Teratogenic Effects Of Ethanol Research Articles

The ameliorative effects of choline on ethanol-induced cell death in the neural tube of susceptible BXD strains of mice.

Fetal alcohol spectrum disorders (FASD) are the leading preventable cause of intellectual disability, providing the impetus for evaluating various potential treatments to ameliorate ethanol's teratogenic effects, particularly in the nervous system. One treatment is the dietary supplement choline which has been shown to mitigate at least some of ethanol's teratogenic effects. The present study was designed to investigate the effects of genetics on choline's efficacy in ameliorating cell death in the developing neural tube. Previously, we examined BXD recombinant inbred mice, and their parental C57BL/6 J (B6) and DBA/2 J strains, and identified strains that were sensitive to ethanol's teratogenic actions. Thus, we used these strains to identify response to choline treatment. Timed pregnant mice from 4 strains (B6, BXD51, BXD73, BXD2) were given either ethanol or isocaloric maltose-dextrin (5.8 g/kg in two administrations separated by 2 h) with choline at one of 3 doses: 0, 100 or 250 mg/kg. Subjects were exposed via intragastric gavage on embryonic day 9 and embryos were collected 7 h after the initial ethanol administrations. Cell death was analyzed using TUNEL staining in the developing forebrain and brainstem. Choline ameliorated the ethanol-induced cell death across all 4 strains without causing enhanced cell death in control mice. Choline was effective in both the developing telencephalon and in the brainstem. Both doses diminished cell death, with some differences across strains and brain regions, although the 100 mg/kg dose was most consistent in mitigating ethanol-related cell death. Comparisons across strains showed that there was an effect of strain, particularly in the forebrain at the higher dose. These results show that choline is effective in ameliorating ethanol-induced cell death at this early stage of nervous system development. However, there were some strain differences in its efficacy, especially at the high dose, providing further evidence of the importance of genetics in influencing the ability of choline to protect against prenatal alcohol exposure.

<i>In vivo</i> influence of S-adenosylmethionine supplementation on ethanol teratogenicity in the rat

Studies have shown that maternal alcohol consumption during pregnancy negatively impacts on prenatal development. Ethanol-induced alteration in S-adenosylmethionine (SAMe) metabolism and subsequent depletion of SAMe may be a mechanism involved in fetal ethanol effects. The aim of the present study was to determine the teratogenicity of ethanol and to investigate protective effects of SAMe. Pregnant Sprague-Dawley rats, weighing 220-250 g were used. Pregnant animals were treated from day 6 through 12 of gestation with ethanol alone (6.4% ethanol) or ethanol plus SAMe (200 mg/kg). The groups for the day-20 and day-12 experiments included: Group I (ethanol liquid diet: unrestricted-fed); Group II (isocaloric maltose-dextrin liquid diet: restricted-fed); and Group III (ethanol liquid diet + SAMe: unrestricted-fed). Embryos and fetuses were recovered on gestational day-12 or day-20, respectively, and were quantitatively and qualitatively assessed for developmental anomalies. The data on continuous variables were analyzed using paired T-Test and categorical embryonic development data were processed using Chi-Square Analysis. In the present study, prenatal growth and developmental retardations such as significantly reduced crown-rump length, anomalies of the central nervous system, the heart, limbs and the skeleton were observed in embryos and fetuses of ethanol treated rats. SAMe supplementation of the ethanol treated pregnant rats significantly improved litter weight, from 20.9 + 1.6 g to 23.6 + 0.8 g, and crown-rump length from 25.8 + 0.8 mm to 27.6 + 0.4 mm, and decreased the incidence of resorptions from 1.9 + 0.6 to 0.5 + 0.8 in day-20 fetuses. It also significantly increased embryonic protein content from 173 + 36 μg to 251 + 29 μg and significantly improved the development of brain vesicles and closure of the posterior neuropore in day-12 embryos. The present study clearly showed that ethanol is teratogenic in vivo . SAMe supplementation of ethanol treated pregnant rats partially ameliorated the teratogenic effects of ethanol. This indicated that the observed ethanol related growth and developmental abnormalities may have been, at least partly, due to ethanol-induced SAMe deficiency. Further studies should consider extending the treatment period to the entire gestational period instead of limiting it to only the days covering the period of organogenesis. Keywords: ethanol teratogenicity, Sprague-Dawley rats, S-adenosylmethionine, embryos, fetuses

Ethanol itself is a holoprosencephaly-inducing teratogen.

