Gene-teratogen interactions influence the penetrance of birth defects by altering Hedgehog signaling strength.

Birth defects result from interactions between genetic and environmental factors, but the mechanisms remain poorly understood. We find that mutations and teratogens interact in predictable ways to cause birth defects by changing target cell sensitivity to Hedgehog (Hh) ligands. These interactions converge on a membrane protein complex, the MMM complex, that promotes degradation of the Hh transducer Smoothened (SMO). Deficiency of the MMM component MOSMO results in elevated SMO and increased Hh signaling, causing multiple birth defects. In utero exposure to a teratogen that directly inhibits SMO reduces the penetrance and expressivity of birth defects in Mosmo-/- embryos. Additionally, tissues that develop normally in Mosmo-/- embryos are refractory to the teratogen. Thus, changes in the abundance of the protein target of a teratogen can change birth defect outcomes by quantitative shifts in Hh signaling. Consequently, small molecules that re-calibrate signaling strength could be harnessed to rescue structural birth defects.

Antiepileptic Drug Teratogenicity and De Novo Genetic Variation Load.

OBJECTIVE: The mechanisms by which antiepileptic drugs (AEDs) cause birth defects (BDs) are unknown. Data suggest that AED-induced BDs may result from a genome-wide increase of de novo variants in the embryo, a mechanism that we investigated. METHODS: Whole exome sequencing data from child-parent trios were interrogated for de novo single-nucleotide variants/indels (dnSNVs/indels) and de novo copy number variants (dnCNVs). Generalized linear models were applied to assess de novo variant burdens in children exposed prenatally to AEDs (AED-exposed children) versus children without BDs not exposed prenatally to AEDs (AED-unexposed unaffected children), and AED-exposed children with BDs versus those without BDs, adjusting for confounders. Fisher exact test was used to compare categorical data. RESULTS: Sixty-seven child-parent trios were included: 10 with AED-exposed children with BDs, 46 with AED-exposed unaffected children, and 11 with AED-unexposed unaffected children. The dnSNV/indel burden did not differ between AED-exposed children and AED-unexposed unaffected children (median dnSNV/indel number/child [range] = 3 [0-7] vs 3 [1-5], p = 0.50). Among AED-exposed children, there were no significant differences between those with BDs and those unaffected. Likely deleterious dnSNVs/indels were detected in 9 of 67 (13%) children, none of whom had BDs. The proportion of cases harboring likely deleterious dnSNVs/indels did not differ significantly between AED-unexposed and AED-exposed children. The dnCNV burden was not associated with AED exposure or birth outcome. INTERPRETATION: Our study indicates that prenatal AED exposure does not increase the burden of de novo variants, and that this mechanism is not a major contributor to AED-induced BDs. These results can be incorporated in routine patient counseling. ANN NEUROL 2020;87:897-906.

Warfarin-exposed zebrafish embryos resembles human warfarin embryopathy in a dose and developmental-time dependent manner - From molecular mechanisms to environmental concerns.

Warfarin is the most worldwide used anticoagulant drug and rodenticide. Since it crosses placental barrier it can induce warfarin embryopathy (WE), a fetal mortality in neonates characterized by skeletal deformities in addition to brain hemorrhages. Although the effects of warfarin exposure in aquatic off target species were already described, the particular molecular toxicological mechanisms during early development are still unclear. Here, we used zebrafish (Danio rerio) to describe and compare the developmental effects of warfarin exposure (0, 15.13, 75.68 and 378.43 mM) on two distinct early developmental phases (embryos and eleuthero-embryos). Although exposure to both developmental phases induced fish mortality, only embryos exposed to the highest warfarin level exhibited features mimicking mammalian WE, e.g. high mortality, higher incidence of hemorrhages and altered skeletal development, among other effects. To gain insights into the toxic mechanisms underlying warfarin exposure, the transcriptome of embryos exposed to warfarin was explored through RNA-Seq and compared to that of control embryos. 766 differentially expressed (564 up- and 202 down-regulated) genes were identified. Gene Ontology analysis revealed particular cellular components (cytoplasm, extracellular matrix, lysosome and vacuole), biological processes (mainly amino acid and lipid metabolism and response to stimulus) and pathways (oxidative stress response and apoptosis signaling pathways) being significantly overrepresented in zebrafish embryos upon warfarin exposure. Protein-protein interaction further evidenced an altered redox system, blood coagulation and vasculogenesis, visual phototransduction and collagen formation upon warfarin exposure. The present study not only describes for the first time the WE in zebrafish, it provides new insights for a better risk assessment, and highlights the need for programming the rat eradication actions outside the fish spawning season to avoid an impact on off target fish community. The urge for the development of more species-specific anticoagulants for rodent pest control is also highlighted.

