Long-lasting implications of embryonic exposure to alcohol: Insights from zebrafish research.

The harmful consumption of ethanol is associated with significant health problems and social burdens. This drug activates a complex network of reward mechanisms and habit formation learning that is supposed to contribute to the consumption of increasingly high and frequent amounts, ultimately leading to addiction. In the context of fetal alcohol spectrum disorders, fetal alcohol syndrome (FAS) is a consequence of the harmful use of alcohol during pregnancy, which affects the embryonic development of the fetus. FAS can be easily reproduced in zebrafish by exposing the embryos to different concentrations of ethanol in water. In this regard, the aim of the present review is to discuss the late pathological implications in zebrafish exposed to ethanol at the embryonic stage, providing information in the context of human fetal alcoholic spectrum disorders. Experimental FAS in zebrafish is associated with impairments in the metabolic, morphological, neurochemical, behavioral, and cognitive domains. Many of the pathways that are affected by ethanol in zebrafish have at least one ortholog in humans, collaborating with the wider adoption of zebrafish in studies on alcohol disorders. In fact, zebrafish present validities required for the study of these conditions, which contributes to the use of this species in research, in addition to studies with rodents.

Prenatal ethanol induces an anxiety phenotype and alters expression of dynorphin & nociceptin/orphanin FQ genes.

Animal models have suggested that prenatal ethanol exposure (PEE) alters the κ opioid receptor system. The present study investigated the brain expression of dynorphin and nociceptin/orphanin FQ related genes and assessed anxiety-like behavior in the light-dark box (LDB), shelter-seeking and risk-taking behaviors in the concentric square field (CSF) test, and ethanol-induced locomotion in the open field (OF), in infant or adolescent Wistar rats that were exposed to PEE (0.0 or 2.0 g/kg, intragastrically, gestational days 17-20). We measured brain mRNA levels of prodynorphin (PDYN), κ opioid receptors (KOR), the nociceptin/orphanin FQ opioid peptide precursor prepronociceptin (ppN/OFQ) and nociceptine/orphanin FQ receptors (NOR). Prenatal ethanol exposure upregulated PDYN and KOR mRNA levels in the ventral tegmental area (VTA) in infant and adolescent rats and KOR mRNA levels in the prefrontal cortex in infant rats. The changes in gene expression in the VTA were accompanied by a reduction of DNA methylation at the PDYN gene promoter, and by a reduction of DNA methylation at the KOR gene promoter. The PEE-induced upregulation of PDYN/KOR in the VTA was accompanied by lower NOR gene expression in the VTA, and lower PDYN gene expression in the nucleus accumbens. PEE rats exhibited hypolocomotion in the OF, greater avoidance of the white and brightly lit areas in the LDB and CSF, and greater preference for the sheltered area in the CSF test. These results suggest that PEE upregulates the dynorphin system, resulting in an anxiety-prone phenotype and triggering compensatory responses in the nociceptin/orphanin FQ system. These findings may help elucidate the mechanisms that underlie the effects of PEE and suggest that the dynorphin and nociceptin/orphanin FQ systems may be possible targets for the prevention and treatment of PEE-induced alterations.

Cellular and molecular oxidative stress-related effects in uterine myometrial and trophoblast-decidual tissues after perigestational alcohol intake up to early mouse organogenesis.

The placenta plays a major role in embryo-fetal defects and intrauterine growth retardation after maternal alcohol consumption. Our aims were to determine the oxidative status and cellular and molecular oxidative stress effects on uterine myometrium and trophoblast-decidual tissue following perigestational alcohol intake at early organogenesis. CF-1 female mice were administered with 10% alcohol in drinking water for 17 days prior to and up to day 10 of gestation. Control females received ethanol-free water. Treated mice had smaller implantation sites compared to controls (p < 0.05), diminished maternal vascular lumen, and irregular/discontinuous endothelium of decidual vessels. The trophoblast giant cell layer was disorganized and presented increased abnormal nuclear frequency. The myometrium of treated females had reduced nitrite content, increased superoxide dismutase activity, and reduced glutathione (GSH) content (p < 0.05). However, the trophoblast-decidual tissue of treated females had increased nitrite content (p < 0.05), increased GSH level (p < 0.001), increased thiobarbituric acid-reactive substance concentration (p < 0.001), higher 3-nitrotyrosine immunoreaction, and increased apoptotic index (p < 0.05) compared to controls. In summary, perigestational alcohol ingestion at organogenesis induced oxidative stress in the myometrium and trophoblast-decidual tissue, mainly affecting cells and macromolecules of trophoblast and decidual tissues around early organogenesis, in CF-1 mouse, and suggests that oxidative-induced abnormal early placental formation probably leads to risk of prematurity and fetal growth impairment at term.

Fetal alcohol syndrome, chemo-biology and OMICS: ethanol effects on vitamin metabolism during neurodevelopment as measured by systems biology analysis.