Ethanol is a teratogen, inducing a variety of structural defects in developing humans and animals that are exposed in utero. Mechanisms of ethanol teratogenicity in specific defects are not well understood. Oxidative metabolism of ethanol by alcohol dehydrogenase or cytochrome P450 2E1 has been implicated in some of ethanol’s teratogenic effects, either via production of acetaldehyde or competitive inhibition of retinoic acid synthesis. Generalized oxidative stress in response to ethanol may also play a role in its teratogenicity. Among the developmental defects that ethanol has been implicated in is holoprosencephaly, a failure to define the midline of the forebrain and midface that is associated with a deficiency in Sonic hedgehog pathway function. Etiologically, holoprosencephaly is thought to arise from a complex combination of genetic and environmental factors. We have developed a gene-environment interaction model of holoprosencephaly in mice, in which mutation of the Sonic hedgehog coreceptor, Cdon, synergizes with transient in utero exposure to ethanol. This system was used to address whether oxidative metabolism is required for ethanol’s teratogenic activity in holoprosencephaly. We report here that t-butyl alcohol, which is neither a substrate nor an inhibitor of alcohol dehydrogenases or Cyp2E1, is a potent inducer of holoprosencephaly in Cdon mutant mice. Additionally, antioxidant treatment did not prevent ethanol- or t-butyl alcohol-induced HPE in these mice. These findings are consistent with the conclusion that ethanol itself, rather than a consequence of its metabolism, is a holoprosencephaly-inducing teratogen.

The BAF (BRG1/BRM-Associated Factor) chromatin-remodeling complex exhibits ethanol sensitivity in fetal neural progenitor cells and regulates transcription at the miR-9-2 encoding gene locus.

Fetal alcohol spectrum disorders are a leading cause of intellectual disability worldwide. Previous studies have shown that developmental ethanol exposure results in loss of microRNAs (miRNAs), including miR-9, and loss of these miRNAs, in turn, mediates some of ethanol's teratogenic effects in the developing brain. We previously found that ethanol increased methylation at the miR-9-2 encoding gene locus in mouse fetal neural stem cells (NSC), advancing a mechanism for epigenetic silencing of this locus and consequently, miR-9 loss in NSCs. Therefore, we assessed the role of the BAF (BRG1/BRM-Associated Factor) complex, which disassembles nucleosomes to facilitate access to chromatin, as an epigenetic mediator of ethanol's effects on miR-9. Chromatin immunoprecipitation and DNAse I-hypersensitivity analyses showed that the BAF complex was associated with both transcriptionally accessible and heterochromatic regions of the miR-9-2 locus, and that disintegration of the BAF complex by combined knockdown of BAF170 and BAF155 resulted in a significant decrease in miR-9. We hypothesized that ethanol exposure would result in loss of BAF-complex function at the miR-9-2 locus. However, ethanol exposure significantly increased mRNA transcripts for maturation-associated BAF-complex members BAF170, SS18, ARID2, BAF60a, BRM/BAF190b, and BAF53b. Ethanol also significantly increased BAF-complex binding within an intron containing a CpG island and in the terminal exon encoding precursor (pre)-miR-9-2. These data suggest that the BAF complex may adaptively respond to ethanol exposure to protect against a complete loss of miR-9-2 in fetal NSCs. Chromatin remodeling factors may adapt to the presence of a teratogen, to maintain transcription of critical miRNA regulatory pathways.