Maternal dietary exposure to selenium nanoparticle led to malformation in offspring.

Selenium (Se) is an essential element and its biological activity is related to its speciation. It is also well-known that in excess it can cause teratogenesis in fish and birds. In this study we compared dietary toxicity of elemental selenium nanoparticles (SeNPs) with selenite and selenomethionine (Se-Met). Japanese medaka (Oryzias latipes) was used as a laboratory model to determine Se effects on adults and their offspring. Adult females were individually exposed using a dry diet fortified with 0, 10 or 20 µg/g of the three Se species for 7 days and then allowed to breed for 3 days. Fertilization rate and the proportion of malformed offspring were examined. The three Se diets led to significant increase in maternal tissue Se concentration in the order of Se-Met >>selenite > SeNP. However, in terms of proportion of malformed offspring, the effect of Se-Met = selenite > SeNP. The malformations included pericardial edema and craniofacial changes, which were typical for Se toxicity. The mismatch of maternal ovary Se concentration and proportion of malformed offspring suggested total Se concentration is a poor predictor of toxicity and teratogenesis. Comparing expression of four genes related to oxidative stress in maternal tissue also showed that there were significant differences in expression patterns between three Se diets in the order of selenite = SeNP > Se-Met. Our results showed that SeNPs cause similar toxicity as other Se species but require further study to elucidate the underlying mechanism.

Investigation of 7-dehydrocholesterol reductase pathway to elucidate off-target prenatal effects of pharmaceuticals: a systematic review.

Mendelian diseases contain important biological information regarding developmental effects of gene mutations that can guide drug discovery and toxicity efforts. In this review, we focus on Smith-Lemli-Opitz syndrome (SLOS), a rare Mendelian disease characterized by compound heterozygous mutations in 7-dehydrocholesterol reductase (DHCR7) resulting in severe fetal deformities. We present a compilation of SLOS-inducing DHCR7 mutations and the geographic distribution of those mutations in healthy and diseased populations. We observed that several mutations thought to be disease causing occur in healthy populations, indicating an incomplete understanding of the condition and highlighting new research opportunities. We describe the functional environment around DHCR7, including pharmacological DHCR7 inhibitors and cholesterol and vitamin D synthesis. Using PubMed, we investigated the fetal outcomes following prenatal exposure to DHCR7 modulators. First-trimester exposure to DHCR7 inhibitors resulted in outcomes similar to those of known teratogens (50 vs 48% born-healthy). DHCR7 activity should be considered during drug development and prenatal toxicity assessment.

Inhibition of the 3-hydroxy-3-methyl-glutaryl-CoA reductase induces orofacial defects in zebrafish.

BACKGROUND: Orofacial clefts (OFCs) are common birth defects, which include a range of disorders with a complex etiology affecting formation of craniofacial structures. Some forms of syndromic OFCs are produced by defects in the cholesterol pathway. The principal enzyme of the cholesterol pathway is the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). Our aim is to study whether defects of HMGCR function would produce orofacial malformation similar to those found in disorders of cholesterol synthesis. METHODS: We used zebrafish hmgcrb mutants and HMGCR inhibition assay using atorvastatin during early and late stages of orofacial morphogenesis in zebrafish. To describe craniofacial phenotypes, we stained cartilage and bone and performed in situ hybridization using known craniofacial markers. Also, we visualized neural crest cell migration in a transgenic fish. RESULTS: Our results showed that mutants displayed loss of cartilage and diminished orofacial outgrowth, and in some cases palatal cleft. Late treatments with statin show a similar phenotype. Affected-siblings displayed a moderate phenotype, whereas early-treated embryos had a minor cleft. We found reduced expression of the downstream component of Sonic Hedgehog-signaling gli1 in ventral brain, oral ectoderm, and pharyngeal endoderm in mutants and in late atorvastatin-treated embryos. CONCLUSION: Our results suggest that HMGCR loss-of-function primarily affects postmigratory cranial neural crest cells through abnormal Sonic Hedgehog signaling, probably induced by reduction in metabolites of the cholesterol pathway. Malformation severity correlates with the grade of HMGCR inhibition, developmental stage of its disruption, and probably with availability of maternal lipids. Together, our results might help to understand the spectrum of orofacial phenotypes found in cholesterol synthesis disorders. Birth Defects Research (Part A) 106:814-830, 2016. © 2016 Wiley Periodicals, Inc.

