Loss of tumor protein 53 protects against alcohol-induced facial malformations in mice and zebrafish.

BACKGROUND: Alcohol exposure during the gastrulation stage of development causes the craniofacial and brain malformations that define fetal alcohol syndrome. These malformations, such as a deficient philtrum, are exemplified by a loss of midline tissue and correspond, at least in part, to regionally selective cell death in the embryo. The tumor suppressor protein Tp53 is an important mechanism for cell death, but the role of Tp53 in the consequences of alcohol exposure during the gastrulation stage has yet to be examined. The current studies used mice and zebrafish to test whether genetic loss of Tp53 is a conserved mechanism to protect against the effects of early developmental stage alcohol exposure. METHODS: Female mice, heterozygous for a mutation in the Tp53 gene, were mated with Tp53 heterozygous males, and the resulting embryos were exposed during gastrulation on gestational day 7 (GD 7) to alcohol (two maternal injections of 2.9 g/kg, i.p., 4 h apart) or a vehicle control. Zebrafish mutants or heterozygotes for the tp53zdf1  M214K mutation and their wild-type controls were exposed to alcohol (1.5% or 2%) beginning 6 h postfertilization (hpf), the onset of gastrulation. RESULTS: Examination of GD 17 mice revealed that eye defects were the most common phenotype among alcohol-exposed fetuses, occurring in nearly 75% of the alcohol-exposed wild-type fetuses. Tp53 gene deletion reduced the incidence of eye defects in both the heterozygous and mutant fetuses (to about 35% and 20% of fetuses, respectively) and completely protected against alcohol-induced facial malformations. Zebrafish (4 days postfertilization) also demonstrated alcohol-induced reductions of eye size and trabeculae length that were less common and less severe in tp53 mutants, indicating a protective effect of tp53 deletion. CONCLUSIONS: These results identify an evolutionarily conserved role of Tp53 as a pathogenic mechanism for alcohol-induced teratogenesis.

Quantifying Proximal Collecting Tubule Deficiency in Angiotensin-Converting Enzyme Inhibitor and Angiotensin II Receptor Blocker Fetopathy.

INTRODUCTION: Angiotensin-Converting Enzyme Inhibitors and Angiotensin II Receptor Blockers (AAs) are used for several indications, with cessation recommended in pregnancy due to toxic effects. AA fetopathy phenotype is similar to renal tubular dysgenesis including reduced proximal convoluted tubules (PCTs). Our study aimed to quantify the reduction of PCTs in fetuses and infants with prenatal exposure to AAs. MATERIALS AND METHODS: We identified 5 fetal AA exposure cases that underwent autopsy at our institution between 2011 and 2018 and compared with 5 gestational age-matched controls. Immunohistochemistry with CD10 and epithelial membrane antigen (EMA) was utilized. RESULTS: CD10 and EMA identified a median PCT density of 19.0% ± 12.3% in AA fetopathy patients, significantly less than controls (52.8% ± 4.4%; p < 0.0001). One case with in utero cessation had a PCT density of 34.2% ± 0.2%. Among other AA fetopathy findings, 1 case demonstrated unilateral renal vein thrombosis and 4 had hypocalvaria. CONCLUSIONS: We have quantified the reduction in AA fetopathy PCT density, and demonstrated in utero cessation may recover PCT differentiation. Future studies may benefit from calculating PCT percentage as a potential biomarker to correlate with post-natal renal function and maternal factors including medication type, dosage, duration, and time from medication cessation.

Role of Cbp, p300 and Akt in valproic acid induced neural tube defects in CD-1 mouse embryos.