Dietary choline levels modify the effects of prenatal alcohol exposure in rats

Prenatal alcohol exposure can cause a range of physical and behavioral alterations; however, the outcome among children exposed to alcohol during pregnancy varies widely. Some of this variation may be due to nutritional factors. Indeed, higher rates of fetal alcohol spectrum disorders (FASD) are observed in countries where malnutrition is prevalent. Epidemiological studies have shown that many pregnant women throughout the world may not be consuming adequate levels of choline, an essential nutrient critical for brain development, and a methyl donor. In this study, we examined the influence of dietary choline deficiency on the severity of fetal alcohol effects. Pregnant Sprague-Dawley rats were randomly assigned to receive diets containing 40, 70, or 100% recommended choline levels. A group from each diet condition was exposed to ethanol (6.0g/kg/day) from gestational day 5 to 20 via intubation. Pair-fed and ad lib lab chow control groups were also included. Physical and behavioral development was measured in the offspring. Prenatal alcohol exposure delayed motor development, and 40% choline altered performance on the cliff avoidance task, independent of one another. However, the combination of low choline and prenatal alcohol produced the most severe impairments in development. Subjects exposed to ethanol and fed the 40% choline diet exhibited delayed eye openings, significantly fewer successes in hindlimb coordination, and were significantly overactive compared to all other groups. These data suggest that suboptimal intake of a single nutrient can exacerbate some of ethanol's teratogenic effects, a finding with important implications for the prevention of FASD.

Involvement of Wnt pathway in ethanol-induced inhibition of mouse embryonic stem cell differentiation.

Ethanol has been reported to have toxicity on embryonic stem cells (ESCs). The present study aims to address the teratogenic effects of ethanol on the growth and cardiac differentiation of ESCs. Mouse embryonic stem D3 cells were employed. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays were used to determine cytotoxicity. Quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting were used to analyze the expressions of cardiac differentiation-related and Wnt signaling factors. The beating profile of cardiomyocytes was recorded to assess cardiac differentiation. Ethanol induced growth inhibition in both undifferentiated and differentiated ESCs after 5 days of exposure. Ethanol inhibited the loss of pluripotent gene expressions including Nanog, Sox2 and Oct4. The expressions of cardiac markers, Nkx2.5, Mef2c, Tbx5, dHand, αMHC, Cx43 and troponin C1, were suppressed by ethanol treatment. Furthermore, ethanol delayed cardiac differentiation of ESCs till 11 days of differentiation. The expressions of Wnt-related regulators, β-catenin and its target cyclin D1, were downregulated by ethanol. Wnt pathway agonist wnt3a could greatly rescue ethanol-induced inhibition of cardiac differentiation and Wnt-pathway-related protein expressions. These finding suggested that ethanol suppresses mouse ESC differentiation largely by inhibiting Wnt signaling pathway.

Phospholipase D is a target for inhibition of astroglial proliferation by ethanol

The proliferation of astrocytes during early brain development is driven by growth factors and is accompanied by the activation of phospholipase D (PLD). Ethanol disrupts PLD signaling in astrocytes, a process which may contribute to delayed brain growth of fetuses exposed to alcohol during pregnancy. We here report that insulin-like growth factor 1 (IGF-1) is a strong mitogen for rat astrocytes (EC50 0.2 μg/ml) and a strong stimulator of astroglial PLD activity; both effects are inhibited by ethanol and 1-butanol, but not t-butanol, suggesting participation of PLD. Downregulation of PLD1 and exposure to the PLD1 inhibitor VU0359595 attenuated PLD activity and strongly reduced the mitogenic activity of serum and IGF-1. The PLD2 inhibitor VU0285655-1 also reduced PLD activity but had lesser effects on IGF-1-driven proliferation. PLD2 down-regulation affected serum – but not IGF-1-induced proliferation. In separate experiments, alcohol treatment of murine astrocytes taken from PLD-deficient animals revealed an insensitivity of PLD1−/− cells to 1-butanol whereas PLD2−/− cells were not affected. We conclude that astroglial proliferation induced by IGF-1 is critically dependent on the PLD signaling pathway, with a stronger contribution from PLD1 than PLD2. The teratogenic effects of ethanol may be explained, at least in part, by disruption of the IGF1-PLD signaling pathway.

Sonic hedgehog expression is disrupted following in ovo ethanol exposure during early chick eye development