Fetal alcohol syndrome (FAS) is a prenatal disease characterized by fetal morphological and neurological abnormalities originating from exposure to alcohol. Although FAS is a well-described pathology, the molecular mechanisms underlying its progression are virtually unknown. Moreover, alcohol abuse can affect vitamin metabolism and absorption, although how alcohol impairs such biochemical pathways remains to be elucidated. We employed a variety of systems chemo-biology tools to understand the interplay between ethanol metabolism and vitamins during mouse neurodevelopment. For this purpose, we designed interactomes and employed transcriptomic data analysis approaches to study the neural tissue of Mus musculus exposed to ethanol prenatally and postnatally, simulating conditions that could lead to FAS development at different life stages. Our results showed that FAS can promote early changes in neurotransmitter release and glutamate equilibrium, as well as an abnormal calcium influx that can lead to neuroinflammation and impaired neurodifferentiation, both extensively connected with vitamin action and metabolism. Genes related to retinoic acid, niacin, vitamin D, and folate metabolism were underexpressed during neurodevelopment and appear to contribute to neuroinflammation progression and impaired synapsis. Our results also indicate that genes coding for tubulin, tubulin-associated proteins, synapse plasticity proteins, and proteins related to neurodifferentiation are extensively affected by ethanol exposure. Finally, we developed a molecular model of how ethanol can affect vitamin metabolism and impair neurodevelopment.

Placental hypoxia and foetal development versus alcohol exposure in pregnancy.

AIMS: To examine the causes of variability in the effect of maternal drinking on the foetus, with particular reference to the pattern, frequency and duration of the period of drinking, differences in maternal, foetal and placental metabolism of ethanol/acetaldehyde, and genetic factors. METHODS: Narrative review of published studies of the pathogenesis of foetal alcohol syndrome (FAS) with emphasis in the development of the central nervous system. RESULTS: Animal models suggest that acetaldehyde, the primary hepatic oxidative metabolite of ethanol, reaches the foetus either by placental production or by placental transference, which in turn could affect foetal growth and development. The most likely hypothesis regarding the decrease of foetal growth is via hypoxia and increased oxidative/nitrative stress, which interfere with cellular processes that require oxygen in order to function adequately, such as placental transport. CONCLUSION: There seems to be an association between the teratogenic effect, hypoxia and oxidative stress, the molecular mechanism involved (e.g. apoptosis) and the range of effects. The review sums ups the evidence that could explain some of the abnormalities in the brain development that could be related to behavioural problems observed in individuals with FAS/foetal alcohol spectrum disorder. This suggests that alcohol consumption produces failures in the normal migration of radial cells, from which the rest of the brain cells would eventually develop.

Proton magnetic resonance spectroscopy in children with fetal alcohol spectrum disorders.

OBJECTIVE: To analyze the metabolic constitution of brain areas through proton magnetic resonance spectroscopy in children affected with fetal alcohol spectrum disorder compared with normal children. METHOD: The sample of this case-control study included eight boys with epidemiologic history of in utero exposure to alcohol (median age 13.6+/-3.8 years) who were diagnosed with fetal alcohol spectrum disorder, and eight controls (median age 12.1+/-3,4 years). An 8 cm(3) single voxel approach was used, with echo time 30 ms, repetition time 1500 ms, and 128 acquisitions in a 1.5T scanner, and four brain areas were analyzed: anterior cingulate, left frontal lobe, left striatum, and left cerebellar hemisphere. Peaks and ratios of metabolites N-acetylaspartate, choline, creatine, and myo-inositol were measured. RESULTS: Children with fetal alcohol spectrum disorder showed a decrease in choline/creatine ratio (p=0.020) in left striatum and an increase in myo-inositol/creatine ratio (p=0.048) in left cerebellum compared with controls. There was no statistically significant difference in all peaks and ratios from the anterior cingulate and frontal lobe between the two groups. CONCLUSION: This study found evidence that the left striatum and left cerebellum are affected by intrauterine exposure to alcohol. Additional studies with larger samples are necessary to expand our knowledge of the effects of fetal exposure to alcohol.

Proton magnetic resonance spectroscopy in children with fetal alcohol spectrum disorders / Espectroscopia por ressonância magnética de prótons em crianças com transtornos do espectro alcoólico fetal

OBJECTIVE: To analyze the metabolic constitution of brain areas through proton magnetic resonance spectroscopy in children affected with fetal alcohol spectrum disorder compared with normal children. METHOD: The sample of this case-control study included eight boys with epidemiologic history of in utero exposure to alcohol (median age 13.6±3.8 years) who were diagnosed with fetal alcohol spectrum disorder, and eight controls (median age 12.1±3,4 years). An 8 cm³ single voxel approach was used, with echo time 30 ms, repetition time 1500 ms, and 128 acquisitions in a 1.5T scanner, and four brain areas were analyzed: anterior cingulate, left frontal lobe, left striatum, and left cerebellar hemisphere. Peaks and ratios of metabolites N-acetylaspartate, choline, creatine, and myo-inositol were measured. RESULTS: Children with fetal alcohol spectrum disorder showed a decrease in choline/creatine ratio (p=0.020) in left striatum and an increase in myo-inositol/creatine ratio (p=0.048) in left cerebellum compared with controls. There was no statistically significant difference in all peaks and ratios from the anterior cingulate and frontal lobe between the two groups. CONCLUSION: This study found evidence that the left striatum and left cerebellum are affected by intrauterine exposure to alcohol. Additional studies with larger samples are necessary to expand our knowledge of the effects of fetal exposure to alcohol.

