Protein Content and Methyl Donors in Maternal Diet Interact to Influence the Proliferation Rate and Cell Fate of Neural Stem Cells in Rat Hippocampus

Maternal diet during pregnancy and early postnatal life influences the setting up of normal physiological functions in the offspring. Epigenetic mechanisms regulate cell differentiation during embryonic development and may mediate gene/environment interactions. We showed here that high methyl donors associated with normal protein content in maternal diet increased the in vitro proliferation rate of neural stem/progenitor cells isolated from rat E19 fetuses. Gene expression on whole hippocampi at weaning confirmed this effect as evidenced by the higher expression of the Nestin and Igf2 genes, suggesting a higher amount of undifferentiated precursor cells. Additionally, protein restriction reduced the expression of the insulin receptor gene, which is essential to the action of IGFII. Inhibition of DNA methylation in neural stem/progenitor cells in vitro increased the expression of the astrocyte-specific Gfap gene and decreased the expression of the neuron-specific Dcx gene, suggesting an impact on cell differentiation. Our data suggest a complex interaction between methyl donors and protein content in maternal diet that influence the expression of major growth factors and their receptors and therefore impact the proliferation and differentiation capacities of neural stem cells, either through external hormone signals or internal genomic regulation.     

Stress–response pathways are altered in the hippocampus of chronic alcoholics

The chronic high-level alcohol consumption seen in alcoholism leads to dramatic effects on the hippocampus, including decreased white matter, loss of oligodendrocytes and other glial cells, and inhibition of neurogenesis. Examining gene expression in post mortem hippocampal tissue from 20 alcoholics and 19 controls allowed us to detect differentially expressed genes that may play a role in the risk for alcoholism or whose expression is modified by chronic consumption of alcohol. We identified 639 named genes whose expression significantly differed between alcoholics and controls at a False Discovery Rate (FDR) ≤ 0.20; 52% of these genes differed by at least 1.2-fold. Differentially expressed genes included the glucocorticoid receptor and the related gene FK506 binding protein 5 (FKBP5), UDP glycosyltransferase 8 (UGT8), urea transporter (SLC14A1), zinc transporter (SLC39A10), Interleukin 1 receptor type 1 (IL1R1), thioredoxin interacting protein (TXNIP), and many metallothioneins. Pathways related to inflammation, hypoxia, and stress showed activation, and pathways that play roles in neurogenesis and myelination showed decreases. The cortisol pathway dysregulation and increased inflammation identified here are seen in other stress-related conditions such as depression and post-traumatic stress disorder and most likely play a role in addiction. Many of the detrimental effects on the hippocampus appear to be mediated through NF-κB signaling. Twenty-four of the differentially regulated genes were previously identified by genome-wide association studies of alcohol use disorders; this raises the potential interest of genes not normally associated with alcoholism, such as suppression of tumorigenicity 18 (ST18), BCL2-associated athanogene 3 (BAG3), and von Willebrand factor (VWF).     

Ethanol treatment of lymphoblastoid cell lines from alcoholics and non-alcoholics causes many subtle changes in gene expression

To elucidate the effects of a controlled exposure to ethanol on gene expression, we studied lymphoblastoid cell lines (LCLs) from 21 alcoholics and 21 controls. We cultured each cell line for 24 h with and without 75 mM ethanol and measured gene expression using microarrays. Differences in expression between LCLs from alcoholics and controls included 13 genes previously identified as associated with alcoholism or related traits, including KCNA3, DICER1, ZNF415, CAT, SLC9A9, and PPARGC1B. The paired design allowed us to detect very small changes due to ethanol treatment: ethanol altered the expression of 37% of the probe sets (51% of the unique named genes) expressed in these LCLs, most by modest amounts. Ninety-nine percent of the named genes expressed in the LCLs were also expressed in brain. Key pathways affected by ethanol include cytokine, TNF, and NFκB signaling. Among the genes affected by ethanol were ANK3, EPHB1, SLC1A1, SLC9A9, NRD1, and SH3BP5, which were reported to be associated with alcoholism or related phenotypes in 2 genome-wide association studies. Genes that either differed in expression between alcoholics and controls or were affected by ethanol exposure are candidates for further study.     