The eye is particularly sensitive to ethanol's teratogenic effects. Our previous work, using a chick embryo model system, has shown that ethanol acts rapidly to perturb vital processes of early eye development producing defects of the lens and retina. Ethanol-induced disruption of the midline ventral telencephalon, a key site for expression of ocular morphogens such as sonic hedgehog (Shh), was further established. Consequently, in this study we have examined the effects of ethanol on the Shh pathway during the period of optic vesicle/optic cup formation. Chick embryos were injected in ovo with 125μL of a 20% ethanol solution directly into the yolk-sac at HH-stage 7, resulting in peak ethanol uptake of 0.294g/dL. Subsequent molecular analysis at 12, 24 and 48h post-treatment revealed that ethanol had no affect on Shh transcription, while, a significant reduction in the expression of the active signalling Shh protein was found. Surprisingly, none of the downstream Shh pathway members (Ptc, Gli1 and Gli3) were significantly altered by ethanol exposure. Overall, our results indicate that ethanol's disruption of Shh may be mediated through some alternative mechanism independent of the classical signalling pathway. However, the precise role of Shh in relation to ethanol teratogenicity continues to be debated. Thus, in conclusion, our findings are discussed in relation to the varied and often conflicting reports of ethanol-induced Shh perturbation found in the literature.

The effects of a single memantine treatment on behavioral alterations associated with binge alcohol exposure in neonatal rats

BackgroundThe third trimester in human fetal development represents a critical time of brain maturation referred to as the “brain growth spurt”. This period occurs in rats postnatally, and exposure to ethanol during this time can increase the risk of impairments on a variety of cognitive and motor tasks. It has been proposed that one potential mechanism for the teratogenic effects of ethanol is NMDA receptor-mediated excitotoxicity during periods of ethanol withdrawal. In neonatal rats, antagonism of NMDA receptors during ethanol withdrawal, with drugs such as MK-801 and eliprodil, has been shown to mitigate some of the behavioral deficits induced by developmental ethanol exposure. The current study examined whether memantine, an NMDA receptor antagonist and a drug used clinically in Alzheimer's patients, would attenuate impairments associated with binge ethanol exposure in neonatal rats. MethodsOn postnatal day 6, rats were exposed to 6g/kg ethanol via intubation with controls receiving an isocaloric maltose dextrin solution. Twenty-one hours following the ethanol binge, rats received intraperitoneal injections of memantine at 0, 10, 15, or 20mg/kg. Ethanol's teratogenic effects were assessed using multiple behavioral tasks: open field activity, parallel bars and spatial discrimination reversal learning. ResultsEthanol-treated rats were overactive in the open field and were impaired on both reversal learning and motor performance. Administration of 15 or 20mg/kg memantine during withdrawal significantly attenuated ethanol's adverse effects on motor coordination, but did not significantly alter activity levels or improve the spatial learning deficits associated with neonatal alcohol exposure. ConclusionThese results indicate that a single memantine administration during ethanol withdrawal can mitigate motor impairments but not spatial learning impairments or overactivity observed following a binge ethanol exposure during development in the rat.

Agrin function associated with ocular development is a target of ethanol exposure in embryonic zebrafish

Alcohol (ethanol) is a teratogen known to affect the developing eyes, face, and brain. Among the ocular defects in fetal alcohol spectrum disorder (FASD) are microphthalmia and optic nerve hypoplasia. Employing zebrafish as an FASD model provides an excellent system to analyze the molecular basis of prenatal ethanol exposure-induced defects because embryos can be exposed to ethanol at defined developmental stages and affected genetic pathways can be examined. We have previously shown that disruption of agrin function in zebrafish embryos produces microphthalmia and optic nerve hypoplasia. Zebrafish embryos were exposed to varying concentrations of ethanol in the absence or presence of morpholino oligonucleotides (MOs) that disrupt agrin function. In situ hybridization was used to analyze ocular gene expression as a consequence of ethanol exposure and agrin knockdown. Morphologic analysis of zebrafish embryos was also conducted. Acute ethanol exposure induces diminished agrin gene expression in zebrafish eyes and, importantly, combined treatment with subthreshold levels of agrin MO and ethanol produces pronounced microphthalmia, markedly reduces agrin gene expression, and perturbs Pax6a and Mbx gene expression. Microphthalmia produced by combined agrin MO and ethanol treatment was rescued by sonic hedgehog (Shh) mRNA overexpression, suggesting that ethanol-mediated disruption of agrin expression results in disrupted Shh function. These studies illustrate the strong potential for using zebrafish as a model to aid in defining the molecular basis for ethanol's teratogenic effects. The results of this work suggest that agrin expression and function may be a target of ethanol exposure during embryogenesis.