Valproic acid (VPA), an antiepileptic and mood-stabilizing drug, is prescribed to women of reproductive age. VPA is associated with a 1-2% increase in neural tube defects in offspring following gestational exposure and results in epigenetic modifications induced by perturbations in transcription cofactors. Cbp and p300, two transcription cofactors, play key roles in embryonic neural development. p300 is a downstream target of Akt, a protein kinase B associated with cell survival and anti-apoptotic mechanisms, as part of the Akt-p300 axis. We examined the effects of in utero VPA exposure on Cbp, p300, and Akt in gestational day (GD)9, GD10 and GD13 CD-1 mouse embryos following a teratogenic maternal dose of 400 mg/kg. Embryos were collected at 0, 1, 3 and 6 h post-dosing on GD9, 24 h post-dosing on GD10 and on GD13. GD10 embryos were grouped according to the status of neural tube closure in control, closed and open groups. GD13 heads were grouped as control, exposed but non-exencephalic and exencephalic. Our data indicate that Cbp, p300 and Akt mRNA levels were downregulated at 1 and 3 h post-exposure in GD9 embryos while Cbp and p300 protein levels remained stable. Akt protein levels were significantly increased 1 h post-exposure. No significant changes were observed in either mRNA or protein expression in embryos with closed or open neural tubes compared to the control group at GD10. Downregulated expression of Cbp, p300, and Akt may play a key role in VPA-induced neural tube defects considering their vitally important role in embryonic development.

The Molecular Mechanisms of Thalidomide Teratogenicity and Implications for Modern Medicine.

Thalidomide is a teratogen that affects many organs but primarily induces limb truncations like phocomelia. Rodents are thalidomide resistant. In the 1950s, this has led to misinterpretations of animal tests and to the fatal assumption that the drug was safe for pregnant women to use against morning sickness. The result was one of the biggest scandals in medical history: 10.000 and more infants with birth defects in Europe. Nonetheless, thalidomide still has its place in modern medicine as it has strong therapeutic potential: it has been approved by the FDA for multiple myeloma and erythema nodosum leprosum, and its anti-inflammatory, immunomodulatory and antiangiogenic activities are considered in many other refractory diseases. The aim is to develop derivatives that are not teratogenic but maintain the therapeutic potential. This requires detailed knowledge about the underlying molecular mechanisms. Much progress has been made in deciphering the teratogenic mechanisms in the last decade. Here, we summarize these mechanisms, explain thalidomide resistance of rodents, and discuss possible mechanisms that could explain why the drug primarily targets the developing limb in the embryo. We also summarize the most important therapeutic mechanisms. Finally, we discuss which therapeutic and teratogenic mechanisms do and do not overlap, and if there is a chance for the development of non-teratogenic thalidomide derivatives with therapeutic potential.

Role of mutation and pharmacologic block of human KCNH2 in vasculogenesis and fetal mortality: partial rescue by transforming growth factor-ß.

BACKGROUND: N629D KCNH2 is a human missense long-QT2 mutation. Previously, we reported that the N629D/N629D mutation embryos disrupted cardiac looping, right ventricle development, and ablated IKr activity at E9.5. The present study evaluates the role of KCNH2 in vasculogenesis. METHODS AND RESULTS: N629D/N629D yolk sac vessels and aorta consist of sinusoids without normal arborization. Isolated E9.5 +/+ first branchial arches showed normal outgrowth of mouse ERG-positive/α-smooth muscle actin coimmunolocalized cells; however, outgrowth was grossly reduced in N629D/N629D. N629D/N629D aortas showed fewer α-smooth muscle actin positive cells that were not coimmunolocalized with mouse ERG cells. Transforming growth factor-ß treatment of isolated N629D/N629D embryoid bodies partially rescued this phenotype. Cultured N629D/N629D embryos recapitulate the same cardiovascular phenotypes as seen in vivo. Transforming growth factor-ß treatment significantly rescued these embryonic phenotypes. Both in vivo and in vitro, dofetilide treatment, over a narrow window of time, entirely recapitulated the N629D/N629D fetal phenotypes. Exogenous transforming growth factor-ß treatment also rescued the dofetilide-induced phenotype toward normal. CONCLUSIONS: Loss of function of KCNH2 mutations results in defects in cardiogenesis and vasculogenesis. Because many medications inadvertently block the KCNH2 potassium current, these novel findings seem to have clinical relevance.

Cadmium-induced embryopathy: nitric oxide rescues teratogenic effects of cadmium.

