DPPA4 increases aggressiveness of pituitary neuroendocrine tumors by enhancing cell stemness.

BACKGROUND: Pituitary neuroendocrine tumors, PitNETs, are often aggressive and precipitate in distant metastases that are refractory to current therapies. However, the molecular mechanism in PitNETs' aggressiveness is not well understood. Developmental pluripotency-associated 4 (DPPA4) is known as a stem cell regulatory gene and overexpressed in certain cancers, but its function in the context of PitNETs' aggressiveness is not known. METHODS: We employed both rat and human models of PitNETs. In the rat pituitary tumor model (RPT), we used prenatal-alcohol-exposed (PAE) female Fischer rats which developed aggressive PitNETs following estrogen treatment, while in the human pituitary tumor (HPT) model, we used aggressively proliferative cells from pituitary tumors of patients undergone surgery. Various molecular, cellular, and epigenetic techniques were used to determine the role of DPPA4 in PitNETs' aggressiveness. RESULTS: We show that DPPA4 is overexpressed in association with increased cell stemness factors in aggressive PitNETs of PAE rats and of human patients. Gene-editing experiments demonstrate that DPPA4 increases the expression of cell stemness and tumor aggressiveness genes and promotes proliferation, colonization, migration, and tumorigenic potential of PitNET cells. ChIP assays and receptor antagonism studies reveal that DPPA4 binds to canonical WINTs promoters and increases directly or indirectly the Wnt/ß-catenin control of cell stemness, tumor growth, and aggressiveness of PitNETs. Epigenetic studies show involvement of histone methyltransferase in alcohol activation of DPPA4. CONCLUSIONS: These findings support a role of DPPA4 in tumor stemness and aggressiveness and provide a preclinical rationale for modulating this stemness regulator for the treatment of PitNETs.

Altered circadian expression of clock genes and clock-regulatory epigenetic modifiers in saliva of children with fetal alcohol spectrum disorders.

Prenatal alcohol-exposed (AE) infants and children often demonstrate disrupted sleep patterns, including more frequent awakenings, reduced total sleep time, and more night-to-night sleep variability. Despite the strong connection between sleep patterns and circadian rhythmicity, relatively little is known about circadian rhythm disruptions in individuals with AE. Recently, several reports demonstrated that evaluating the expression patterns of human clock genes in biological fluids could reveal an individual's circadian phenotype. Human saliva offers an emerging and easily available physiological sample that can be collected non-invasively for core-clock gene transcript analyses. We compared the expression patterns of core-clock genes and their regulatory genes in salivary samples of children aged 6-10 years-old with and without AE during the light cycle between ZT0-ZT11. We isolated the RNA from the samples and measured the expression patterns of core clock genes and clock regulating genes using the human specific primers with quantitative real-time PCR. Analysis of core clock genes expression levels in saliva samples from AE children indicates significantly altered levels in expression of core-clock BMAL1, CLOCK, PER1-3 and CRY1,2, as compared to those in age-matched control children. We did not find any sex difference in levels of clock genes in AE and control groups. Cosinor analysis was used to evaluate the rhythmic pattern of these clock genes, which identified circadian patterns in the levels of core clock genes in the control group but absent in the AE group. The gene expression profile of a salivary circadian biomarker ARRB1 was rhythmic in saliva of control children but was arhythmic in AE children. Altered expression patterns were also observed in clock regulatory genes: NPAS2, NFL3, NR1D1, DEC1, DEC2, and DBP, as well as chromatin modifiers: MLL1, P300, SIRT1, EZH2, HDAC3, and ZR1D1, known to maintain rhythmic expression of core-clock genes. Overall, these findings provide the first evidence that AE disturbs the circadian patten expression of core clock genes and clock-regulatory chromatin modifiers in saliva.

Neuroimmune Interactions in Fetal Alcohol Spectrum Disorders: Potential Therapeutic Targets and Intervention Strategies.

