Teratogenic Mechanisms Research Articles

Fungicide prochloraz induces oxidative stress and DNA damage in vitro

Prochloraz is widely used in horticulture and agriculture, e.g. as a post-harvest anti-mold treatment. Prochloraz is a known endocrine disruptor causing developmental toxicity with multiple mechanisms of action. However, data are scarce concerning other toxic effects. Since oxidative stress response, with formation of reactive oxygen species (ROS), is a common mechanism for different toxic endpoints, e.g. genotoxicity, carcinogenicity and teratogenicity, the aim of this study was to investigate if prochloraz can induce oxidative stress and/or DNA damage in human cells. A cell culture based in vitro model was used to study oxidative stress response by prochloraz, as measured by the activity of the nuclear factor erythroid 2-related factor 2 (Nrf2), a key molecule in oxidative defense mechanisms. It was observed that prochloraz induced oxidative stress in cultured human adrenocortical H295R and hepatoma HepG2 cells at non-toxic concentrations. Further, we used Comet assay to investigate the DNA damaging potential of prochloraz, and found that non-toxic concentrations of prochloraz induced DNA damage in HepG2 cells. These are novel findings, contradicting previous studies in the field of prochloraz and genotoxicity. This study reports a new mechanism by which prochloraz may exert toxicity. Our findings suggest that prochloraz might have genotoxic properties.

Thalidomide-induced limb abnormalities in a humanized CYP3A mouse model.

Thalidomide is a teratogen in humans but not in rodents. It causes multiple birth defects including malformations of limbs, ears, and other organs. However, the species-specific mechanism of thalidomide teratogenicity is not completely understood. Reproduction of the human teratogenicity of thalidomide in rodents has previously failed because of the lack of a model reflecting human drug metabolism. In addition, because the maternal metabolic effect cannot be eliminated, the migration of unchanged thalidomide to embryos is suppressed, and the metabolic activation is insufficient to develop teratogenicity. Previously, we generated transchromosomic mice containing a human cytochrome P450 (CYP) 3A cluster in which the endogenous mouse Cyp3a genes were deleted. Here, we determined whether human CYP3A or mouse Cyp3a enzyme expression was related to the species difference in a whole embryo culture system using humanized CYP3A mouse embryos. Thalidomide-treated embryos with the human CYP3A gene cluster showed limb abnormalities, and human CYP3A was expressed in the placenta, suggesting that human CYP3A in the placenta may contribute to the teratogenicity of thalidomide. These data suggest that the humanized CYP3A mouse is a useful model to predict embryonic toxicity in humans.

In Vitro Developmental Toxicology Screens: A Report on the Progress of the Methodology and Future Applications

There has been increasing focus on generation and assessment of in vitro developmental toxicology models for assessing teratogenic liability of chemicals. The driver for this focus has been to find reliable in vitro assays that will reduce or replace the use of in vivo tests for assessing teratogenicity. Such efforts may be eventually applied in testing pharmaceutical agents where a developmental toxicology assay or battery of assays may be incorporated into regulatory testing to replace one of the two species currently used in teratogenic assessment. Such assays may be eventually applied in testing a broader spectrum of chemicals, supporting efforts aligned with Tox21 strategies and responding to REACH legislation. This review describes the developmental toxicology assays that are of focus in these assessments: rodent whole embryo culture, zebrafish embryo assays, and embryonic stem cell assays. Progress on assay development as well as future directions of how these assays are envisioned to be applied for broader safety testing of chemicals are discussed. Altogether, the developmental model systems described in this review provide rich biological systems that can be utilized in better understanding teratogenic mechanisms of action of chemotypes and are promising in providing proactive safety assessment related to developmental toxicity. Continual advancements in refining/optimizing these in vitro assays are anticipated to provide a robust data set to provide thoughtful assessment of how whole animal teratogenicity evaluations can be reduced/refined in the future.

Harvesting clues from genome wide transcriptome analysis for exploring thalidomide mediated anomalies in eye development of chick embryo: Nitric oxide rectifies the thalidomide mediated anomalies by swinging back the system to normal transcriptome pattern.