Although Cadmium (Cd) is a well-known heavy metal pollutant and teratogen, the mechanism behind Cd-mediated teratogenicity remains unknown. Previously, we have reported of the protective role of Nitric oxide (NO), a key signaling molecule in the embryonic developmental process, against Thalidomide-induced teratogenicity. The objective of this study was to obtain a mechanistic in-sight of the antiteratogenic potential of NO against Cd-mediated teratogenicity. To achieve this goal, we first studied the effect of Cd on the vasculature of developing embryos and then we investigated whether Cd mediated its effects by interfering with the redox regulation of NO signaling in the early development milieu. We used a chick embryonic model to determine the time and dose-dependent effects of Cd and NO recovery against Cd assault. The effects of Cd and NO recovery were assessed using various angiogenic assays. Redox and NO levels were also measured. Results demonstrated that exposure to Cd at early stage of development caused multiple birth defects in the chick embryos. Exposure to Cd suppressed endogenous NO levels and cGMP signaling, inhibiting angioblast activation and subsequently impairing yolk sac vascular development. Furthermore, Cd-induced superoxide and lipid peroxidation mediated activation of proapoptotic markers p21 and p53 in the developing embryo. Cd also caused the down-regulation of FOXO1, and up-regulation of FOXO3a and Caspase 3-mediated apoptosis. Addition of exogenous NO through a NO donor was able to blunt Cd-mediated effects and restore normal vascular and embryonic development. In conclusion, Cd-mediated teratogenicity occurs as a result of impaired NO-cGMP signaling, increased oxidative stress, and the activation of apoptotic pathways. Subsequent addition of exogenous NO through NO donor negated Cd-mediated effects and protected the developing embryo.

Comparative effects of neonatal diethylstilbestrol on external genitalia development in adult males of two mouse strains with differential estrogen sensitivity.

The effect of neonatal exposure to diethylstilbestrol (DES), a potent synthetic estrogen, was examined to evaluate whether the CD-1 (estrogen insensitive, outbred) and C57 (estrogen sensitive, inbred) mouse strains differ in their response to estrogen disruption of male ExG differentiation. CD-1 and C57BL/6 litters were injected with sesame oil or DES (200 ng/g/5 µl in sesame oil vehicle) every other day from birth to day 10. Animals were sacrificed at the following time points: birth, 5, 10 and 60 days postnatal. Neonatally DES-treated mice from both strains had many ExG abnormalities that included the following: (a) severe truncation of the prepuce and glans penis, (b) an abnormal urethral meatus, (c) ventral tethering of the penis, (d) reduced os penis length and glans width, (e) impaired differentiation of cartilage, (f) absence of urethral flaps, and (g) impaired differentiation of erectile bodies. Adverse effects of DES correlated with the expression of estrogen receptors within the affected tissues. While the effects of DES were similar in the more estrogen-sensitive C57BL/6 mice versus the less estrogen-sensitive CD-1 mice, the severity of DES effects was consistently greater in C57BL/6 mice. We suggest that many of the effects of DES, including the induction of hypospadias, are due to impaired growth and tissue fusion events during development.

Metabolic characterization of all-trans-retinoic acid (ATRA)-induced craniofacial development of murine embryos using in vivo proton magnetic resonance spectroscopy.

AIM: To characterize the abnormal metabolic profile of all-trans-retinoic acid (ATRA)-induced craniofacial development in mouse embryos using proton magnetic resonance spectroscopy (1H-MRS). METHODS: Timed-pregnant mice were treated by oral gavage on the morning of embryonic gestation day 11 (E11) with all-trans-retinoic acid (ATRA). Dosing solutions were adjusted by maternal body weight to provide 30, 70, or 100 mg/kg RA. The control group was given an equivalent volume of the carrier alone. Using an Agilent 7.0 T MR system and a combination of surface coil coils, a 3 mm×3 mm×3 mm 1H-MRS voxel was selected along the embryonic craniofacial tissue. 1H-MRS was performed with a single-voxel method using PRESS sequence and analyzed using LCModel software. Hematoxylin and eosin was used to detect and confirm cleft palate. RESULT: 1H-MRS revealed elevated choline levels in embryonic craniofacial tissue in the RA70 and RA100 groups compared to controls (P<0.05). Increased choline levels were also found in the RA70 and RA100 groups compared with the RA30 group (P<0.01). High intra-myocellular lipids at 1.30 ppm (IMCL13) in the RA100 group compared to the RA30 group were found (P<0.01). There were no significant changes in taurine, intra-myocellular lipids at 2.10 ppm (IMCL21), and extra-myocellular lipids at 2.30 ppm (EMCL23). Cleft palate formation was observed in all fetuses carried by mice administered 70 and 100 mg/kg RA. CONCLUSIONS: This novel study suggests that the elevated choline and lipid levels found by 1H-MRS may represent early biomarkers of craniofacial defects. Further studies will determine performance of this test and pathogenetic mechanisms of craniofacial malformation.