Fetal alcohol spectrum disorders (FASD) are a set of abnormalities caused by prenatal exposure to ethanol and are characterized by developmental defects in the brain that lead to various overt and non-overt physiological abnormalities. Growing evidence suggests that in utero alcohol exposure induces functional and structural abnormalities in gliogenesis and neuron-glia interactions, suggesting a possible role of glial cell pathologies in the development of FASD. However, the molecular mechanisms of neuron-glia interactions that lead to the development of FASD are not clearly understood. In this review, we discuss glial cell pathologies with a particular emphasis on microglia, primary resident immune cells in the brain. Additionally, we examine the involvement of several neuroimmune molecules released by glial cells, their signaling pathways, and epigenetic mechanisms responsible for FASD-related alteration in brain functions. Growing evidence suggests that extracellular vesicles (EVs) play a crucial role in the communication between cells via transporting bioactive cargo from one cell to the other. This review emphasizes the role of EVs in the context of neuron-glia interactions during prenatal alcohol exposure. Finally, some potential applications involving nutritional, pharmacological, cell-based, and exosome-based therapies in the treatment of FASD are discussed.

miRNA-383 and miRNA-384 Suppress Proopiomelanocortin Gene Expression in the Hypothalamus: Effects of Early Life Ethanol Exposure.

INTRODUCTION: Early life ethanol exposure is known to program hypothalamic proopiomelanocortin (POMC) neurons to express a reduced level of POMC and its control of stress axis functions throughout the life span. In this study, we tested whether miRNAs contribute to the ethanol-induced suppression of Pomc gene expression during the developmental period. METHODS: In in vivo studies, POMC-EGFP male mice were fed with 2.5 g/kg ethanol using milk formula (AF), pair-fed isocaloric milk formula, or left in the litter during postnatal days (PNDs) 2-6. In in vitro studies, mHypoA-POMC/GFP cells were treated with ethanol (50 mM) for a 24-h period. Hypothalamic tissues or cell extracts were used for measurement of miRNAs and POMC mRNA. RESULTS: Determination of genome-wide microRNA expression profile identified 40 miRNAs significantly altered in hypothalamic tissues of AF mice. In silico analysis further identified miRNA-383, -384, and -488 have putative binding sites at the POMC 3'UTR. However, only miR-383 and miR-384 are identified to be responsive to ethanol. Administration of miR-383 or -384 inhibitor oligos suppressed ethanol-stimulated miR-383 or -384 expression and restored Pomc mRNA and protein expression in AF mice. mHypoA-POMC/GFP cells when treated with ethanol showed elevated levels of miR-383 and miR-384 and reduced level of Pomc mRNA. Treatment with miR-383 or -384 mimic oligos reduced the level of Pomc mRNA, while treatment with miR-383 or -384 inhibitor oligos increased the level of Pomc mRNA. Reporter assay further confirms the binding specificity of miR-383 and miR-384 to Pomc 3'UTR. CONCLUSION: These data suggest that miR-383 and miR-384 suppress Pomc gene expression and may contribute to the ethanol-induced alteration of the stress axis functions.

Fetal alcohol exposure impairs learning and memory functions and elevates levels of various biochemical markers of Alzheimer's disease in the brain of 12-month-old rats.

BACKGROUND: Alcohol drinking during pregnancy often adversely affects brain development among offspring, inducing persistent central nervous system dysfunction. However, it is unknown whether fetal alcohol exposure (FAE) promotes the biochemical characteristics of Alzheimer's disease in offspring. METHODS: We used a first- and second-trimester human equivalent rat model of FAE that involves feeding a liquid diet containing 6.7% v/v ethanol from gestational days 7 through 21 in Fischer-344 rats. Control rats were fed an isocaloric liquid diet or rat chow ad libitum. Pups were weaned on postnatal day 21 and housed by sex. They were used for behavioral and biochemical studies at about 12 months of age. Only one male or one female offspring from a litter was included in each experimental group. RESULTS: Fetal alcohol-exposed offspring had poorer learning and memory functions than controls. The experimental animals, both male and female, also had elevated levels of acetylcholinesterase (AChE) activity, hyperphosphorylated-tau protein, ß-amyloid (Aß) and Aß1-42 proteins, ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), and Unc-5 netrin receptor C (UNC5C) proteins in the cerebral cortex and hippocampus at 12 months of age. CONCLUSIONS: These findings show that FAE increases the expression of some of the biochemical and behavioral phenotypes of Alzheimer's disease.