Thalidomide, the notorious teratogen is known to cause various developmental abnormalities, among which a range of eye deformations are very common. From the clinical point of view, it is necessary to pinpoint the mechanisms of teratogens that tune the gene expression. However, to our knowledge, the molecular basis of eye deformities under thalidomide treatmenthas not been reported so far. Present study focuses on the possible mechanism by which thalidomide affects eye development and the role of Nitric Oxide in recovering thalidomide-mediated anomalies of eye development using chick embryo and zebrafish models with transcriptome analysis. Transcriptome analysis showed that 403 genes were up-regulated and 223 genes were down-regulated significantly in thalidomide pre-treated embryos. 8% of the significantly modulated genes have been implicated in eye development including Pax6, OTX2, Dkk1 and Shh. A wide range of biological process and molecular function was affected by thalidomide exposure. Biological Processes including structural constituent of eye lens and Molecular functions such as visual perception and retinal metabolic process formed strong annotation clustersindicating the adverse effects of thalidomide on eye development and function. Here, we have discussed the whole embryo transcriptome with the expression of PAX6, SOX2, and CRYAAgenes from developing eyes. Our experimental data showing structural and functional aspects includingeye size, lens transparency and optic nerve activity and bioinformatics analyses of transcriptome suggest that NO could partially protect thalidomide treated embryos from its devastating effects on eye development and function.

Localization of Viral Antigens Improves Understanding of Congenital Rubella Syndrome Pathophysiology

Rubella virus (RV) is responsible for benign postnatal and deadly antenatal infections. Its involvement in developmental defects in children following congenital transmission was first made by Sir Norman McAlister Gregg in 1941. Indeed, the maternal primary infection results in an RV viremia and placental transfer. If it occurs before 12 weeks of gestation, during organogenesis, it can result in severe fetal damage causing miscarriage or malformations primarily affecting the cardiovascular system, eyes, ears and the central nervous system; it is fatal in nearly one third of cases during the first year of life (Tondury and Smith, 1966). This severe RV infection acquired in utero early in pregnancy is named as Congenital Rubella Syndrome or CRS. In 2014, 141 CRS cases were reported from 114 countries mostly from South-East Asia (n = 86/141), although reporting is probably inconsistent according to the authors themselves (Grant et al., 2015). This number had been estimated as closer to 100,000 cases each year in developing countries alone (Cutts and Vynnycky, 1999). To better understand the pathogenesis of CRS in the absence of an animal model, the first histopathological explorations were conducted in some of the 20,000 fetuses infected during the 1964–1965 epidemics in the US when about 12.5 M Rubella cases were reported (National Communicable Disease Center, 1969). Although these studies identified histological lesions in the heart, blood vessels, crystalline lens, ears, brain, teeth and liver consistent with clinical features and sequelae of CRS, they did not solved the mechanisms of virus teratogenicity (Tondury and Smith, 1966, Esterly and Oppenheimer, 1969). In particular, until recently, the exact nature of the cell types infected with RV remained unknown.

Excessive feedback of Cyp26a1 promotes cell non-autonomous loss of retinoic acid signaling

Teratogenic levels of retinoic acid (RA) signaling can cause seemingly contradictory phenotypes indicative of both increases and decreases of RA signaling. However, the mechanisms underlying these contradictory phenotypes are not completely understood. Here, we report that using a hyperactive RA receptor to enhance RA signaling in zebrafish embryos leads to defects associated with gain and loss of RA signaling. While the gain-of-function phenotypes arise from an initial increase in RA signaling, using genetic epistasis analysis we found that the loss-of-function phenotypes result from a clearing of embryonic RA that requires a rapid and dramatic increase in cyp26a1 expression. Thus, the sensitivity of cyp26a1 expression to increased RA signaling causes an overcompensation of negative feedback and loss of embryonic RA signaling. Additionally, we used blastula transplantation experiments to test if Cyp26a1, despite its cellular localization, can limit RA exposure to neighboring cells. We find that enhanced Cyp26a1 expression limits RA signaling in the local environment, thus providing the first direct evidence that Cyp26 enzymes can have cell non-autonomous consequences on RA levels within tissues. Therefore, our results provide novel insights into the teratogenic mechanisms of RA signaling and the cellular mechanisms by which Cyp26a1 expression can shape a RA gradient.