MicroRNA-34a is dispensable for p53 function as teratogenesis inducer.

The tumor suppressor protein p53 is a powerful regulator of the embryo's susceptibility to diverse teratogenic stimuli, functioning both as a teratogenesis inducer and suppressor. However, the targets that p53 engages to fulfill its functions remain largely undefined. We asked whether the microRNA (miRNA) miR-34 family, identified as one of the main targets of p53, mediates its function as a teratogenesis inducer. For this, pregnant ICR-, p53- and miR-34a-deficient mice, as well as rats, were exposed to 5-aza-2'-deoxycytidine (5-aza), a teratogen inducing limb reduction anomalies (LRA) of the hindlimbs in mice and either the hindlimbs or forelimbs in rats. Using hind- and forelimb buds of 5-aza-exposed embryos, we identified that the miR-34 family members are the most upregulated miRNAs in mouse and rat limb buds, with their increase level being significantly higher in limb buds destined for LRA. We showed that p53 mediates the 5-aza-induced miR-34 transcription followed by met proto-oncogene and growth-arrest-specific 1 target suppression in embryonic limb buds. We demonstrated that p53 regulates the teratogenic response to 5-aza acting as a teratogenesis inducer albeit miR-34a deletion does not affect the susceptibility of mice to 5-aza. Overall, our study thoroughly characterizes the expression and regulation of miR-34 family in teratogen-resistant and teratogen-sensitive embryonic structures and discusses the involvement of epigenetic miRNA-mediated pathway(s) in induced teratogenesis.

Evaluation of fetal skeletal malformations in deoxynivalenol-treated mice using microarray analysis.

Deoxynivalenol (DON [vomitoxin]), one of trichothecene mycotoxins produced by the fungus Fusarium, is commonly detected in cereal foods across the world. DON induces diverse toxic effects in humans and animals, including emesis and diarrhea, anorexia, and immunotoxicity, and impaired maternal reproduction and fetal development. Recently, the teratogenic potential of DON has been shown and has received much attention. DON can cause various skeletal deformities in fetuses, but the underlying mechanisms have not yet been fully examined. In this study, fetal skeletal malformations in DON-treated maternal mice were thoroughly investigated using microarray assay. The results showed that DON administration caused various skeletal defects in fetuses, including misaligned or fused sternebrae and vertebrae, divided or fused ribs and polydactyly, hemivertebrae, short toes, and tail anomalies. Microarray analysis showed that 282 genes, including 148 downregulated and 134 upregulated genes, were abnormally expressed in fetal vertebral bones after maternal DON exposure. These identified genes can be classified into several categories: skeletal development, carcinogenesis, nervous disorders, sperm development and embryogenesis, and inflammation. Of these, 6 genes, mostly related to bone development, were intentionally selected for further validation using real-time reverse transcription-Polymerase Chain Reaction (RT-PCR). It was confirmed that the mRNA expression of 4 genes, i.e., fibrillin-1, Col9A2, 3'-phosphoadenosine 5'-phosphosulfate synthase 2, and Pax1, was upregulated significantly by DON administration, whereas that of 2 other genes, Runx2 and parathyroid hormone-like hormone, was downregulated significantly. Taken together, the results of our study suggest that altered expression of these 6 genes plays a critical role in fetal skeletal deformities induced by DON and thus they are worthy of further investigation.

Fumonisin FB1 treatment acts synergistically with methyl donor deficiency during rat pregnancy to produce alterations of H3- and H4-histone methylation patterns in fetuses.