Epigenetic insight into effects of prenatal alcohol exposure on stress axis development: Systematic review with meta-analytic approaches.

We conducted a systematic review with meta-analytic elements using publicly available Gene Expression Omnibus (GEO) datasets to determine the role of epigenetic mechanisms in prenatal alcohol exposure (PAE)-induced hypothalamic-pituitary-adrenal (HPA) axis dysfunctions in offspring. Several studies have demonstrated that PAE has long-term consequences on HPA axis functions in offspring. Some studies determined that alcohol-induced epigenetic alterations during fetal development persist in adulthood. However, additional research is needed to understand the major epigenetic events leading to alcohol-induced teratogenesis of the HPA axis. Our network analysis of GEO datasets identified key pathways relevant to alcohol-mediated histone modifications, DNA methylation, and miRNA involvement associated with PAE-induced alterations of the HPA axis. Our analysis indicated that PAE perturbated the epigenetic machinery to activate corticotrophin-releasing hormone, while it suppressed opioid, glucocorticoid receptor, and circadian clock genes. These results help to further our understanding of the epigenetic basis of alcohol's effects on HPA axis development.

Beta 2 adrenergic receptor and mu opioid receptor interact to potentiate the aggressiveness of human breast cancer cell by activating the glycogen synthase kinase 3 signaling.

BACKGROUND: Opioid and beta-adrenergic receptors are recently shown to cross talk via formation of receptor heterodimers to control the growth and proliferation of breast cancer cells. However, the underlying cell signaling mechanism remained unclear. METHODS: To determine the effect of the interaction of the two systems in breast cancer, we employed triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468, CRISPR or chemical inhibition or activation of beta-adrenergic receptors (B2AR) and mu-opioid receptors (MOR) gene, and PCR array technology and studied aggressive tumor phenotype and signaling cascades. RESULTS: We show here that in triple-negative breast cancer cells, the reduction in expression B2AR and MOR by genetic and pharmacological tools leads to a less aggressive phenotype of triple-negative breast cancer cells in vitro and in animal xenografts. Genomic analysis indicates the glycogen synthase kinase 3 (GSK3) pathway as a possible candidate messenger system involved in B2AR and MOR cross talk. GSK3 inactivation in MDA-MB-231 and MDA-MB-468 cells induced similar phenotypic changes as the inhibition of B2AR and/or MOR, while a GSK3 activation by wortmannin reversed the effects of B2AR and/or MOR knockdown on these cells. GSK3 inactivation also prevents B2AR agonist norepinephrine or MOR agonist DAMGO from affecting MDA-MB-231 and MDA-MB-468 cell proliferation. CONCLUSIONS: These data confirm a role of B2AR and MOR interaction in the control of breast cancer cell growth and identify a possible role of the GSK3 signaling system in mediation of these two receptors' cross talk. Screening for ligands targeting B2AR and MOR interaction and/or the GSK3 system may help to identify novel drugs for the prevention of triple-negative breast cancer cell growth and metastasis.

Transgenerational inheritance of fetal alcohol effects on proopiomelanocortin gene expression and methylation, cortisol response to stress, and anxiety-like behaviors in offspring for three generations in rats: Evidence for male germline transmission.