Reduction of ethanol-induced ocular abnormalities in mice through dietary administration of N-acetylcysteine

N-Acetylcysteine (NAC) is a derivative of the amino acid l-cysteine, which, previously, has been shown to protect against ethanol-induced apoptosis during early development. Ongoing research demonstrates that NAC is also proving clinically beneficial in reducing oxidative stress–mediated lung, liver, and kidney damage, with protection likely resulting from a NAC-mediated increase in glutathione levels. In the present study, the hypothesis that coadministration of NAC and ethanol by means of liquid diet on days 7 and 8 of pregnancy in mice would reduce ethanol's teratogenicity was tested. For this work, adult nonpregnant female mice were acclimated to a liquid diet containing ethanol for 16 days, withdrawn from the ethanol, bred, and then returned to the liquid diet containing 4.8% ethanol and/or either 0.5 or 1-mg NAC/mL diet on their seventh and eighth days of pregnancy. At the concentrations used, the mice received NAC dosages of approximately 300 or 600 mg/kg/day and achieved peak blood ethanol concentrations (BEC) that averaged approximately 200 mg/dL. There was no difference in BEC between the ethanol-alone and ethanol plus 600 mg/kg NAC group. After maternal euthanasia, gestational day (GD) 14 fetuses were removed, fixed, weighed, and examined for the presence and severity of ocular abnormalities, a readily assessed endpoint that results from GD 7 and 8 ethanol exposures. Although the lower dosage of NAC (300 mg/kg) resulted in a decrease in the incidence of ocular defects in both the left and right eyes, this reduction was not statistically significant. However, doubling the NAC concentration did yield a significant change; as compared with the group treated with ethanol alone, the incidence of ocular abnormalities was diminished by 22%. These results show the potential of an orally administered compound with proven clinical efficacy to reduce ethanol's teratogenic effects and support the premise that oxidative damage plays an important mechanistic role in fetal alcohol spectrum disorders.

Ethanol Teratogenesis in Five Inbred Strains of Mice

Previous studies have demonstrated individual differences in susceptibility to the detrimental effects of prenatal ethanol exposure. Many factors, including genetic differences, have been shown to play a role in susceptibility and resistance, but few studies have investigated the range of genetic variation in rodent models. We examined ethanol teratogenesis in 5 inbred strains of mice: C57BL/6J (B6), Inbred Short-Sleep, C3H/Ibg, A/Ibg, and 129S6/SvEvTac (129). Pregnant dams were intubated with either 5.8 g/kg ethanol (E) or an isocaloric amount of maltose-dextrin (MD) on day 9 of pregnancy. Dams were sacrificed on day 18 and fetuses were weighed, sexed, and examined for gross morphological malformations. Every other fetus within a litter was then either placed in Bouin's fixative for subsequent soft-tissue analyses or eviscerated and placed in ethanol for subsequent skeletal analyses. B6 mice exposed to ethanol in utero had fetal weight deficits and digit, kidney, brain ventricle, and vertebral malformations. In contrast, 129 mice showed no teratogenesis. The remaining strains showed varying degrees of teratogenesis. Differences among inbred strains demonstrate genetic variation in the teratogenic effects of ethanol. Identifying susceptible and resistant strains allows future studies to elucidate the genetic architecture underlying prenatal alcohol phenotypes.

Choline supplementation attenuates learning deficits associated with neonatal alcohol exposure in the rat: Effects of varying the timing of choline administration

Despite the harmful effects of fetal alcohol exposure, some pregnant women continue to drink alcohol. Thus, it is imperative to pursue safe, effective treatments for children with fetal alcohol spectrum disorders. Using an animal model, our laboratory has demonstrated that choline, an essential nutrient, effectively reduces the severity of some fetal alcohol effects, even when administered after the ethanol insult is complete. The present study investigated whether there is a critical developmental period when choline is most effective in attenuating ethanol's teratogenic effects. Sprague–Dawley rats were exposed to 5.25 g/kg/day ethanol during the third trimester equivalent brain growth spurt (postnatal days (PD) 4–9) via intubation. A non-intubation control group and a sham intubation control group were included. Following ethanol exposure, pups received subcutaneous injections of saline vehicle or choline chloride (100 mg/kg/day) from PD 11–20, PD 21–30, or PD 11–30. Beginning on PD 45, subjects were tested on a Morris water maze spatial learning task. Performance of both the ethanol-exposed group that did not receive choline and the ethanol-exposed group treated with choline from PD 21–30 was significantly impaired compared to controls during acquisition of the Morris water maze task. Performance of ethanol-exposed groups treated with choline from PD 11–20 or PD 11–30 was intermediate, not differing significantly from any other groups. However, during the probe trial, ethanol exposure produced significant deficits in spatial memory which were mitigated by all choline treatments, regardless of the timing of administration. These findings suggest that choline's therapeutic window may be very large, or spans across the two developmental periods examined in this study. Importantly, these findings indicate that choline supplementation may effectively reduce some alcohol-related learning impairments, even when administered in later childhood.