Ethanol-induced disruption of Golgi apparatus morphology,primary neurite number and cellular orientation in developing cortical neurons

Prenatal ethanol exposure disrupts cortical neurite initiation and outgrowth, but prior studies have reported both ethanol-dependent growth promotion and inhibition. To resolve this ambiguity and better approximate in vivo conditions, we quantitatively analyzed neuronal morphology using a new, whole hemisphere explant model. In this model, Layer 6 (L6) cortical neurons migrate, laminate and extend neurites in an organotypic fashion. To selectively label L6 neurons, we performed ex utero electroporation of a GFP expression construct at embryonic day 13 and allowed the explants to develop for 2 days in vitro. Explants were exposed to (400 mg/dL) ethanol for either 4 or 24 h prior to fixation. Complete 3-D reconstructions were made of >80 GFP-positive neurons in each experimental condition. Acute responses to ethanol exposure included compaction of the Golgi apparatus accompanied by elaboration of supernumerary primary apical neurites, as well as a modest (∼15%) increase in higher order apical neurite length. With longer exposure time, ethanol exposure leads to a consistent, significant disorientation of the cell (cell body, primary apical neurite, and Golgi) with respect to the pial surface. The effects on cellular orientation were accompanied by decreased expression of cytoskeletal elements, microtubule-associated protein 2 and F-actin. These findings indicate that upon exposure to ethanol, developing L6 neurons manifest disruptions in Golgi apparatus and cytoskeletal elements which may in turn trigger selective and significant perturbations to primary neurite formation and neuronal polarity.     

Cell Cycle and Mismatch Repair Genes as Potential Biomarkers in Arabidopsis thaliana Seedlings Exposed to Silver Nanoparticles

The expression of cell cycle genes and DNA mismatch repair (MMR) genes were analyzed in Arabidopsis thaliana seedlings exposed to 0, 0.2, 0.5 and 1 mg/L of silver nanoparticles for 24, 48 and 72 h using real-time PCR. Significant up-regulation of AtPCNA1 was observed after 24 h exposure to 0.2 and 0.5 mg/L of silver nanoparticles. AtPCNA2 gene was up-regulated after 24, 48 and 72 h exposure to 0.5 and 1 mg/L of silver nanoparticles. AtMLH1 gene was up-regulated after 48 h exposure to 0.5 and 1 mg/L of silver nanoparticles and down-regulated after 72 h. Down-regulation of AtMSH2, AtMSH3, AtMSH6 and AtMSH7 mRNA was observed after exposure to all concentrations of silver nanoparticles for different time periods. Exposure to silver ions showed no significant change in the expression levels of AtPCNA and MMR genes. The results show that AtPCNA and MMR genes could be used as potential molecular biomarkers.     

Prenatal Stress Exposure and Post-traumatic Stress Disorder Associated With Risk of Postpartum Alcohol Misuse Among Women Veterans

ObjectivesMaternal alcohol misuse during the postpartum period is associated with negative maternal and infant outcomes. This study examined whether greater stress exposure in the year before the baby's birth and maternal post-traumatic stress disorder (PTSD) were associated with postpartum alcohol misuse among a sample of women veterans. Maternal PTSD was also examined as a moderator of the association between stress exposure and postpartum alcohol misuse.MethodsData were drawn from the Center for Maternal and Infant Outcomes Research in Translation study, a multisite prospective cohort study of pregnant and postpartum women veterans. Interviews were conducted within 12 weeks after birth. At this post-birth interview, women reported whether they experienced stressful events (e.g., loss of job, military deployment, separation/divorce) in the year before birth. PTSD diagnosis and postpartum scores on the Alcohol Use Disorders Identification Test (AUDIT-C) were derived from the Department of Veterans Affairs medical records.ResultsModels testing main and interaction effects showed a statistically significant association of both PTSD (p = .02) and stress exposure (p = .04), as well as significant interaction of PTSD and stress exposure (p = .03) with AUDIT-C scores postpartum, after controlling for marital status, age, and race. Specifically, compared with women without PTSD, those with PTSD had higher overall AUDIT-C scores postpartum, regardless of stress exposure. For women without PTSD, more stress exposure before birth was associated with higher AUDIT-C scores during the postpartum phase.ConclusionsPTSD diagnosis and life stressors before infant birth predicted maternal alcohol misuse during the postpartum period. Identifying such risk factors is an initial step in preventing alcohol misuse, with the goal of enhancing postpartum health for the birthing parent and infant.     