Previously it has been shown that fetal alcohol exposure increases the stress response partly due to lowering stress regulatory proopiomelanocortin (Pomc) gene expression in the hypothalamus via epigenetic mechanisms for multiple generations in mixed-breed rats. In this study we assess the induction of heritable epigenetic changes of Pomc-related variants by fetal alcohol exposure in isogenic Fischer 344 rats. Using transgenerational breeding models and fetal alcohol exposure procedures, we determined changes in hypothalamic Pomc gene expression and its methylation levels, plasma corticosterone hormone response to restraint stress, and anxiety-like behaviors using elevated plus maze tests in fetal alcohol-exposed offspring for multiple generations in isogenic Fischer rats. Fetal alcohol-exposed male and female rat offspring showed significant deficits in POMC neuronal functions with increased Pomc gene methylation and reduced expression. These changes in POMC neuronal functions were associated with increased plasma corticosterone response to restraint stress and increased anxiety-like behavior. These effects of fetal alcohol exposure persisted in the F1, F2, and F3 progeny of the male germline but not of the female germline. These data suggest that fetal alcohol exposure induces heritable changes in Pomc-related variants involving stress hyperresponsiveness and anxiety-like behaviors which perpetuate into subsequent generations through the male germline via epigenetic modifications.

Beta 2 Adrenergic Receptor Antagonist Propranolol and Opioidergic Receptor Antagonist Naltrexone Produce Synergistic Effects on Breast Cancer Growth Prevention by Acting on Cancer Cells and Immune Environment in a Preclinical Model of Breast Cancer.

Cancer progression is known to be promoted by increased body stress caused by elevated beta-adrenergic and opioidergic nervous system activities. The effects of ß2-adrenergic blocker propranolol (PRO) and µ-opioid receptor antagonist naltrexone (NTX) were tested using a preclinical model of human breast cancer. These drugs, individually, and more potently when combined, inhibited the cell growth and progression of breast cancer cells in vitro in cultures, and in vivo in rat xenografts. The antitumor activities of these drugs were associated with direct cell intrinsic effects, including increased cell growth arrest, elevated levels of apoptotic proteins, and reduced production of epithelial-mesenchymal transition factors by the tumor cells, as well as effects on innate immune activation and reduced inflammatory cytokine levels in plasma. These data suggest that the combined treatments of PRO and NTX produce impressive antitumor effects in the preclinical breast cancer model, and thereby may provide a new combinatorial treatment strategy with more clinical treatment modalities.

Sex-Determining Region Y Controls the Effects of Fetal Alcohol Exposure on Proopiomelanocortin Gene Expression.

Fetal alcohol exposure (FAE) causes various neurodevelopmental deficits in offspring, including reduced expression of the stress regulatory proopiomelanocortin (Pomc) gene and an elevated stress response for multiple generations via the male germline. Male germline-specific effects of FAE on the Pomc gene raises the question if the sex-determining region Y (SRY) may have a role in regulating Pomc gene expression. Using a transgenerational model of FAE in Fischer 344 rats, we determined the role of SRY in the regulation of the Pomc gene. FAEs, like on the Pomc gene, reduced Sry gene expression in sperm and the mediobasal hypothalamus (MBH) in male adult offspring. Fetal alcohol-induced inhibition of Sry gene expression was associated with increased Sry promoter DNA methylation. Additionally, fetal alcohol effects on the Sry gene persisted for three generations in the male germline but not in the female germline. Sry gene knockdown reduced the Pomc gene expression. Sry recruitment onto the Pomc promoter was found to be reduced in the hypothalamus of fetal alcohol-exposed rats compared to control rats. Pomc promoter luciferase activity was increased following Sry overexpression. A site-directed mutagenesis study revealed that SRY binding sites are required for POMC promoter transcription activity. Overall, these findings suggest that SRY plays a stimulatory role in the regulation of Pomc gene expression and may potentially contribute to the fetal alcohol-induced changes in the level of Pomc gene expression for multiple generations.

Preconception Alcohol Exposure Increases the Susceptibility to Diabetes in the Offspring.