Reprotoxicity of the antifoulant chlorothalonil in ascidians: an ecological risk assessment.

Chlorothalonil is a widely used biocide in antifouling paint formulation that replaces tin-based compounds after their definitive ban. Although chlorothalonil inputs into the marine environment have significantly increased in recent years, little is known about its effect on marine animals and in particular on their reproductive processes. In this line, the aim of the present study was to investigate the effects of chlorothalonil exposure on the gamete physiology, fertilization rate and transmissible damage to offspring in the marine invertebrate Ciona intestinalis (ascidians). To identify a possible mechanism of action of chlorothalonil, electrophysiological techniques were used to study the impact on oocyte membrane excitability and on the electrical events occurring at fertilization. The pre-exposure of spermatozoa and oocytes to chlorothalonil did not affect the fertilization rate but caused damage to the offspring by inducing larval malformation. The highest toxicity was observed when fertilization was performed in chlorothalonil solutions with the lowest EC50 value. In particular, it was observed that low chlorothalonil concentrations interfered with embryo development and led to abnormal larvae, whereas high concentrations arrested embryo formation. In mature oocytes, a decrease in the amplitudes of the sodium and fertilization currents was observed, suggesting an involvement of plasma membrane ion currents in the teratogenic mechanism of chlorothalonil action. The risk estimation confirmed that the predicted no-effect concentration (PNEC) exceeded the predicted effect concentration (PEC), thus indicating that chlorothalonil may pose a risk to aquatic species.

Abstract 2279: Adenine nucleotide translocase-2 is a key molecule in the enhancement of TRAIL sensitivity by flavonoids

Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine which belongs to the tumor necrosis factor family. TRAIL binds to death receptor (DR) 4 and DR5 and selectively induces apoptosis in various malignant tumors, but not in normal cells. Therefore, TRAIL is an attractive cancer therapeutic agent. However, several malignant tumors have resistance to TRAIL and overcoming this resistance is essential for chemotherapy using the TRAIL pathway. Anticancer agents such as bortezomib, etoposide, doxorubicin, and irinotecan have been reported to upregulate DR5 and sensitize cancer cells to TRAIL in vitro and in vivo. Since these agents cause various adverse effects, we and others have screened dietary compounds inducing DR5 expression and identified several flavonoids as DR5 inducers. For instance, dietary flavonoid apigenin and quercetin enhance TRAIL sensitivity by upregulating DR5. However, the mechanisms by which these flavonoids upregulate DR5 are unknown. To elucidate the mechanisms by which these flavonoids induce DR5, we explored the proteins binding to apigenin and quercetin, but not the flavonoid genistein which did not induce DR5. We used magnetic FG beads, which recently revealed the target of thalidomide and the mechanism of thalidomide teratogenicity. We identified ANT2 as a binding protein of flavonoids, apigenin and quercetin, upregulating DR5. As with the treatment of apigenin and quercetin, knockdown of ANT2 enhanced TRAIL-induced apoptosis by upregulating DR5. Moreover, knockdown of ANT2 attenuated the enhancement of TRAIL-mediated apoptosis by apigenin and quercetin. In the present study, we show that ANT2 is a target of flavonoids which upregulate DR5 and sensitize malignant tumor cells to TRAIL. Citation Format: Masakatsu Oishi, Yosuke Iizumi, Takashi Ueda, Saya Ito, Terukazu Nakamura, Fumiya Hongo, Yoshio Naya, Tsuneharu Miki, Toshiyuki Sakai. Adenine nucleotide translocase-2 is a key molecule in the enhancement of TRAIL sensitivity by flavonoids. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2279. doi:10.1158/1538-7445.AM2014-2279