Chronic ethanol increases fetal cerebral blood flow specific to the ethanol‐sensitive cerebellum under normoxaemic, hypercapnic and acidaemic conditions: ovine model

Cerebral hypoxia has been proposed as a mechanism by which prenatal ethanol exposure causes fetal alcohol spectrum disorder (FASD) in children, but no study had tested this hypothesis using a chronic exposure model that mimicks a common human exposure pattern. Pregnant sheep were exposed to ethanol, 0.75 or 1.75 g kg(-1) (to create blood ethanol concentrations of 85 and 185 mg dl(-1), respectively), or saline 3 days per week in succession (a 'binge drinking' model) from gestational day (GD) 109 until GD 132. Fetuses were instrumented on GD 119-120 and studied on GD 132. The 1.75 g kg(-1) dose resulted in a significant increase in fetal biventricular output (measured by radiolabelled microsphere technique) and heart rate, and a reduction of mean arterial pressure and total peripheral resistance at 1 h, the end of ethanol infusion. The arterial partial pressure of CO(2) was increased, arterial pH was decreased and arterial partial pressure of O(2) did not change. Fetal whole-brain blood flow increased by 37% compared with the control group at 1 h, resulting in increased cerebral oxygen delivery. The elevation in brain blood flow was region specific, occurring preferentially in the ethanol-sensitive cerebellum, increasing by 44% compared with the control group at 1 h. There were no changes in the lower dose group. Assessment of regional differences in the teratogenic effects of ethanol by stereological cell-counting technique showed a reduced number of cerebellar Purkinje cells in response to the 1.75 g kg(-1) dose compared with the control brains. However, no such differences in neuronal numbers were observed in the hippocampus or the olfactory bulb. We conclude that repeated exposure to moderate doses of ethanol during the third trimester alters fetal cerebral vascular function and increases blood flow in brain regions that are vulnerable to ethanol in the presence of acidaemia and hypercapnia, and in the absence of hypoxia.

Maternal Oral Intake Mouse Model for Fetal Alcohol Spectrum Disorders: Ocular Defects as a Measure of Effect

This work was conducted in an effort to establish an oral intake model system in which the effects of ethanol insult that occur during early stages of embryogenesis can be easily examined and in which agents that may modulate ethanol's teratogenicity can be readily tested in vivo. The model system described utilizes the alcohol deprivation effect to obtain teratogenic levels of maternal ethanol intake on days 7 and 8 of pregnancy in C57Bl/6J mice. Ocular defects including microphthalmia and uveal coloboma, which have previously been shown to result from ethanol administered by gavage or via intraperitoneal injection on these days, served as the developmental end point for this study. The ocular defects are readily identifiable and their degree of severity is expected to correlate with concurrently developing defects of the central nervous system (CNS). Female C57Bl/6J mice were maintained on an ethanol-containing (4.8% v/v) liquid diet for 14 days and then mated during a subsequent abstinence period. Mice were then reexposed to ethanol on days 7 and 8 of pregnancy only. Control as well as ethanol-exposed dams were killed on their 14th day of pregnancy. Fetuses were then weighed, measured for crown rump length, photographed, and analyzed for ocular abnormalities. Globe size, palpebral fissure length, and pupil size and shape were noted for both the right and left eyes of all fetuses and informative comparisons were made. This exposure paradigm resulted in peak maternal blood alcohol concentrations that ranged from 170 to 220 mg/dL on gestational day (GD) 8. Compared with the GD 14 fetuses from the normal control group, the pair-fed, acquisition controls, as well as the ethanol-exposed fetuses, were developmentally delayed and had reduced weights. Confirming previous studies, comparison of similarly staged control and treated GD 8 embryos illustrated reductions in the size of the forebrain in the latter. Subsequent ocular malformations were noted in 33% of the right eyes and 25% of the left eyes of the 103 GD 14 ethanol-exposed fetuses examined. This incidence of defects is twice that observed in the control groups. Additionally, it was found that the palpebral fissure length is directly correlated with globe size. The high incidence of readily identifiable ocular malformations produced by oral ethanol intake in this model and their relevance to human fetal alcohol spectrum disorders (FASD) makes this an excellent system for utilization in experiments involving factors administered to the embryo that might alter ethanol's teratogenic effects. Additionally, the fact that early ethanol insult yields ocular and forebrain abnormalities that are developmentally associated allows efficient specimen selection for subsequent detailed analyses of CNS effects in this in vivo mammalian FASD model.