Heavy alcohol drinking alters glucose metabolism, but the inheritability of this effect of alcohol is not well understood. We used an animal model of preconception alcohol exposure in which adult female rats were given free access to 6.7% alcohol in a liquid diet and water for about 4 weeks, went without alcohol for 3 weeks, and then were bred to generate male and female offspring. Control animals were either ad lib-fed rat chow or pair-fed an isocaloric liquid diet during the time of alcohol-feeding in the experimental animals. Our results show that the female rats fed with alcohol in the liquid diet, but not with the isocaloric liquid diet, prior to conception had an altered stress gene network involving glucose metabolism in oocytes when compared with those in ad lib-fed chow diet controls. The offspring born from preconception alcohol-fed mothers showed significant hyperglycemia and hypoinsulinemia when they were adults. These rats also showed increased levels of inflammatory cytokines and cellular apoptosis in the pancreas, altered insulin production and actions in the liver, and a reduced number of proopiomelanocortin neurons in the hypothalamus. Replenishment of proopiomelanocortin neurons in these animals normalized the abnormal glucose to restore homeostasis. These data suggest that preconception alcohol exposures alter glucose homeostasis by inducing proopiomelanocortin neuronal functional abnormalities. Our findings provide a novel insight into the impact of high doses of alcohol on the female gamete that may cause inheritance of an increased susceptibility to diabetes.

Alcohol Increases Exosome Release from Microglia to Promote Complement C1q-Induced Cellular Death of Proopiomelanocortin Neurons in the Hypothalamus in a Rat Model of Fetal Alcohol Spectrum Disorders.

Microglia, a type of CNS immune cell, have been shown to contribute to ethanol-activated neuronal death of the stress regulatory proopiomelanocortin (POMC) neuron-producing ß-endorphin peptides in the hypothalamus in a postnatal rat model of fetal alcohol spectrum disorders. We determined whether the microglial extracellular vesicle exosome is involved in the ethanol-induced neuronal death of the ß-endorphin neuron. Extracellular vesicles were prepared from hypothalamic tissues collected from postnatal rats (both males and females) fed daily with 2.5 mg/kg ethanol or control milk formula for 5 d or from hypothalamic microglia cells obtained from postnatal rats, grown in cultures for several days, and then challenged with ethanol or vehicle for 24 h. Nanoparticle tracking analysis and transmission electron microscopy indicated that these vesicles had the size range and shape of exosomes. Ethanol treatments increased the number and the ß-endorphin neuronal killing activity of microglial exosomes both in vivo and in vitro Proteomics analyses of exosomes of cultured microglial cells identified a large number of proteins, including various complements, which were elevated following ethanol treatment. Proteomics data involving complements were reconfirmed using quantitative protein assays. Ethanol treatments also increased deposition of the complement protein C1q in ß-endorphin neuronal cells in both in vitro and in vivo systems. Recombinant C1q protein increased while C1q blockers reduced ethanol-induced C3a/b, C4, and membrane attack complex/C5b9 formations; ROS production; and ultimately cellular death of ß-endorphin neurons. These data suggest that the complement system involving C1q-C3-C4-membrane attack complex and ROS regulates exosome-mediated, ethanol-induced ß-endorphin neuronal death.SIGNIFICANCE STATEMENT Neurotoxic action of alcohol during the developmental period is recognized for its involvement in fetal alcohol spectrum disorders, but the lack of clear understanding of the mechanism of alcohol action has delayed the progress in therapeutic intervention of this disease. Proopiomelanocortin neurons known to regulate stress, energy homeostasis, and immune functions are reported to be killed by developmental alcohol exposure because of activation of microglial immune cells in the brain. While microglia are known to use extracellular vesicles to communicate with neurons for maintaining homeostasis, we show here that ethanol exposure during the developmental period hijacks this system to spread apoptotic factors, including complement protein C1q, to induce the membrane attack complex and reactive super-oxygen species for proopiomelanocortin neuronal killing.

Transgenerational inheritance of fetal alcohol exposure adverse effects on immune gene interferon-ϒ.