Effects of thyroxine exposure on the Twist 1 +/- phenotype: A test of gene-environment interaction modeling for craniosynostosis.

BACKGROUND: Craniosynostosis, the premature fusion of one or more of the cranial sutures, is estimated to occur in 1:1800 to 2500 births. Genetic murine models of craniosynostosis exist, but often imperfectly model human patients. Case, cohort, and surveillance studies have identified excess thyroid hormone as an agent that can either cause or exacerbate human cases of craniosynostosis. METHODS: Here we investigate the influence of in utero and in vitro exogenous thyroid hormone exposure on a murine model of craniosynostosis, Twist 1 +/-. RESULTS: By 15 days post-natal, there was evidence of coronal suture fusion in the Twist 1 +/- model, regardless of exposure. With the exception of craniofacial width, there were no significant effects of exposure; however, the Twist 1 +/- phenotype was significantly different from the wild-type control. Twist 1 +/- cranial suture cells did not respond to thyroxine treatment as measured by proliferation, osteogenic differentiation, and gene expression of osteogenic markers. However, treatment of these cells did result in modulation of thyroid associated gene expression. CONCLUSION: Our findings suggest the phenotypic effects of the genetic mutation largely outweighed the effects of thyroxine exposure in the Twist 1 +/- model. These results highlight difficultly in experimentally modeling gene-environment interactions for craniosynostotic phenotypes. Birth Defects Research (Part A) 106:803-813, 2016. © 2016 Wiley Periodicals, Inc.

In silico screening of the impact of hERG channel kinetic abnormalities on channel block and susceptibility to acquired long QT syndrome.

Accurate diagnosis of predisposition to long QT syndrome is crucial for reducing the risk of cardiac arrhythmias. In recent years, drug-induced provocative tests have proved useful to unmask some latent mutations linked to cardiac arrhythmias. In this study we expanded this concept by developing a prototype for a computational provocative screening test to reveal genetic predisposition to acquired long-QT syndrome (aLQTS). We developed a computational approach to reveal the pharmacological properties of I(Kr) blocking drugs that are most likely to cause aLQTS in the setting of subtle alterations in I(Kr) channel gating that would be expected to result from benign genetic variants.Weused themodel to predict themost potentially lethal combinations of kinetic anomalies and drug properties. In doing so, we also implicitly predicted ideal inverse therapeutic properties of K channel openers that would be expected to remedy a specific defect. We systematically performed "in silico mutagenesis" by altering discrete kinetic transition rates of the Fink et al. Markov model of human I(Kr) channels, corresponding to activation, inactivation, deactivation and recovery from inactivation of I(Kr) channels. We then screened and identified the properties of I(Kr) blockers that caused acquired long QT and therefore unmasked mutant phenotypes formild,moderate and severe variants. Mutant I(Kr) channels were incorporated into the O'Hara et al. human ventricular action potential (AP) model and subjected to simulated application of a wide variety of I(Kr)-drug interactions in order to identify the characteristics that selectively exacerbate the AP duration (APD) differences between wild-type and I(Kr) mutated cells. Our results show that drugs with disparate affinities to conformation states of the I(Kr) channel are key to amplify variants underlying susceptibility to acquired long QT syndrome, an effect that is especially pronounced at slow frequencies. Finally, we developed a mathematical formulation of the M54T MiRP1 latent mutation and simulated a provocative test. In this setting, application of dofetilide dramatically amplified the predicted QT interval duration in the M54T hMiRP1 mutation compared to wild-type.

Prenatal ethanol exposure-induced adrenal developmental abnormality of male offspring rats and its possible intrauterine programming mechanisms.

