratogenic effect of D-amphetamine sulphate: histodifferentiation and electrocardiogram pattern of mouse embryonic heart

Pregnant albino mice (ICR random-bred strain) received daily injections of 50 or 100 mg/kg body weight of D-amphetamine sulphate between days 9 and 11 of gestation. Parallel control animals were injected with saline solution. Treated mice were sacrificed on day 15 or 19 of gestation. The embryos were examined for gross malformations and direct embryonic ECG recordings were made; they were weighed, and their hearts were carefully dissected. Microscopic sections of the heart were stained with hematoxylin and eosin. It was found that high doses of D-amphetamine raised the incidence of mortality in the treated pregnant mice up to 40%. The resorption rate in the survivors was high (up to 58%) following the high dose of the drug. Up to 15% of the embryos from the treated groups showed gross malformations, including skeletal and eye malformations and exencephaly. The electrocardiogram (ECG) recordings in most 19-day-old embryos from both treated groups showed a pattern with prolonged Q-T interval (PQT), similar to that of control embryos in the intermediate developmental stages (days 14-16 of gestation). The ECG of control embryos (from day 17 on) resembled that of prenatal fetuses. Microscopically, the hearts of treated embryos showed a large number of undifferentiated cardiac myoblasts. It can be inferred that high doses of D-amphetamine affect embryonic development generally and delay the histodifferentiation of the myocardium, resulting in incomplete maturation of the cardiac muscles, thus leading to the immature ECG pattern, with PQT intervals.     

Adenoviral supply of active transforming growth factor-beta1 (TGF-beta1) did not prevent lethality in transforming growth factor-beta1-knockout embryos.

In TGF-beta1-knockout mice, TGF-beta1-null conceptuses die during embryonic development with a penetrance of lethality that depends on the mouse genetic background. Studies have suggested that transplacental passage of maternal TGF-beta1 could account for the rescue of some TGF-beta1-null embryos. Herein, we have used an adenovirus-based gene delivery system and a strain of mice where most TGF-beta1-null conceptuses die prior to parturition, to investigate whether an increase in maternal TGF-beta1 during pregnancy would rescue TGF-beta1-null embryos. A single intravenous injection of an adenovirus containing a modified version of TGF-beta1 cDNA (Ad-TGF-beta1S223/S225), coding for a biologically active form of the cytokine, induced a 20-fold increase in plasma TGF-beta1 (active and latent forms) levels for up to 3 months in adult mice. Similar levels of TGF-beta1 were detected in 13-day post co?tum (dpc) embryos from Ad-TGF-beta1-treated mothers, demonstrating an efficient maternal/fetal transfer of the cytokine. However, no increase in the frequencies of TGF-beta1-null neonates nor in day 11.5 dpc TGF-beta1-null conceptuses was observed despite elevated levels of TGF-beta1 delivered throughout gestation. In addition, we show that the high levels of TGF-beta1-titrated in the plasma from Ad-TGF-beta1S223/S225-treated mice were partly the consequence of a stimulation of an autocrine production by exogenous bioactive TGF-beta1. These results indicate that transplacental passage of TGF-beta1 was not effective in rescuing TGF-beta1-null conceptuses from embryonic lethality.     

Phenytoin teratogenicity and midgestational pharmacokinetics in mice.

Mice of the A/J and C57BL/6J (C57) strains were dosed with phenytoin (PHT) every 48 hr throughout pregnancy by gastric intubation to test the hypothesis that maternal plasma PHT concentration may be the significant factor in determining PHT reproductive and developmental toxicity. Serial serum samples were obtained from each mouse from gestation day (GD) 10-GD 12 for determination of individual dam PHT pharmacokinetics. Maximum PHT concentration and PHT AUC (area under-the-time-concentration curve) were regressed to laparotomy and fetal evaluation endpoints to determine whether significant association existed. Although serum PHT concentrations exceeded levels associated with teratogenicity (greater than 10 micrograms/ml), few major malformations were induced in either strain. However, in the A/J strain, there was a significant increased incidence of hydrocephaly and open eyelid. Regression of pharmacokinetic parameters with embryo and maternal endpoints indicated significant associations between gestational weight gain and maximum concentration measured (Cmax) or AUC in both strains. This association was also found for fetal weight in the C57 strain. In the A/J strain, the induction of decreased ossification of the sternebrae was also associated with maternal PHT concentration; however, linear regression of hydrocephaly and open eyelid to PHT concentration was not statistically significant. These results suggest that maternal plasma PHT concentration may be a quantifiable determinant of certain aspects of PHT developmental toxicity in the mouse.