BACKGROUND: Alcohol exposures in utero have been shown to alter immune system functions in the offspring which persists into adulthood. However, it is not apparent why the in utero alcohol effect on the immune system persists into adulthood of fetal alcohol-exposed offspring. The objective of this study was to determine the long-term effects of fetal alcohol exposure on the production of interferon-ϒ (IFN-ϒ), a cytokine known to regulate both innate and adaptive immunity. METHODS: Isogenic Fisher 344 rats were bred to produce pregnant dams, which were fed with a liquid diet containing 6.7% alcohol between gestation days 7 and 21 and pair-fed with an isocaloric liquid diet or fed ad libitum with rat chow; their male and female offspring were used for the study. F1-F3 generation rats were used when they were 2 to 3 months old. Fetal alcohol exposure effects on the Ifn-ɣ gene was determined by measuring the gene promoter methylation and mRNA and protein expression in the spleen. Additionally, transgenerational studies were conducted to evaluate the germline-transmitted effects of fetal alcohol exposure on the Ifn-ɣ gene. RESULTS: Fetal alcohol exposure reduced the expression of Ifn-ɣ mRNA and IFN-ϒ protein while it increased the proximal promoter methylation of the Ifn-ɣ gene in both male and female offspring during the adult period. Transgenerational studies revealed that the reduced levels of Ifn-ɣ expression and increased levels of its promoter methylation persisted only in F2 and F3 generation males derived from the male germ line. CONCLUSION: Overall, these findings provide the evidence that fetal alcohol exposures produce an epigenetic mark on the Ifn-ɣ gene that passes through multiple generations via the male germ line. These data provide the first evidence that the male germ line transmits fetal alcohol exposure's adverse effects on the immune system.

Persistent Changes in Stress-Regulatory Genes in Pregnant Women or Children Exposed Prenatally to Alcohol.

BACKGROUND: We have recently shown that binge or heavy levels of alcohol drinking increase deoxyribonucleic acid (DNA) methylation and reduce gene expression of proopiomelanocortin (POMC) and period 2 (PER2) in adult human subjects (Gangisetty et al., Alcohol Clin Exp Res, 43, 2019, 212). One hypothesis would be that methylation of these 2 genes is consistently associated with alcohol exposure and could be used as biomarkers to predict risk of prenatal alcohol exposure (PAE). Results of the present study provided some support for this hypothesis. METHODS: We conducted a series of studies to determine DNA methylation changes in stress regulatory genes proopiomelanocortin (POMC) and period 2 (PER2) using biological samples from 3 separate cohorts of patients: (i) pregnant women who consumed moderate-to-high levels of alcohol or low/unexposed controls, (ii) children with PAE and non-alcohol-exposed controls, and (iii) children with PAE treated with or without choline. RESULTS: We found pregnant women who consumed moderate-to-high levels of alcohol and gave birth to PAE children had higher DNA methylation of POMC and PER2. PAE children also had increased methylation of POMC and PER2. The differences in the gene methylation of PER2 and POMC between PAE and controls did not differ by maternal smoking status. PAE children had increased levels of stress hormone cortisol and adrenocorticotropic hormone. Choline supplementation reduced DNA hypermethylation and increased expression of POMC and PER2 in children with PAE. CONCLUSIONS: These data suggest that PAE significantly elevates DNA methylation of POMC and PER2 and increases levels of stress hormones. Furthermore, these results suggest the possibility that measuring DNA methylation levels of PER2 and POMC in biological samples from pregnant women or from children may be useful for identification of a woman or a child with PAE.

Alcohol exposure alters pre-mRNA splicing of antiapoptotic Mcl-1L isoform and induces apoptosis in neural progenitors and immature neurons.

Alternative splicing and expression of splice variants of genes in the brain may lead to the modulation of protein functions, which may ultimately influence behaviors associated with alcohol dependence and neurotoxicity. We recently showed that ethanol exposure can lead to pre-mRNA missplicing of Mcl-1, a pro-survival member of the Bcl-2 family, by downregulating the expression levels of serine/arginine rich splicing factor 1 (SRSF1). Little is known about the physiological expression of these isoforms in neuronal cells and their role in toxicity induced by alcohol exposure during the developmental period. In order to investigate the impact of alcohol exposure on alternative splicing of Mcl-1 pre-mRNA and its role in neurotoxicity, we developed a unique primary human neuronal culture model where neurospheres (hNSPs), neural progenitors (hNPCs), immature neurons, and mature neurons were cultured from the matching donor fetal brain tissues. Our data suggest that neural progenitors and immature neurons are highly sensitive to the toxic effects of ethanol, while mature neuron cultures showed resistance to ethanol exposure. Further analysis of Mcl-1 pre-mRNA alternative splicing by semi-quantitative and quantitative analysis revealed that ethanol exposure causes a significant decrease in Mcl-1L/Mcl-1S ratio in a dose and time dependent manner in neural progenitors. Interestingly, ectopic expression of Mcl-1L isoform in neural progenitors was able to recover the viability loss and apoptosis induced by alcohol exposure. Altogether, these observations suggest that alternative splicing of Mcl-1 may play a crucial role in neurotoxicity associated with alcohol exposure in the developing fetal brain.