Fetal adrenal developmental status is the major determinant of fetal tissue maturation and offspring growth. We have previously proposed that prenatal ethanol exposure (PEE) suppresses fetal adrenal corticosterone (CORT) synthesis. Here, we focused on PEE-induced adrenal developmental abnormalities of male offspring rats before and after birth, and aimed to explore its intrauterine programming mechanisms. A rat model of intrauterine growth retardation (IUGR) was established by PEE (4g/kg·d). In PEE fetus, increased serum CORT concentration and decreased insulin-like growth factor 1 (IGF1) concentration, with lower bodyweight and structural abnormalities as well as a decreased Ki67 expression (proliferative marker), were observed in the male fetal adrenal cortex. Adrenal glucocorticoid (GC)-metabolic activation system was enhanced while gene expression of IGF1 signaling pathway with steroidogenic acute regulatory protein (StAR), 3ß-hydroxysteroid dehydrogenase (3ß-HSD) was decreased. Furthermore, in the male adult offspring of PEE, serum CORT level was decreased but IGF1 was increased with partial catch-up growth, and Ki67 expression demonstrated no obvious change. Adrenal GC-metabolic activation system was inhibited, while IGF1 signaling pathway and 3ß-HSD was enhanced with the steroidogenic factor 1 (SF1), and StAR was down-regulated in the adult adrenal. Based on these findings, we propose a "two-programming" mechanism for PEE-induced adrenal developmental toxicity: "the first programming" is a lower functional programming of adrenal steroidogenesis, and "the second programming" is GC-metabolic activation system-related GC-IGF1 axis programming.

Embryonic catalase protects against ethanol embryopathies in acatalasemic mice and transgenic human catalase-expressing mice in embryo culture.

Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat(b)/J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug=GD 1), exposed for 24h to 2 or 4mg/mL EtOH or vehicle, and evaluated for functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (p<0.001). Maternal pretreatment of C57BL/6 WT dams with 50kU/kg PEG-catalase (PEG-cat) 8h prior to embryo culture, which increases embryonic catalase activity, blocked all EtOH embryopathies (p<0.001). Vehicle-exposed aCat mouse embryos had lower yolk sac diameters compared to WT controls, suggesting that endogenous ROS are embryopathic. EtOH was more embryopathic in aCat embryos than WT controls, evidenced by reduced head length and somite development (p<0.01), and trends for reduced anterior neuropore closure, turning and crown-rump length. Maternal pretreatment of aCat dams with PEG-Cat blocked all EtOH embryopathies (p<0.05). These data suggest that embryonic catalase is a determinant of risk for EtOH embryopathies.

Importance of a multidisciplinary approach and monitoring in fetal warfarin syndrome.

Warfarin is a synthetic oral anticoagulant that crosses the placenta and can lead to a number of congenital abnormalities known as fetal warfarin syndrome. Our aim is to report on the follow-up from birth to age 8 years of a patient with fetal warfarin syndrome. He presented significant respiratory dysfunction, as well as dental and speech and language complications. The patient was the second child of a mother who took warfarin during pregnancy due to a metallic heart valve. The patient had respiratory dysfunction at birth. On physical examination, he had a hypoplastic nose, pectus excavatum, and clubbing of the fingers. Nasal fibrobronchoscopy showed upper airway obstruction due to narrowing of the nasal cavities. He underwent surgical correction with Max Pereira graft, zetaplasty, and osteotomies for the piriform aperture. At dental evaluation, he had caries and delayed eruption of the upper incisors. Speech and language assessment revealed high palate, mouth breathing, little nasal patency, and shortened upper lip. Auditory long latency and cognitive-related potential to auditory stimuli demonstrated functional changes in the cortical auditory pathways. We believe that the frequency of certain findings observed in our patient may be higher in fetal warfarin syndrome than is appreciated, since a significant number result in abortions, stillbirths, or children evaluated in the first year of life without a follow-up. Thus, a multidisciplinary approach and long-term monitoring of these patients may be necessary.

Epigenetic changes and disturbed neural development in a human embryonic stem cell-based model relating to the fetal valproate syndrome.

