Chanchal Mandal

Chronic ethanol exposure increases goosecoid (GSC) expression in human embryonic carcinoma cell differentiation.

Fetal alcohol spectrum disorder (FASD) is a set of developmental malformations caused by excess alcohol consumption during pregnancy. Using an in vitro system, we examined the role that chronic ethanol (EtOH) exposure plays in gene expression changes during the early stage of embryonic differentiation. We demonstrated that EtOH affected the cell morphology, cell cycle progression and also delayed the down‐regulation of OCT4 and NANOG during differentiation. Gene expression profiling and pathway analysis demonstrated that EtOH deregulates many genes and pathways that are involved in early embryogenesis. Follow‐up analyzes revealed that EtOH exposure to embryoid bodies (EBs) induced the expression of an organizer‐specific gene, goosecoid (GSC), in comparison to controls. Moreover, EtOH treatment altered several important genes that are involved in embryonic structure formation, nervous system development, and placental and embryonic vascularization, which are all common processes that FASD can disrupt. Specifically, EtOH treatment let to a reduction in ALDOC, ENO2 and CDH1 expression, whereas EtOH treatment induced the expression of PTCH1, EGLN1, VEGFA and DEC2 in treated EBs. We also found that folic acid (FA) treatment was able to correct the expression of the majority of genes deregulated by EtOH exposure during early embryo development. Finally, the present study identified a gene set including GSC, which was deregulated by EtOH exposure that may contribute to the etiology of fetal alcohol syndrome (FAS). We also reported that EtOH‐induced GSC expression is mediated by Nodal signaling, which may provide a new avenue for analyzing the molecular mechanisms behind EtOH teratogenicity in FASD individuals. Copyright

PCDHB14- and GABRB1-like nervous system developmental genes are altered during early neuronal differentiation of NCCIT cells treated with ethanol

Ethanol (EtOH) exposure during embryonic development causes dysfunction of the central nervous system (CNS). Here, we examined the effects of chronic EtOH on gene expression during early stages of neuronal differentiation. Human embryonic carcinoma (NCCIT) cells were differentiated into neuronal precursors/lineages in the presence or absence of EtOH and folic acid. Gene expression profiling and pathway analysis demonstrated that EtOH deregulates many genes and pathways that are involved in early brain development. EtOH exposure downregulated several important genes, such as PCDHB14, GABRB1, CTNND2, NAV3, RALDH1, and OPN5, which are involved in CNS development, synapse assembly, synaptic transmission, and neurotransmitter receptor activity. GeneGo pathway analysis revealed that the deregulated genes mapped to disease pathways that were relevant to fetal alcohol spectrum disorders (FASD, such as neurotic disorders, epilepsy, and alcohol-related disorders). In conclusion, these findings suggest that the impairment of the neurological system or suboptimal synapse formation resulting from EtOH exposure could underlie the neurodevelopmental disorders in individuals with FASD.

Profiling ethanol-targeted transcription factors in human carcinoma cell-derived embryoid bodies

Fetal alcohol spectrum disorder is a collective term that represents fetal abnormalities associated with maternal alcohol consumption. Prenatal alcohol exposure and related anomalies are well characterized, but the molecular mechanism behind this phenomenon is not yet understood. Few insights have been gained from genetic and epigenetic studies of fetal alcohol spectrum disorder. Our aim was to profile the important molecular regulators of ethanol-related alterations of the genome. For this purpose, we have analyzed the gene expression pattern of human carcinoma cell-derived embryoid bodies in the absence or presence of ethanol. A cDNA microarray analysis was used to profile mRNA expression in embryoid bodies at day 7 with or without ethanol treatment. A total of 493 differentially expressed genes were identified in response to 50 mM ethanol exposure. Of these, 111 genes were up-regulated, and 382 were down-regulated. Gene ontology term enrichment analysis revealed that these genes are involved in important biological processes: neurological system processes, cognition, behavior, sensory perception of smell, taste and chemical stimuli and synaptic transmission. Similarly, the enrichment of disease-related genes included relevant categories such as neurological diseases, developmental disorders, skeletal and muscular disorders, and connective tissue disorders. Furthermore, we have identified a group of 26 genes that encode transcription factors. We validated the relative gene expression of several transcription factors using quantitative real time PCR. We hope that our study substantially contributes to the understanding of the molecular mechanisms underlying the pathology of alcohol-mediated anomalies and facilitates further research.