SCOPE: Prenatal folate and methyl donor malnutrition lead to epigenetic alterations that could enhance susceptibility to disease. Methyl-deficient diet (MDD) and fumonisin FB1 are risk factors for neural tube defects and cancers. Evidence indicates that FB1 impairs folate metabolism. METHODS AND RESULTS: Folate receptors and four heterochromatin markers were investigated in rat fetuses liver derived from dams exposed to MDD and/or FB1 administered at a dose twice higher than the provisional maximum tolerable daily intake (PMTDI = 2 µg/kg/day). Even though folate receptors transcription seemed up-regulated by methyl depletion regardless of FB1 treatment, combined MDD/FB1 exposure might reverse this up-regulation since folate receptors transcripts were lower in the MDD/FB1 versus MDD group. Methyl depletion decreased H4K20me3. Combined MDD/FB1 decreased H4K20me3 even more and increased H3K9me3. The elevated H3K9me3 can be viewed as a defense mechanism inciting the cell to resist heterochromatin disorganization. H3R2me2 and H4K16Ac varied according to this mechanism even though statistical significance was not consistent. CONCLUSION: Considering that humans are exposed to FB1 levels above the PMTDI, this study is relevant because it suggests that low doses of FB1 interact with MDD thus contributing to disrupt the epigenetic landscape.

Regulation of cardiovascular development by adenosine and adenosine-mediated embryo protection.

Few signaling molecules have as much potential to influence the developing mammal as the nucleoside adenosine. Adenosine levels increase rapidly with tissue hypoxia and inflammation. Adenosine antagonists include the methylxanthines caffeine and theophylline. The receptors that transduce adenosine action are the A1, A2a, A2b, and A3 adenosine receptors (A1AR, A2aAR, A2bAR, and A3AR). We examined how adenosine acts via A1ARs to influence embryo development. Transgenic mice were studied along with embryo cultures. Embryos lacking A1ARs were markedly growth retarded following intrauterine hypoxia exposure. Studies of mice selectively lacking A1AR in the heart identify the heart as a key site of adenosine's embryo-protective effects. Studies of isolated embryos showed that adenosine plays a key role in modulating embryo cardiac function, especially in the setting of hypoxia. When pregnant mice were treated during embryogenesis with the adenosine antagonist caffeine, adult mice had abnormal heart function. Adenosine acts via A1ARs to play an essential role in protecting the embryo against intrauterine stress, and adenosine antagonists, including caffeine, may be an unwelcome exposure for the embryo.

Reproductive toxicity of Rhizoma Sparganii (Sparganium stoloniferum Buch.-Ham.) in mice: mechanisms of anti-angiogenesis and anti-estrogen pharmacologic activities.

ETHNOPHARMACOLOGICAL RELEVANCE: Indications and preliminary studies of Rhizoma Sparganii (RS) suggest its pharmacological mechanism is involved with endocrine/angiogenesis functions. We therefore studied its potential toxicity on reproduction in mice. MATERIALS AND METHODS: Reproductive toxicity of 100, 200 and 400 mg/kg RS extract were studied in pregnant ICR mice and its offspring. The embryos' fibroblast growth factor-1 (FGF-1), vascular endothelial growth factor (VEGF) and estrogen receptor-α (ER-α) were evaluated as targets of endocrine/angiogenesis by immunohistochemical test. RESULTS: The offspring of treated mice (100, 200 and 400 mg/kg RS extract) during their pregnancy had various pathological conditions, suggesting an abnormal FGF signaling phenomenon during pregnancy. Embryos from the 400 mg/kg group had significantly depressed levels of FGF-1 (P < 0.01) and VEGF (P < 0.05) expression levels as compared to controls by immunohistochemical test. Dysplasia in the heart (12.9%), craniofacial region (18.3%) and vertebrae (32.5%) presented in embryos of the 400 mg/kg group. Furthermore, the ER-α expression was inversely proportional to FGF-1 levels in the same embryo (P < 0.01). CONCLUSIONS: These results implicate a FGF signaling abnormality in vivo and indicate that RS has anti-angiogenesis and anti-estrogen toxicity effects in pregnant rodents.