Fine mapping and expression of candidate genes within the chromosome 10 QTL region of the high and low alcohol-drinking rats

The high and low alcohol-drinking (HAD and LAD) rats were selectively bred for differences in alcohol intake. The HAD/LAD rats originated from the N/Nih heterogeneous stock developed from intercrossing eight inbred rat strains. The HAD × LAD F2 were genotyped, and a powerful analytical approach, using ancestral recombination and F2 recombination, was used to narrow a quantitative trait loci (QTL) for alcohol drinking to a 2-cM region on distal chromosome 10 that was in common in the HAD1/LAD1 and HAD2/LAD2 analyses. Quantitative real-time PCR was used to examine mRNA expression of six candidate genes (Crebbp, Trap1, Gnptg, Clcn7, Fahd1, and Mapk8ip3) located within the narrowed QTL region in the HAD1/LAD1 rats. Expression was examined in five brain regions, including the nucleus accumbens, amygdala, caudate putamen, hippocampus, and prefrontal cortex. All six genes showed differential expression in at least one brain region. Of the genes tested in this study, Crebbp and Mapk8ip3 may be the most promising candidates with regard to alcohol drinking.     

Effects of third trimester-equivalent ethanol exposure on Cl co-transporter expression,network activity,and GABAergic transmission in the CA3 hippocampal region of neonatal rats

Fetal alcohol spectrum disorders are often associated with structural and functional hippocampal abnormalities, leading to long-lasting learning and memory deficits. The mechanisms underlying these abnormalities are not fully understood. Here, we investigated whether ethanol exposure during the 3rd trimester-equivalent period alters spontaneous network activity that is involved in neuronal circuit development in the CA3 hippocampal region. This activity is driven by GABA_A receptors, which can have excitatory actions in developing neurons as a consequence of greater expression of the Cl⁻ importer, NKCC1, with respect to expression of the Cl⁻ exporter, KCC2, resulting in high [Cl⁻]_i. Rat pups were exposed to ethanol vapor from postnatal day (P) 2–16 (4 h/day). Weight gain was significantly reduced in pups exposed to ethanol compared to control at P15 and 16. Brain slices were prepared immediately after the end of the 4-h exposure on P4-16 and experiments were also performed under ethanol-free conditions at the end of the exposure paradigm (P17-22). Ethanol exposure did not significantly affect expression of KCC2 or NKCC1, nor did it affect network activity in the CA3 hippocampal region. Ethanol exposure significantly decreased the frequency (at P9-11) and increased the amplitude (at P5-8 and P17-21) of GABA_A receptor-mediated miniature postsynaptic currents. These data suggest that repeated in vivo exposure to ethanol during the 3rd trimester-equivalent period alters GABAergic transmission in the CA3 hippocampal region, an effect that could lead to abnormal circuit maturation and perhaps contribute to the pathophysiology of fetal alcohol spectrum disorders.     

Maternal fish oil supplementation ameliorates maternal high-fructose diet-induced dyslipidemia in neonatal mice with suppression of lipogenic gene expression in livers of postpartum mice

Maternal fructose consumption during pregnancy and lactation is associated with metabolic dysregulation in offspring. We tested the hypothesis that fish oil (FO) supplementation during pregnancy and lactation improves fructose-induced metabolic dysregulation in postpartum dams and offspring mice. We therefore aimed to determine the effects of FO supplementation on metabolic disruption in neonatal mice and dams induced by a maternal high-fructose diet (HFrD). The weight of the offspring of dams fed with HFrD on postnatal day 5 was significantly low, but this was reversed by adding FO to the maternal diet. Feeding dams with HFrD significantly increased plasma concentrations of triglycerides, uric acid, and total cholesterol, and decreased free fatty acid concentrations in offspring. Maternal supplementation with FO significantly suppressed HFrD-induced hypertriglyceridemia and hyperuricemia in the offspring. Maternal HFrD induced remarkable mRNA expression of the lipogenic genes Srebf1, Fasn, Acc1, Scd1, and Acly in the postpartum mouse liver without affecting hepatic and plasma lipid levels. Although expression levels of lipogenic genes were higher in the livers of postpartum dams than in those of nonmated mice, HFrD feeding increased the hepatic lipid accumulation in nonmated mice but not in postpartum dams. These findings suggest that although hepatic lipogenic activity is higher in postpartum dams than nonmated mice, the lipid consumption is enhanced in postpartum dams during pregnancy and lactation. Maternal FO supplementation obviously suppressed the expression of these lipogenic genes. These findings coincide with reduced plasma triglyceride concentrations in the offspring. Therefore, dietary FO apparently ameliorated maternal HFrD-induced dyslipidemia in offspring by suppressing maternal lipogenic gene expression and/or neonatal plasma levels of uric acid.     