Reduction of Nfia gene expression and subsequent target genes by binge alcohol in the fetal brain

The objective of the present study was to investigate the changes in gene expression in the fetal brain (forebrain and hippocampus) caused by maternal binge alcohol consumption. Pregnant C57BL/6J mice were treated intragastrically with distilled phosphate-buffered saline (PBS) or ethanol (2.9 g/kg) from embryonic day (ED) 8-12. Microarray analysis revealed that a significant number of genes were altered at ED 18 in the developing brain. Specifically, in hippocampus, nuclear factor one alpha (Nfia) and three N-methyl-D-aspartate (Nmda) receptors (Nmdar1, Nmdar2b, and Nmdar2d) were down-regulated. The transcription factor Nfia controls gliogenesis, cell proliferation and Nmda-induced neuronal survival by regulating the expression of target genes. Some of the Nfia-target gene (Aldh1a, Folh1, Gjb6, Fgf1, Neurod1, Sept4, and Ntsr2) expressions were also altered as expected. These results suggest that the altered expression of Nfia and Nmda receptors may be associated with the etiology of fetal alcohol syndrome (FAS). The data presented in this report will contribute to the understanding of the molecular mechanisms associated with the effects of alcohol in FASD individuals.

Maternal alcohol consumption and altered miRNAs in the developing fetus: Context and future perspectives

Alcohol is a teratogenic agent that can cause a wide range of developmental disorders, and sometimes, the effects persist throughout an individuals lifetime. Researchers have shown the involvement of epigenetic mechanisms in alcohol‐mediated disorders. Non‐coding RNAs are one of the major sources of epigenetic modifications, especially microRNAs. The association of microRNAs with alcohol consumption leads to a new focus on finding the molecular mechanisms of alcohol toxicity. It has been suggested that alcohol alters the relative expression of microRNAs and regulates target mRNA expression in both in vitro and in vivo models. Currently, we lack information regarding the relationship between altered microRNA expression and disease phenotypes in alcohol‐mediated disorders. In this review, we tried to gather all of the available information about the alcohol‐mediated dysregulation of microRNA expression in utero. We hope that our efforts will help future researchers identify major microRNAs in the field of prenatal alcohol toxicity and related therapeutics.

RNA Sequencing Reveals the Alteration of the Expression of Novel Genes in Ethanol-Treated Embryoid Bodies

Fetal alcohol spectrum disorder is a collective term representing fetal abnormalities associated with maternal alcohol consumption. Prenatal alcohol exposure and related anomalies are well characterized, but the molecular mechanism behind this phenomenon is not well characterized. In this present study, our aim is to profile important genes that regulate cellular development during fetal development. Human embryonic carcinoma cells (NCCIT) are cultured to form embryoid bodies and then treated in the presence and absence of ethanol (50 mM). We employed RNA sequencing to profile differentially expressed genes in the ethanol-treated embryoid bodies from NCCIT vs. EB, NCCIT vs. EB+EtOH and EB vs. EB+EtOH data sets. A total of 632, 205 and 517 differentially expressed genes were identified from NCCIT vs. EB, NCCIT vs. EB+EtOH and EB vs. EB+EtOH, respectively. Functional annotation using bioinformatics tools reveal significant enrichment of differential cellular development and developmental disorders. Furthermore, a group of 42, 15 and 35 transcription factor-encoding genes are screened from all of the differentially expressed genes obtained from NCCIT vs. EB, NCCIT vs. EB+EtOH and EB vs. EB+EtOH, respectively. We validated relative gene expression levels of several transcription factors from these lists by quantitative real-time PCR. We hope that our study substantially contributes to the understanding of the molecular mechanism underlying the pathology of alcohol-mediated anomalies and ease further research.