A newborn with Pierre Robin sequence after preconceptional mitoxantrone exposure of a female with multiple sclerosis.

Multiple sclerosis is a common disease of young adults in which accidental and unplanned pregnancies under disease modifying or immunosuppressive therapies may occur. The experience with mitoxantrone (MIX) especially in the first trimenon is very limited, until now only one case of a pregnant woman with MS who was exposed to MIX in early pregnancy and delivered a growth restricted but healthy child was published. We report a case of a secondary progressive MS patient who was exposed periconceptionally to MIX and delivered a child with Pierre Robin Sequence (PRS), a syndrome with the main features of glossoptosis, micrognathia, and palate clefts. PRS is a very rare defect and therefore a causal relation with MIX seems possible.

Effects of quinazolinones on Balb/C mice embryonic livers.

Heterocyclic compounds such as quinazolinones have variety of biological and pharmacological properties (anticancer, antiinflammatory, antimicrobial, antimalaria, etc.). Effects of two new quinazolinones viz., 4(3H)-quinazolinone-2-propyl-2-phenylethyl (QPPE) and 4(3H)quinazolinone-2-ethyl-2-phenylethyl (QEPE) were investigated on Balb/C mice embryos livers-the major organ of metabolism and detoxification of drugs and toxins. Histological and pathological studies demonstrated QPPE and QEPE as producers of toxic metabolites after biotransformation, creating necrosis, fatty changes, increase in the number of band cells, hepatocytes' diameters and alkaline phosphatase, in addition to sinusoid dilation, hemorrhages and hyperemia. Transmission electron micrographs showed lipid droplets in hepatocytes' cytoplasm, necrosis, vacuolization, cytoplasm disintegration, disfigured and swollen mitochondria, irregular and abnormal nuclei, nuclei with heterochromatin, condensed chromatins, myelin figures and autophages in injured hepatocytes. In conclusion, QPPE and QEPE make toxic components after biotransformation injuring membranes and creating inflammatory reactions. They also disturb metabolism of lipids pathways and cause the appearances of lipid droplets in hepatocytes.

Embryotoxicity and biotransformation responses in zebrafish exposed to water-soluble fraction of crude oil.

The toxic effects of water-soluble fraction (WSF) of crude oil (API27, Petrobras Campos Basin, Brazil) were evaluated during the early life stages of zebrafish, as well as its biotransformation in juvenile fish. Embryonic development was studied during 96 h. Reduced heartbeat rate, weak pigmentation, tail defects, and embryo mortality were observed for all of the tested concentrations of the WSF. Activities of the biotransformation enzymes were induced at the highest concentrations, showing that these enzymes played a role in its elimination. As shown in this study the crude oil WSF altered the normal embryonic development of fish.

Teratogenic effects of thalidomide: molecular mechanisms.

Fifty years ago, prescription of the sedative thalidomide caused a worldwide epidemic of multiple birth defects. The drug is now used in the treatment of leprosy and multiple myeloma. However, its use is limited due to its potent teratogenic activity. The mechanism by which thalidomide causes limb malformations and other developmental defects is a long-standing question. Multiple hypotheses exist to explain the molecular mechanism of thalidomide action. Among them, theories involving oxidative stress and anti-angiogenesis have been widely supported. Nevertheless, until recently, the direct target of thalidomide remained elusive. We identified a thalidomide-binding protein, cereblon (CRBN), as a primary target for thalidomide teratogenicity. Our data suggest that thalidomide initiates its teratogenic effects by binding to CRBN and inhibiting its ubiquitin ligase activity. In this review, we summarize the biology of thalidomide, focusing on the molecular mechanisms of its teratogenic effects. In addition, we discuss the questions still to be addressed.

MicroRNA expression changes during zebrafish development induced by perfluorooctane sulfonate.