Prevenção da deficiência mental: conhecimento e percepção dos profissionais de saúde

This article aimed to evaluate the prevention of mental disability in primary healthcare services in Maringá, Paraná, Brazil. The sample consisted of 90 male and female physicians from different fields, namely gynecology and obstetrics, pediatrics, general practice, and family health, as well as 66 male and female nurses. A multiple-choice questionnaire was filled out by the subjects themselves from August to December 2003. Qualitative variables were compared using the chi-square test at 5% significance level. Partial data relating to both the perception and knowledge of health professionals concerning mental disability were as follows: 75% were unable to choose the correct alternative on prevalence; 25% did not know how the genome contributes to etiology; 37% were unaware of prevention for mental disability; 28% were not confident in providing orientation on the teratogenic effect of ethanol; 35% demonstrated insecurity in orienting patients on amniocentesis. The data showed that participants had an unsatisfactory perception of the relevance of mental disability within the overall population disease profile, and that they need more information on the respective genetic and environmental issues.

Rapid Induction of Apoptosis in Gastrulating Mouse Embryos by Ethanol and Its Prevention by HB‐EGF

Ethanol exposure during gastrulation and early neurulation induces apoptosis within certain embryonic cell populations, leading to craniofacial and neurological defects. There is currently little information about the initial kinetics of ethanol-induced apoptosis, and interest in the ability of endogenous survival factors to moderate apoptosis is growing. Ethanol alters intracellular signaling, leading to cell death in chick embryos, suggesting that apoptosis could occur rapidly and that signaling pathways activated by survival factors might reduce apoptosis. Pregnant mice were intubated with 1, 2, or 4 g/kg ethanol on day 7.5 of embryogenesis (E7.5) 1, 3, or 6, hours before harvesting gastrulation-stage embryos. Control animals received maltose/dextran. Blood alcohol concentrations (BAC) were determined by gas chromatography. E7.5 embryos isolated from untreated dams were cultured in vitro for 1 or 3 hr with 0 or 400 mg% ethanol and 0 or 5 nM heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF). Apoptosis was quantified using fluorescence microscopy to detect annexin V binding and DNA fragmentation [terminal deoxynucleotidyl transferase-mediated dUTP-X nick end labeling (TUNEL)] in whole-mount or sectioned embryos. Both annexin V binding and TUNEL were elevated (p < 0.05) in embryos exposed in utero to 1 g/kg ethanol for 3 hours, increasing linearly with time and ethanol concentration. Apoptosis increased (p < 0.05) in all germ cell layers. Mice treated with 4 g/kg sustained BAC of 400 mg% for nearly 3 hours, significantly increasing apoptosis within the first hour. Cultured embryos exposed to 400 mg% ethanol displayed 2- to 3-fold more TUNEL than vehicle-treated embryos (p < 0.05); however, exogenous HB-EGF prevented apoptosis. Ethanol rapidly produced apoptosis in gastrulation-stage embryos, consistent with induction by intracellular signaling. The ethanol-induced apoptotic pathway was blocked by the endogenous survival factor, HB-EGF. Differences in the expression of survival factors within individual embryos could be partly responsible for variations in the teratogenic effects of ethanol among offspring exposed prenatally.