Ocular Involvement in Fetal Alcohol Spectrum Disorder: A Review

Fetal Alcohol Syndrome (FAS), the most severe manifestation of Fetal Alcohol Spectrum Disorder (FASD) is considered the leading non-hereditary cause of mental retardation and neurological deficit in the Western world. There lie a huge associated human cost to both FASD victims and their families and a considerable financial burden. This problem is being tackled on many fronts including community awareness programs, biomarker development for fetal alcohol exposure, research into preventative treatments and the development of more robust diagnostic systems for the early detection of FASD. Although ethanol can affect many of the major systems of the body, the eye is a primary target. Ocular aberrations including optic nerve hypoplasia, tortuosity of retinal vessels, coloboma and microphthalmia are frequently observed in children diagnosed with FAS. In this regard, ocular involvement in FAS has gained importance, particularly in relation to early diagnosis and identification of FAS. Furthermore, our considerable knowledge of the molecular mechanisms underlying eye development has provided a powerful tool for the investigation of the teratogenic actions of ethanol. In this review, we initially provide an overview of FASD in terms of historical background, epidemiology and current status. Next, we explore the role of ocular involvement in FASD and the use of eye measurements in the diagnosis of FAS. Lastly, we review how current knowledge of early eye development can be used to gain new insights into the molecular mechanisms of ethanol teratogenicity with particular reference to the sonic hedgehog pathway.

Chronic prenatal ethanol exposure alters expression of central and peripheral insulin signaling molecules in adult guinea pig offspring

Maternal ethanol consumption during pregnancy can produce a range of teratogenic outcomes in offspring. The mechanism of ethanol teratogenicity is multi-faceted, but may involve alterations in insulin and insulin-like growth factor (IGF) signaling pathways. These pathways are not only important for metabolism, but are also critically involved in neuronal survival and plasticity, and they can be altered by chronic prenatal ethanol exposure (CPEE). The objective of this study was to test the hypothesis that CPEE alters expression of insulin and IGF signaling molecules in the prefrontal cortex and liver of adult guinea pig offspring. Pregnant Dunkin-Hartley-strain guinea pigs received ethanol (4 g/kg maternal body weight/day) or isocaloric-sucrose/pair-feeding (nutritional control) throughout gestation. Fasting blood glucose concentration was measured in male and female offspring at postnatal day 150–200, followed by euthanasia, collection of prefrontal cortex and liver, and RNA extraction. IGF-1, IGF-1 receptor (IGF-1R), IGF-2, IGF-2 receptor (IGF-2R), insulin receptor substrate (IRS)-1, IRS-2, and insulin receptor (INSR) mRNA expression levels were measured in tissues using quantitative real-time PCR. The mean maternal blood ethanol concentration was 281 ± 15 mg/dL at 1 h after the second divided dose of ethanol on GD 57. CPEE resulted in increased liver weight in adult offspring, but produced no difference in fasting blood glucose concentration compared with nutritional control. In the liver, CPEE decreased mRNA expression of IGF-1, IGF-1R, and IGF-2, and increased IRS-2 mRNA expression in male offspring only compared with nutritional control. Female CPEE offspring had decreased INSR hepatic mRNA expression compared with male CPEE offspring. In the prefrontal cortex, IRS-2 mRNA expression was increased in CPEE offspring compared with nutritional control. The data demonstrate that CPEE alters both central and peripheral expression of insulin and IGF signaling molecules at the mRNA level, which may be related to metabolic dysregulation in adult offspring. Furthermore, altered insulin and IGF signaling may be a mechanism of ethanol neurobehavioral teratogenicity.

Commentary: catching a conserved mechanism of ethanol teratogenicity.

Due to its profound impact on human development, ethanol (EtOH) teratogenicity is a field of intense study. The complexity of variables that influence the outcomes of embryonic or prenatal EtOH exposure compels the use of animal models in which these variables can be isolated. Numerous model systems have been used in these studies. The zebrafish is a powerful model system, which has seen a recent increase in usage for EtOH studies. Those using zebrafish for alcohol studies often face 2 questions: (i) How physiologically relevant are the doses of EtOH administered to zebrafish embryos? and (ii) Will the mechanisms of EtOH teratogenesis be conserved to other model systems and human? The current article by Flentke and colleagues () helps to shed important light on these questions and clearly demonstrates that the zebrafish will be a valuable model system with which to understand EtOH teratogenicity.