Expression analysis of radiation-responsive genes in human hematopoietic stem/progenitor cells

To clarify the nature of the genes that contribute to the radiosensitivity of human hematopoietic stem/progenitor cells (HSPCs), we analyzed the gene expression profiles detected in HSPCs irradiated with 2 Gy X-rays after culture with or without an optimal combination of hematopoietic cytokines. Highly purified CD34⁺ cells from human placental/umbilical cord blood were used as HSPCs. The cells were exposed to 2 Gy X-irradiation and treated in serum-free medium under five different sets of conditions for 6 h. The gene expression levels were analyzed by cDNA microarray, and then the network of responsive genes was investigated. A comprehensive genetic analysis to search for genes associated with cellular radiosensitivity was undertaken, and we found that expression of the genes downstream of MYC oncogene increased after X-irradiation. In fact, the activation of MYC was observed immediately after X-irradiation, and MYC was the only gene still showing activation at 6 h after irradiation. Furthermore, MYC had a significant impact on the biological response, particularly on the tumorigenesis of cells and the cell cycle control. The activated gene regulator function of MYC resulting from irradiation was suppressed by culturing the HSPCs with combinations of cytokines (recombinant human thrombopoietin + interleukin 3 + stem cell factor), which exerted radioprotective effects. MYC was strongly associated with the radiosensitivity of HSPCs, and further study and clarification of the genetic mechanisms that control the cell cycle following X-irradiation are required.     

Anti-cancer activity of a novel palladium(II) complex on human breast cancer cells in vitro and in vivo

Anti-cancer effects of a newly-synthesized palladium(II) complex, [Pd(sac)(terpy)](sac)·4H₂O (sac = saccharinate, and terpy = 2,2′:6′,2″-terpyridine), were tested against human breast cancer cell lines, MCF-7 and MDA-MB-231. The Pd complex had a strong anti-growth effect in a dose- and time-dependent manner in vitro. This effect was also confirmed by the experiment performed on Balb/c mice in vivo. The IC₅₀ values were 0.09 μM for MDA-MB-231 and 3.05 μM for MCF-7. It was also very effective in disrupting the formation of MDA-MB-231 tubules on matrigel, indicative of a putative anti-invasive activity. It induced apoptosis via the cell death genes of DR4 and DR5. In conclusion, this newly-synthesized Pd (II) complex represents a potentially active novel drug for the breast cancer treatment.     

Oral intake of chicoric acid reduces acute alcohol-induced hepatic steatosis in mice

ObjectiveAcute and chronic consumption of alcohol can alter intestinal barrier function thereby increasing portal endotoxin levels subsequently leading to an activation of toll-like receptor (TLR) 4-dependent signaling cascades, elevated levels of reactive oxygen species and induction of tumor necrosis factor α in the liver. Recent studies suggest that chicoric acid found in Echinacea pupurea, chicory, and other plants, may possess antioxidant and anti-inflammatory effects. The aim of the present study was to determine if chicoric acid can reduce acute alcohol-induced liver damage.MethodsFemale mice were given chicoric acid orally (4 mg/kg body weight) for 4 d before acute ethanol administration (6 g/kg body weight). Furthermore, the effect of chicoric acid on the lipopolysaccharide (LPS)-dependent activation in an in vitro model of Kupffer cells (RAW264.7 macrophages) was assessed.ResultsAcute alcohol ingestion caused a significant increase in hepatic triacylglycerols accumulation, which was associated with increased protein levels of the inducible nitric oxide synthase (iNOS), 4-hydroxynonenal protein adducts, and active plasminogen activator inhibitor 1 protein in the liver. Pretreatment of animals with chicoric acid significantly attenuated these effects of alcohol on the liver. In LPS-treated RAW264.7 macrophages, pretreatment with chicoric acid significantly suppressed LPS-induced mRNA expression of iNOS and tumor necrosis factor α.ConclusionThese data suggest that chicoric acid may reduce acute alcohol-induced steatosis in mice through interfering with the induction of iNOS and iNOS-dependent signaling cascades in the liver.     