OBJETIVO: Analisar a composição metabólica de áreas encefálicas através da espectroscopia de prótons por ressonância magnética em crianças com transtornos do espectro alcoólico fetal e crianças normais. MÉTODO: A amostra deste estudo de casos-controles incluiu 8 meninos com história epidemiológica de exposição fetal ao álcool (idade mediana 13,6±3,8 anos), diagnosticados com transtorno do espectro alcoólico fetal, e 8 controles (idade mediana 12,1±3,4 anos). Utilizou-se voxel único de 8 cm³, tempo de eco 30 ms, tempo de repetição 1.500 ms, 128 aquisições, em scanner de 1,5T para as seguintes áreas: cíngulo anterior, lobo frontal esquerdo, estriado esquerdo e hemisfério cerebelar esquerdo. Estudaram-se os picos e as razões dos metabólitos N-acetilaspartato, colina, creatina e o mio-inositol. RESULTADOS: As crianças acometidas apresentaram diminuição da razão colina/creatina (p=0,020) no estriado esquerdo, e aumento da razão mio-inositol/creatina (p=0,048) no cerebelo esquerdo. Não houve diferença estatisticamente significativa nos valores encontrados no cíngulo anterior e lobo frontal entre os dois grupos. CONCLUSÃO: Este estudo encontrou evidências de que o estriado e o cerebelo esquerdos são acometidos pela exposição intra-uterina ao álcool. Estudos adicionais com amostras maiores são essenciais para expandir nosso conhecimento dos efeitos da exposição fetal ao álcool.

Effects of maternal ethanol intake on immunoexpression of epidermal growth factor in developing rat mandibular molar.

OBJECTIVE: A polyclonal antibody was used to investigate the effects of ethanol ingestion before and during pregnancy, in the expression of EGF on dentinogenesis and amelogenesis of rat mandibular first molar. DESIGN: Ethanol was administered to drinking water (treated group) starting at concentrations of 1% and increasing weekly to 5, 10, 15, 20 and 25% (v/v). During week 7, these rats were mated and continued to receive the 25% alcoholic solution, up to delivery. The control group received tap water. On postnatal days 0, 4 and 9, two offspring of each litter were killed, their hemimandibles removed and prepared for paraffin processing and immunohistochemistry. RESULTS: At postnatal day 0 the EGF immunoreactivity of the inner enamel epithelium and presecretory ameloblasts was weak when compared to controls. At postnatal day 4 EGF immunoreactivity of the secretory ameloblasts and odontoblasts was only moderate compared to controls. At postnatal day 9 EGF staining of the ameloblasts was weak when compared to controls. CONCLUSIONS: These results suggest that, maternal alcoholism interferes with EGF expression during initial dentinogenesis and amelogenesis and in the secretion and maturation of the dentin and enamel, therefore, which may cause a reduction of dentin and enamel formation.

Immunoexpression of epidermal growth factor in odontogenesis of the offspring of alcoholic mice.

Several forms of cell perturbation have been associated with ethanol ingestion. Fetal alcohol syndrome (FAS) as well as diminished maxillofacial development and inhibition of cell regeneration in vitro and in vivo have been described. Epidermal growth factor (EGF) stimulates maxillofacial growth, DNA synthesis, and it is a potent mitogen for a number of various cell types. EGF exerts its effects on cells through binding to a specific cell surface receptor which leads to activation of a thyrosine kinase in the intracellular part of the receptor. The inhibitory effect of alcohol on EGF in the mouse dental follicle was studied in the offspring of alcoholic mothers using immunocytochemistry. Adult female mice were given 22% alcohol in their drinking water and fed a pelleted diet before and during pregnancy. Maternal blood alcohol levels were 262 +/- 1.3 mg/100 ml on gestation day 12.5. The offspring of the alcoholic and control mice were sacrificed on postnatal day 1.5, their mandibles were dissected, weighed and processed by routine immunocytochemistry with the following results. 1) Significant differences were found in mandible weight p < 0.01 after parturition. 2) The tooth germs in the offspring of ethanol treated mice were morphometrically smaller than those of control littermates. 3) Immunoexpression of EGF in the mandibular first molar of the control group was strong and homogeneous while in the experimental group the expression was light and heterogeneous. It is concluded that maternal alcoholism reduces EGF in the offspring.