Fetal alcohol spectrum disorders.

Prenatal alcohol exposure (PAE) is one of the most prevalent and modifiable risk factors for somatic, behavioral, and neurological abnormalities. Affected individuals exhibit a wide range of such features referred to as fetal alcohol spectrum disorders (FASD). These are characterized by a more or less specific pattern of minor facial dysmorphic features, growth deficiency and central nervous system symptoms. Nevertheless, whereas the diagnosis of the full-blown fetal alcohol syndrome does not pose a major challenge, only a tentative diagnosis of FASD can be reached if only mild features are present and/or maternal alcohol consumption during pregnancy cannot be verified. The respective disorders have lifelong implications. The teratogenic mechanisms induced by PAE can lead to various additional somatic findings and structural abnormalities of cerebrum and cerebellum. At the functional level, cognition, motor coordination, attention, language development, executive functions, memory, social perception and emotion processing are impaired to a variable extent. The long-term development is characterized by disruption and failure in many domains; an age-adequate independency is frequently not achieved. In addition to primary prevention, individual therapeutic interventions and tertiary prevention are warranted; provision of extensive education to affected subjects and their caregivers is crucial. Protective environments are often required to prevent negative consequences such as delinquency, indebtedness or experience of physical/sexual abuse.

Teratogenicity: a mechanism based short review on common teratogenic agents

Teratogenicity or reproductive and developmental toxicity has increasingly been recognized as a most important part of overall toxicology. Each compound has its own unique toxicological profile and mechanism of teratogenicity. The present view-point was to explore and review the teratogenic mechanism and common teratogenic effects of teratogenic compounds, biological agents and physical agents. Every year millions of different chemicals are produced and used in the world which contaminate environment and consequently are exposed to human. Many of these chemicals have ability to penetrate into human tissue and developing foetus, negatively impacting the reproductive health of human. Many of the teratogenic effects occur because of biological and physical factors like infection and radiation respectively.

Alteration of bioelectrically-controlled processes in the embryo: a teratogenic mechanism for anticonvulsants

Maternal use of anticonvulsants during the first trimester of pregnancy has been associated with an elevated risk of major congenital malformations in the offspring. Whether the increased risk is caused by the specific pharmacological mechanisms of certain anticonvulsants, the underlying epilepsy, or common genetic or environmental risk factors shared by epilepsy and malformations has been controversial. We hypothesize that anticonvulsant therapies during pregnancy that attain more successful inhibition of neurotransmission might lead to both better seizure control in the mother and stronger alteration of bioelectrically-controlled processes in the embryo that result in structural malformations. We propose that development of pharmaceuticals that do not alter cell resting transmembrane voltage levels could result in safer drugs.

Effects of macrolide antibiotics on rat embryonic heart function in vitro.

The Swedish Medical Product Agency (MPA) has listed erythromycin as a suggested human teratogen, causing cardiovascular malformations. It is further suggested that this may be a class effect of macrolide antibiotics. The proposed teratogenic mechanism is blockade of the human ether-á-go-go-related (hERG)/IKr current in the embryonic heart causing bradycardia and arrhythmia resulting in altered cardiac blood flow and/or embryonic hypoxia. To test this hypothesis, we examined the effect of three macrolide antibiotics on the function of the rat embryonic heart. Gestational day 13 rat embryos in vitro were exposed to erythromycin (25-500 μM), clarithromycin (25-500 μM), or azithromycin (100 μM to 1 mM) for 3 hr. The effect on the embryonic heart was monitored every hour. The results showed that erythromycin and clarithromycin caused a concentration-dependent bradycardia. Twenty-five micromolar was a no-effect concentration for erythromycin and was close to a no-effect concentration for clarithromycin. Azithromycin only caused significant bradycardia at 1 mM. Additional studies were performed with the embryos cultured at 40°C instead of 38°C, to mimic fever. The increased temperature increased the number of arrhythmias but did not worsen the drug-induced bradycardia. The results support the concept that erythromycin and clarithromycin can adversely affect the embryonic heart but only at concentrations well outside expected embryonic exposure in the human.