Maternal Exposure to Polycyclic Aromatic Hydrocarbons and 5'-CpG Methylation of Interferon-γ in Cord White Blood Cells

Background: Maternal factors are implicated in the onset of childhood asthma. Differentiation of naïve CD4⁺ T lymphocytes into pro-allergic T-helper 2 cells induces interleukin (IL)4 expression and inhibits interferon (IFN)γ expression accompanied by concordant methylation changes in the promoters of these genes. However, it has yet to be established whether maternal exposure to polycyclic aromatic hydrocarbons (PAHs) can alter these gene promoters epigenetically during fetal development.Objectives: In this study we sought to elucidate the relationship between maternal PAH exposure and promoter methylation status of IFNγ and IL4.Methods: We assessed the effects of benzo[a]pyrene (BaP), a representative airborne PAH, on the methylation status of the IFNγ and IL4 promoters in Jurkat cells and two lung adenocarcinoma cell lines, and on gene expression. In addition, we evaluated methylation status of the IFNγ promoter in cord white blood cells from 53 participants in the Columbia Center for Children’s Environmental Health cohort. Maternal PAH exposure was estimated by personal air monitoring during pregnancy.Results: In vitro exposure of the cell models to low, noncytotoxic doses (0.1 and 1 nM) of BaP elicited increased promoter hypermethylation and reduced expression of IFNγ, but not IL4. IFNγ promoter methylation in cord white blood cells was associated with maternal PAH exposure in the cohort study subsample.Conclusion: Consistent with the results for the cell lines, maternal exposure to PAHs was associated with hypermethylation of IFNγ in cord blood DNA from cohort children. These findings support a potential role of epigenetics in fetal reprogramming by PAH-induced environmental diseases.     

Effect of Maternal Immune Status on Responsiveness of Bacillus Calmette-Guérin Vaccination in Mouse Neonates

ObjectivesBacillus Calmette-Guérin (BCG) vaccination has proven to be efficient in immunologically naïve infants; however, it has not been investigated that maternal natural exposure to Mycobacterium and/or BCG vaccine could influence the characteristics of immune responses to BCG in newborns. In this study, we analyzed whether the maternal immune status to M tuberculosis (M tb) can affect neonatal immunity to BCG using a mouse model.MethodsNeonates were obtained from mice that were previously exposed to live BCG, to live M avium, or to heat-killed M tb H37Rv, and from naïve control mothers. One week after birth, the neonates were divided into two subgroups: one group immunized with live BCG via the subcutaneous route and the other group of neonates sham-treated. Interferon-gamma (IFNγ) secretion in response to in vitro stimulation with heat-killed BCG or purified protein derivative (PPD) was examined. Protection against M tb infection was evaluated by challenging mice nasally with live M tb H37Rv followed by counting colonies from spleen and lung homogenates.ResultsBCG-immunized neonates showed increased IFNγ secretion in response to heat-killed BCG or PPD. All mice in BCG-immunized neonates subgroups showed reduced bacterial burden (colony forming unit) in the lungs when compared with control naive neonate mice. However, no statistically significant difference was observed when comparing BCG-immunized mice born from mothers previously exposed to M avium or immunized with either heat-killed H37Rv or live BCG and mice born from naïve mothers.ConclusionThe maternal immune status to M tb does not appear to impact on the immunogenicity of BCG vaccine in their progeny in our experimental conditions.     

Replication of genome wide association studies on hepatocellular carcinoma susceptibility loci of STAT4 and HLA-DQ in a Korean population

A recent genome-wide association study (GWAS) for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) identified two loci (rs7574865 in STAT4 and rs9275319 in HLA-DQ) in a Chinese population. We attempted to replicate the associations between the two SNP loci and the risk of HCC in a Korean population. The rs7574865 in STAT4 and rs9275319 in HLA-DQ were genotyped in a total of 3838 Korean subjects composed of 287 HBV-related hepatocellular carcinoma patients, 671 chronic hepatitis B virus (CHB) patients, and 2880 population controls using TaqMan genotyping assay. Gene expression was measured by microarray. A logistic regression analysis revealed that rs7574865 in STAT4 and rs9275319 in HLA-DQ were associated with the risk of CHB (OR = 1.25, P = 0.0002 and OR = 1.57, P = 1.44 × 10⁻¹⁰, respectively). However, these loci were no association with the risk of HBV-related HCC among CHB patients. In the gene expression analyses, although no significant differences in mRNA expression of nearby genes according to genotypes were detected, a significantly decreased mRNA expression in HCC subjects was observed in STAT4, HLA-DQA1, and HLA-DQB1. Although the genetic effects of two HCC susceptibility loci were not replicated, the two loci were found to exert susceptibility effects on the risk of CHB in a Korean population. In addition, the decreased mRNA expression of STAT4, HLA-DQA1, and HLA-DQB1 in HCC tissue might provide a clue to understanding their role in the progression to HCC.