Exposure to the antiepileptic drug valproic acid (VPA) during gestation causes neurofunctional and anatomic deficits in later life. At present, there are little human data on how early neural development is affected by chemicals. We used human embryonic stem cells, differentiating to neuroectodermal precursors, as a model to investigate the modes of action of VPA. Microarray expression profiling, qPCR of specific marker genes, immunostaining and the expression of green fluorescent protein under the control of the promoter of the canonical neural precursor cell marker HES5 were used as readouts. Exposure to VPA resulted in distorted marker gene expression, characterized by a relative increase in NANOG and OCT4 and a reduction in PAX6. A similar response pattern was observed with trichostatin A, a potent and specific histone deacetylase inhibitor (HDACi), but not with several other toxicants. Differentiation markers were disturbed by prolonged, but not by acute treatment with HDACi, and the strongest disturbance of differentiation was observed by toxicant exposure during early neural fate decision. The increased acetylation of histones observed in the presence of HDACi may explain the up-regulation of some genes. However, to understand the down-regulation of PAX6 and the overall complex transcript changes, we examined further epigenetic markers. Alterations in the methylation of lysines 4 and 27 of histone H3 were detected in the promoter region of PAX6 and OCT4. The changes in these activating and silencing histone marks provide a more general mechanistic rational for the regulation of developmentally important genes at non-cytotoxic drug concentrations.

A teratologic suppressor role for p53 in benzo[a]pyrene-treated transgenic p53-deficient mice.

DNA damage may mediate birth defects caused by many drugs and environmental chemicals, therefore p53, a tumour suppressor gene that facilitates DNA repair, may be critically embryoprotective. We have studied the effects of the environmental teratogen, benzo[a]pyrene, on pregnant heterozygous p53-deficient mice. Such mice exhibited between 2- to 4-fold higher embryotoxicity and teratogenicity than normal p53-controls. Fetal resorptions reflecting in utero death were genotyped using the polymerase chain reaction and found to be increased 2.6-fold and 3.6-fold respectively with heterozygous and homozygous p53-deficient embryos. These results provide the first direct evidence that p53 may be an important teratological suppressor gene which protects the embryo from DNA-damaging chemicals and developmental oxidative stress.

Mouse mutants from chemically mutagenized embryonic stem cells.

The drive to characterize functions of human genes on a global scale has stimulated interest in large-scale generation of mouse mutants. Conventional germ-cell mutagenesis with N-ethyl-N-nitrosourea (ENU) is compromised by an inability to monitor mutation efficiency, strain and interlocus variation in mutation induction, and extensive husbandry requirements. To overcome these obstacles and develop new methods for generating mouse mutants, we devised protocols to generate germline chimaeric mice from embryonic stem (ES) cells heavily mutagenized with ethylmethanesulphonate (EMS). Germline chimaeras were derived from cultures that underwent a mutation rate of up to 1 in 1,200 at the Hprt locus (encoding hypoxanthine guanine phosphoribosyl transferase). The spectrum of mutations induced by EMS and the frameshift mutagen ICR191 was consistent with that observed in other mammalian cells. Chimaeras derived from ES cells treated with EMS transmitted mutations affecting several processes, including limb development, hair growth, hearing and gametogenesis. This technology affords several advantages over traditional mutagenesis, including the ability to conduct shortened breeding schemes and to screen for mutant phenotypes directly in ES cells or their differentiated derivatives.

Potential relation of aberrant proteolysis of human protein tyrosine kinase 7 (PTK7) chuzhoi by membrane type 1 matrix metalloproteinase (MT1-MMP) to congenital defects.

Membrane PTK7 pseudo-kinase plays an essential role in planar cell polarity and the non-canonical Wnt pathway in vertebrates. Recently, a new N-ethyl-N-nitrosourea-induced mutant named chuzhoi (chz) was isolated in mice. chz embryos have severe birth defects, including a defective neural tube, defective heart and lung development, and a shortened anterior-posterior body axis. The chz mutation was mapped to the Ala-Asn-Pro tripeptide insertion into the junction region between the fifth and the sixth Ig-like domains of PTK7. Unexpectedly, chz reduced membrane localization of the PTK7 protein. We hypothesized and then proved that the chz mutation caused an insertion of an additional membrane type 1 matrix metalloproteinase cleavage site in PTK7 and that the resulting aberrant proteolysis of chz affected the migratory parameters of the cells. It is likely that aberrations in the membrane type 1 matrix metalloproteinase/PTK7 axis are detrimental to cell movements that shape the body plan and that chz represents a novel model system for increasing our understanding of the role of proteolysis in developmental pathologies, including congenital defects.