Teratogenic Mechanisms Associated with Prenatal Medication Exposure

Birth defects may originate through multiple mechanisms and may be caused by a variety of possible exposures, including medications in early pregnancy. In this review, we describe six principal teratogenic mechanisms suspected to be associated with medication use: folate antagonism, neural crest cell disruption, endocrine disruption, oxidative stress, vascular disruption, and specific receptor- or enzyme-mediated teratogenesis. Knowledge about these mechanisms, for some of which evidence is mainly derived from animal models, may not only be relevant for etiologic and post-marketing research, but may also have implications for prescribing behavior for women of reproductive age. Since combinations of seemingly unrelated medications may have effects through similar teratogenic mechanisms, the risk of birth defects may be strongly increased in multi-therapy.

Human Cytochrome P450 Oxidation of 5-Hydroxythalidomide and Pomalidomide, an Amino Analogue of Thalidomide

The sedative and antiemetic drug thalidomide [α-(N-phthalimido)glutarimide] was withdrawn in the early 1960s because of its potent teratogenic effects but was approved for the treatment of lesions associated with leprosy in 1998 and multiple myeloma in 2006. The mechanism of teratogenicity of thalidomide still remains unclear, but it is well-established that metabolism of thalidomide is important for both teratogenicity and cancer treatment outcome. Thalidomide is oxidized by various cytochrome P450 (P450) enzymes, the major one being P450 2C19, to 5-hydroxy-, 5'-hydroxy-, and dihydroxythalidomide. We previously reported that P450 3A4 oxidizes thalidomide to the 5-hydroxy and dihydroxy metabolites, with the second oxidation step involving a reactive intermediate, possibly an arene oxide, that can be trapped by glutathione (GSH) to GSH adducts. We now show that the dihydroxythalidomide metabolite can be further oxidized to a quinone intermediate. Human P450s 2J2, 2C18, and 4A11 were also found to oxidize 5-hydroxythalidomide to dihydroxy products. Unlike P450s 2C19 and 3A4, neither P450 2J2, 2C18, nor 4A11 oxidized thalidomide itself. A recently approved amino analogue of thalidomide, pomalidomide (CC-4047, Actimid), was also oxidized by human liver microsomes and P450s 2C19, 3A4, and 2J2 to the corresponding phthalimide ring-hydroxylated product.

A High-Throughput Screen for Teratogens Using Human Pluripotent Stem Cells

There is need in the pharmaceutical and chemical industries for high-throughput human cell-based assays for identifying hazardous chemicals, thereby reducing the overall reliance on animal studies for predicting the risk of toxic responses in humans. Despite instances of human-specific teratogens such as thalidomide, the use of human cell-teratogenicity assays has just started to be explored. Herein, a human pluripotent stem cell test (hPST) for identifying teratogens is described, benchmarking the in vitro findings to traditional preclinical toxicology teratogenicity studies and when available to teratogenic outcomes in humans. The hPST method employs a 3-day monolayer directed differentiation of human embryonic stem cells. The teratogenic risk of a compound is gauged by measuring the reduction in nuclear translocation of the transcription factor SOX17 in mesendodermal cells. Decreased nuclear SOX17 in the hPST model was strongly correlated with in vivo teratogenicity. Specifically, 71 drug-like compounds with known in vivo effects, including thalidomide, were examined in the hPST. A threshold of 5 μM demonstrated 94% accuracy (97% sensitivity and 92% specificity). Furthermore, 15 environmental toxicants with physicochemical properties distinct from small molecule pharmaceutical agents were examined and a similarly strong concordance with teratogenicity outcomes from in vivo studies was observed. Finally, to assess the suitability of the hPST for high-throughput screens, a small library of 300 kinase inhibitors was tested, demonstrating the hPST platform's utility for interrogating teratogenic mechanisms and drug safety prediction. Thus, the hPST assay is a robust predictor of teratogenicity and appears to be an improvement over existing in vitro models.