Ethanol toxicity affects olfactory receptor genes in forebrain of fetal mice

Toxicity of ethanol can lead to behavioral and cognitive impairments of fetus. The aim of this current study was to investigate the alteration of genes at developing fetal brain by maternal binge alcohol consumption. Genome-wide transcriptional analysis revealed a set of differentially expressed genes (20 upregulated and 21 down-regulated; 1.5-fold cut-off) on embryonic day 15 (ED15) in the developing fetal brain. The gene ontology analysis revealed the associations of these genes with sensory perception, whereas Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that they were correlated with olfactory transduction. Six olfactory receptor genes (Olfr960, Olfr1342, Olfr43, Olfr836, Olfr1262 and Olfr1419) encoding proteins involved in the olfactory transduction pathway showed reduced expression levels. The downregulation of these olfactory receptor genes may cause odor identification defects as well as abnormalities in the olfactory system. Our findings aid in the elucidation of the molecular mechanism underlying the defective olfactory processing that occurs following prenatal alcohol exposure.

Gestational Alcohol Exposure Altered DNA Methylation Status in the Developing Fetus

Ethanol is well known as a teratogenic factor that is capable of inducing a wide range of developmental abnormalities if the developing fetus is exposed to it. Duration and dose are the critical parameters of exposure that affect teratogenic variation to the developing fetus. It is suggested that ethanol interferes with epigenetic processes especially DNA methylation. We aimed to organize all of the available information on the alteration of DNA methylation by ethanol in utero. Thus, we have summarized all published information regarding alcohol-mediated alterations in DNA methylation during gestation. We tried to arrange information in a way that anyone can easily find the alcohol exposure time, doses, sampling time, and major changes in genomic level. Manuscript texts will also represent the correlation between ethanol metabolites and subsequent changes in methylome patterns. We hope that this review will help future researchers to further examine the issues associated with ethanol exposure.

Gene expression signatures after ethanol exposure in differentiating embryoid bodies

During the differentiation process, various epigenetic factors regulate the precise expression of important genes and control cellular fate. During this stage, the differentiating cells become vulnerable to external stimuli. Here, we used an early neural differentiation model to observe ethanol-mediated transcriptional alterations. Our objective was to identify important molecular regulators of ethanol-related alterations in the genome during differentiation. A transcriptomic analysis was performed to profile the mRNA expression in differentiating embryoid bodies with or without ethanol treatment. In total, 147 differentially expressed genes were identified in response to 50mM ethanol. Of these differentially expressed genes, 78 genes were up-regulated and 69 genes were down-regulated. Our analysis revealed a strong association among the transcript signatures of the important modulators which were involved in protein modification, protein synthesis and gene expression. Additionally, ethanol-mediated activation of DNA transcription was observed. We also profiled ethanol-responsive transcription factors (TFs), upstream transcriptional regulators and TF-binding motifs in the differentiating embryoid bodies. In this study, we established a platform that we hope will help other researchers determine the ethanol-mediated changes that occur during cellular differentiation.

In Utero Alcohol Exposure and the Alteration of Histone Marks in the Developing Fetus: An Epigenetic Phenomenon of Maternal Drinking

Ethanol is well known for its teratogenic effects during fetal development. Maternal alcohol consumption allows the developing fetus to experience the detrimental effects of alcohol exposure. Alcohol-mediated teratogenic effects can vary based on the dosage and the length of exposure. The specific mechanism of action behind this teratogenic effect is still unknown. Previous reports demonstrated that alcohol participates in epigenetic alterations, especially histone modifications during fetal development. Additional research is necessary to understand the correlation between major epigenetic events and alcohol-mediated teratogenesis such as that observed in fetal alcohol spectrum disorder (FASD). Here, we attempted to collect all the available information concerning alcohol-mediated histone modifications during gestational fetal development. We hope that this review will aid researchers to further examine the issues associated with ethanol exposure.