Embryonic catalase protects against endogenous and phenytoin-enhanced DNA oxidation and embryopathies in acatalasemic and human catalase-expressing mice.

Oxidative stress and reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)), which is detoxified by catalase, are implicated in fetal death and birth defects. However, embryonic levels of catalase are only ∼ 5% of adult activity, and its protective role is not understood completely. Herein, we used mutant catalase-deficient mice [acatalasemic (aCat)] and transgenic mice expressing human catalase (hCat), which, respectively, exhibited 40-50% reductions and 2-fold elevations in the activities of embryonic and fetal brain catalase, to show that embryonic catalase protects the embryo from both physiological oxidative stress and the ROS-initiating antiepileptic drug phenytoin. Compared to wild-type (WT) catalase-normal controls, both untreated and phenytoin-exposed aCat mice exhibited a 30% increase in embryonic DNA oxidation and a >2-fold increase in embryopathies, both of which were completely blocked by protein therapy with exogenous catalase. Conversely, compared to WT controls, untreated and, to a lesser extent, phenytoin-exposed hCat mice were protected, with untreated hCat embryos exhibiting a 40% decrease in embryonic DNA oxidation and up to a 67% decrease in embryopathies. Embryonic catalase accordingly plays an important protective role, and both physiological and phenytoin-enhanced oxidative stress can be embryopathic.

Genetic basis of susceptibility to teratogen induced birth defects.

Birth defects remain the leading cause of infant death in US. The field of teratology has been focused on the causes and underlying mechanisms of birth defects for decades, yet our understanding of these critical issues remain unacceptably vague. Conclusions from years of animal and human studies made it clear that the vast majority of birth defects have multifactorial origins, with contributions from environmental and genetic factors. The environment comprises not only of the physical, biological, and chemical external environment surrounding the pregnant woman, but it also includes the internal environment of the woman's body that interact with the developing embryo in a complex fashion. The importance of maternal and embryonic genetic factors consisting of countless genetic variants/mutations that exist within every individual contribute to birth defect susceptibility is only now being more fully appreciated. This great complexity of the genome and its diversity within individuals and populations seems to be the principal reason why the same teratogenic exposure can induce severe malformation in one embryo, while fail to do so to other exposed embryos. As the interaction between genetic and environmental factors has long been recognized as the first "Principle of Teratology" by Wilson and Warkany [1965. Teratology: Principles and techniques. Chicago: University of Chicago Press], it is only recently that the appropriate investigative tools have been developed with which to fully investigate this fundamental principle. The introduction of high throughput technologies like whole genome sequencing or genome-wide association studies are promising to deliver an enormous amount of new data that will shed light on the genomic factors that contribute susceptibility to environmental teratogens. In this review, we attempt to summarize the epidemiological and experimental literature concerning birth defects whose phenotypic expression can be clearly related to the interactions between several select environmental factors and those genetic pathways in which they are most likely to have significant modifying effects. © 2011 Wiley-Liss, Inc.

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.

Gestational and family risk factors for carriers of congenital heart defects in southern Brazil.

BACKGROUND: Congenital heart disease (CHD) is a serious threat to public health. Despite this, its etiology is poorly understood and few cardiac teratogens have been defined. The aim of the present study was to identify gestational and family risk factors for CHD in a sample of patients from a pediatric hospital in southern Brazil. METHODS: A prospective and consecutive sample from subjects with or without CHD, hospitalized at a pediatric intensive care unit, was enrolled. All patients with CHD underwent a GTG-banding karyotype. Chromosomal abnormalities were observed in 47 subjects (15.8%), and these were excluded from the study. The final sample consisted of 250 CHD subjects and 303 controls. RESULTS: After statistical analysis, using logistic regression, the variables age, rural location, gestational loss, use of anti-hypertensive medication, antibiotics and alcohol in the first trimester of pregnancy were all independently associated with CHD. These results were similar to those of some studies and different from others. It should be noted, however, that, for several variables, the data in the literature as well as the present study were insufficient to determine risk. CONCLUSIONS: Some differences found may be explained by genetic factors and sociocultural diversity. In contrast, because CHD consists of a heterogeneous group of lesions, the etiology may vary. The standardization of research data and classification of methods for future studies are essential.