Developmental exposure to valproic acid alters the expression of microRNAs involved in neurodevelopment in zebrafish

Congenital malformations are a prevalent cause of infant mortality in the United States and their induction has been linked to a variety of factors, including exposure to teratogens. However, the molecular mechanisms of teratogenicity are not fully understood. MicroRNAs are an important group of small, non-coding RNAs that regulate mRNA expression. MicroRNA roles in early embryonic development are well established, and their disruption during development can cause abnormalities. We hypothesized that developmental exposure to teratogens such as valproic acid alters microRNA expression profiles in developing embryos. Valproic acid is an anticonvulsant and mood-stabilizing drug used to treat epilepsy, bipolar disorder and migraines. To examine the effects of valproic acid on microRNA expression during development, we used zebrafish embryos as a model vertebrate developmental system. Zebrafish embryos were continuously exposed to valproic acid (1mM) or vehicle control (ethanol) starting from 4h post-fertilization (hpf) and sampled at 48 and 96hpf to determine the miRNA expression profiles prior to and after the onset of developmental defects. At 96hpf, 95% of the larvae showed skeletal deformities, abnormal swimming behavior, and pericardial effusion. Microarray expression profiling was done using Agilent zebrafish miRNA microarrays. Microarray results revealed changes in miRNA expression at both time points. Thirteen miRNAs were differentially expressed at 48hpf and 22 miRNAs were altered at 96hpf. Among them, six miRNAs (miR-16a, 18c, 122, 132, 457b, and 724) were common to both time points. Bioinformatic target prediction and examination of published literature revealed that these miRNAs target several genes involved in the normal functioning of the central nervous system. These results suggest that the teratogenic effects of valproic acid could involve altered miRNA expression.

Drugs associated with teratogenic mechanisms. Part II: a literature review of the evidence on human risks

What is the current state of knowledge on the human risks of drugs suspected to be associated with teratogenic mechanisms? Evidence for the presence or absence of human risks of birth defects is scarce or non-existent for the majority of drugs associated with teratogenic mechanisms. Medical drugs suspected to be associated with teratogenic mechanisms are dispensed to a significant proportion of women in the first trimester of pregnancy. However, an overview of the current state of knowledge on the human teratogenic effects of these drugs is lacking. We performed an extensive literature review of studies in the English language which examined the associations between selected drugs and specific birth defects. The literature was identified from MEDLINE and EMBASE from database inception (January 1946 and January 1974, respectively) through December 2012 using 287 terms for the drugs of interest. We only included studies if they specified birth defect subtypes and, specifically for cohort studies, involved live born infants. Of 14 406 potentially relevant articles, 556 full-text articles were assessed for eligibility and 250 met the inclusion criteria. The studies included were divided into four categories according to their design to increase the validity of our study. Epidemiologic studies assessing teratogenic risks were identified for less than half of the drugs included in the review. A substantial variation in study design and data collection methods was observed. When the data collection method is of questionable validity, study quality may be affected considerably. For only 15 drugs of interest, birth defects were assessed in at least 1000 infants in cohort studies, and 13 of these were associated with one or more specific birth defects. The majority of associations observed in case-control studies are as yet unconfirmed. For most drugs and drug groups, however, the numbers of exposed infants studied were too small to draw any conclusions regarding their human teratogenic risks. The validity of our review is limited by the validity and reporting of the studies from which the data were extracted. Some relevant studies might have been missed owing to the exclusion of articles not in the English language and publication bias. It is a cause of concern that the drugs most often dispensed in the first trimester of pregnancy are not necessarily the drugs for which teratogenic risks have been studied. Future studies should focus on those drugs that are most commonly used during pregnancy and for which the teratogenic risks are unknown, such as iron preparations, serotonin receptor agonists or antagonists, drugs used in fertility treatment, dihydrofolate reductase inhibitors. Marleen van Gelder was supported by the Netherlands Organisation for Scientific Research/NWO (grant no. 021.001.008). No competing interests are declared. N/A.