Perfluorooctane sulfonate (PFOS), a kind of widely distributed environmentally organic compound, has been found to cause developmental toxicity. Although microRNAs (miRNAs) play an important role in many metabolic tasks, whether and how they are involved in the process of PFOS-induced toxicity is largely unknown. To address this problem, PFOS-induced changes in miRNAs and target gene expression in zebrafish embryos, and the potential mechanism of PFOS-induced toxic action were studied in this research. Zebrafish embryos were exposed to 1 µg ml(-1) PFOS or DMSO control from 6 h post-fertilization (hpf) to 24 or 120 hpf. Subsequently, RNA was isolated from the embryo pool and the expression profiles of 219 known zebrafish miRNAs were analyzed using microarray. Finally, quantitative real-time polymerase chain reaction was used to validate several miRNAs expression of microarray data. The analysis revealed that PFOS exposure induced significant changes in miRNA expression profiles. A total of 39 and 81 miRNAs showed significantly altered expression patterns after PFOS exposure 24 and 120 hpf. Of the changed miRNAs, 20 were significantly up-regulated and 19 were significantly down-regulated (p < 0.01) at 24 hpf, whereas 41 were significantly up-regulated and 40 were significantly down-regulated (p < 0.01) at 120 hpf. These miRNAs were involved in development, apoptosis and cell signal pathway, cell cycle progression and proliferation, oncogenesis, adipose metabolism and hormone secretion, whereas there is still little functional information available for 32 miRNAs. Our results demonstrate that PFOS exposure alters the expression of a suite of miRNAs and may induce developmental toxicity.

The relationship between fetal growth restriction and small placenta in 6-mercaptopurine exposed rat.

In order to investigate the effect of placental size on fetal intrauterine growth retardation (IURG), we examined the morphology and alterations in the expression of glucose transporter in the placentas of rats exposed to 6-mercaptopurine (6-MP). 6-MP was administered orally at 0 and 60 mg/kg/day on gestation day (GD) 9, 11, 13 or 15, and the placentas were sampled on GDs 17 and 21. The main findings in the treated groups were small placenta caused by mitotic inhibition and apoptosis, fetal resorption and IUGR with or without some malformations. The most sensitive period to 6-MP-induced fetal mortality was found to be in the GD9-treated group, and the small placenta and fetal abnormalities in the GD11-treated group, respectively. However, the litters in a quarter of the dams with the treatment on GD 11 had no fetotoxicity despite 25% decline in the placental weight. Histopathologically, the expression of glucose transporter GLUT3 was increased in the trophoblastic septa in all treated groups, particularly remarkable with proliferation of trophoblasts in the above litters, where the fetal-placental weight ratio was increased. Thus, we consider that the normal fetal growth and development can be maintained caused by adaptive change, even if the placental weight decreased by approximately 25% in 6-MP exposed rats.

Epigenetic modifications in valproic acid-induced teratogenesis.

Exposure to the anticonvulsant drug valproic acid (VPA) in utero is associated with a 1-2% increase in neural tube defects (NTDs), however the molecular mechanisms by which VPA induces teratogenesis are unknown. Previous studies demonstrated that VPA, a direct inhibitor of histone deacetylase, can induce histone hyperacetylation and other epigenetic changes such as histone methylation and DNA demethylation. The objective of this study was to determine if maternal exposure to VPA in mice has the ability to cause these epigenetic alterations in the embryo and thus contribute to its mechanism of teratogenesis. Pregnant CD-1 mice (GD 9.0) were administered a teratogenic dose of VPA (400mg/kg, s.c.) and embryos extracted 1, 3, 6, and 24h after injection. To assess embryonic histone acetylation and histone methylation, Western blotting was performed on whole embryo homogenates, as well as immunohistochemical staining on embryonic sections. To measure DNA methylation changes, the cytosine extension assay was performed. Results demonstrated that a significant increase in histone acetylation that peaked 3h after VPA exposure was accompanied by an increase in histone methylation at histone H3 lysine 4 (H3K4) and a decrease in histone methylation at histone H3 lysine 9 (H3K9). Immunohistochemical staining revealed increased histone acetylation in the neuroepithelium, heart, and somites. A decrease in methylated histone H3K9 staining was observed in the neuroepithelium and somites, METHYLATED histone H3K4 staining was observed in the neuroepithelium. No significant differences in global or CpG island DNA methylation were observed in embryo homogenates. These results support the possibility that epigenetic modifications caused by VPA during early mouse organogenesis results in congenital malformations.