Histochemical Study Of The Effect Of Ethanol On Alkaline Phosphatase In The Femur Of Wistar Rat Foetuses

The teratogenic effects of ethanol include the assaults of the various developmental processes of tissues exposed in utero, and particularly the mineralisation of bones. An experimental investigation of the mechanisms of action of this toxic agent was conducted in the femoral bones of the foetal Wistar rat by the histochemical assessment of the activity of alkaline phosphatase, an enzyme present within the osteoblasts and actively involved in the mineral deposition in bones during development. Enzyme was relatively sparse and less intense activity in the growing end of ethanol treated bone compared to the control. This appears to be a possible indication of the mechanism by which ethanol suppresses osteoblasts functions, hence, the ability to potentiate its toxic and teratogenic effects. KEY WORDS: Ethanol; alkaline phosphatase; Wistar rat fetuses; Bone mineralization. J.Expt. & Clin. Anat. 2(1) 2003: 38-40

Zinc treatment prevents lipopolysaccharide‐induced teratogenicity in mice

During pregnancy, exposure to lipopolysaccharide (LPS) can lead to abortion, preterm delivery, and teratogenicity. The mechanisms underlying these effects are unclear. Both LPS and ethanol are potent inducers of liver metallothionein (MT), a key Zn binding protein. The teratogenic effects of ethanol have been linked to MT-induced changes in maternal-fetal Zn homeostasis, leading tofetal deficiency. This study was designed to assess whether the teratogenic effects of LPS are also related to MT induction and changes in Zn homeostasis. Non-pregnant normal (MT +/+) and MT-null (MT -/-) mice were injected subcutaneously with 0.5 microg/gm LPS and killed over 48 hr. In MT +/+ mice, liver MT concentrations were elevated from 6 hr, and were maximal at 24 hr (30-fold basal), whereas liver Zn levels were also increased from 6 hr. Plasma Zn concentrations decreased by 80% at 6 hr, and were below normal between 6 and 24 hr. In MT -/- mice, plasma Zn levels were increased from basal between 6 and 16 hr. Dams were injected with LPS, saline, or LPS and ZnSO4 (2 microg/gm, MT +/+ only) on Day 8 of gestation (GDS), killed on GD18, and the fetuses examined for malformations. External abnormalities were most prevalent in fetuses from MT +/+ dams exposed to LPS, where 34% of fetuses in each litterwere affected. MT +/+ dams treated with LPS and ZnSO4, and MT -/- dams treated with LPS had litters in which 5.4 and 4.8% of fetuses were abnormal respectively. The findings of this study strongly support the hypothesis that LPS teratogenicity is mediated at least in part by MT-induced changes in maternal Zn homeostasis,which compromises fetal Zn supply.

Genetic Influences on Craniofacial Outcome in an Avian Model of Prenatal Alcohol Exposure

Background: Understanding the basis for ethanol's teratogenic effects may inform the etiology of fetal alcohol syndrome. Here we investigate how genetic background and susceptibility to ethanol-induced neural crest apoptosis contribute to the distinctive craniofacial phenotype observed after prenatal alcohol exposure. Methods: Nine different chick strains were exposed to ethanol at gastrulation. The sensitivity of these embryos to ethanol-induced neural crest apoptosis was reported elsewhere (Debelak and Smith, 2000). Here, these embryos were permitted to develop until embryonic day 10, when facial morphogenesis was largely complete, and cephalometric measurements were made on cleared skulls. Shifts in facial growth were correlated against the severity of ethanol-induced apoptosis in facial precursors. Results: The facial shape produced by ethanol exposure was a function of the embryos’ genetic strain. Three general responses were observed: apparent midfacial flattening (Babcock B300 x Hampshire Red, ISA-Babcock, HyLine W98, and HyLine W36 strains), overall facial expansion (Spafas and Babcock B300 strains), or overall facial hypoplasia (DeKalb strains). When dose and timing of exposure were held constant, the embryo's genetic background predicted the facial outcome. For ethanol-sensitive strains, apoptosis of facial precursor populations was required to produce the facial defects. That some strains had essentially normal faces despite extensive cell death indicated a capacity to recover from the earlier neural crest losses. Conclusions: We propose that ethanol's effects on craniofacial development are multifactoral, and these influences may include susceptibility to apoptosis, regenerative capacity, and compensatory outgrowth of the facial primordia. The embryo's genetic background may modulate these events. The high and low responder chick strains are useful tools to dissect these contributions.