Early life alcohol exposure primes hypothalamic microglia to later-life hypersensitivity to immune stress: possible epigenetic mechanism.

Growing evidence has shown that developmental alcohol exposure induces central nervous system inflammation and microglia activation, which may contribute to long-term health conditions, such as fetal alcohol spectrum disorders. These studies sought to investigate whether neonatal alcohol exposure during postnatal days (PND) 2-6 in rats (third trimester human equivalent) leads to long-term disruption of the neuroimmune response by microglia. Exposure to neonatal alcohol resulted in acute increases in activation and inflammatory gene expression in hypothalamic microglia including tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). Adults with neonatal alcohol pre-exposure (alcohol fed; AF) animals showed an exaggerated peripheral stress hormonal response to an immune challenge (lipopolysaccharides; LPS). In addition, there were significantly more microglia present in the hypothalamus of adult AF animals, and their hypothalamic microglia showed more cluster of differentiation molecule 11b (Cd11b) activation, TNF-α expression, and IL-6 expression in response to LPS. Interestingly, blocking microglia activation with minocycline treatment during PND 2-6 alcohol exposure ameliorated the hormonal and microglial hypersensitivity to LPS in AF adult animals. Investigation of possible epigenetic programming mechanisms by alcohol revealed neonatal alcohol decreased several repressive regulators of transcription in hypothalamic microglia, while concomitantly increasing histone H3 acetyl lysine 9 (H3K9ac) enrichment at TNF-α and IL-6 promoter regions. Importantly, adult hypothalamic microglia from AF animals showed enduring increases in H3K9ac enrichment of TNF-α and IL-6 promoters both at baseline and after LPS exposure, suggesting a possible epigenetic mechanism for the long-term immune disruption due to hypothalamic microglial priming.

Hypermethylation of Proopiomelanocortin and Period 2 Genes in Blood Are Associated with Greater Subjective and Behavioral Motivation for Alcohol in Humans.

BACKGROUND: Epigenetic modifications of a gene have been shown to play a role in maintaining a long-lasting change in gene expression. We hypothesize that alcohol's modulating effect on DNA methylation on certain genes in blood is evident in binge and heavy alcohol drinkers and is associated with alcohol motivation. METHODS: Methylation-specific polymerase chain reaction (PCR) assays were used to measure changes in gene methylation of period 2 (PER2) and proopiomelanocortin (POMC) genes in peripheral blood samples collected from nonsmoking moderate, nonbinging, binge, and heavy social drinkers who participated in a 3-day behavioral alcohol motivation experiment of imagery exposure to either stress, neutral, or alcohol-related cues, 1 per day, presented on consecutive days in counterbalanced order. Following imagery exposure on each day, subjects were exposed to discrete alcoholic beer cues followed by an alcohol taste test (ATT) to assess behavioral motivation. Quantitative real-time PCR was used to measure gene expression of PER2 and POMC gene levels in blood samples across samples. RESULTS: In the sample of moderate, binge, and heavy drinkers, we found increased methylation of the PER2 and POMC DNA, reduced expression of these genes in the blood samples of the binge and heavy drinkers relative to the moderate, nonbinge drinkers. Increased PER2 and POMC DNA methylation was also significantly predictive of both increased levels of subjective alcohol craving immediately following imagery (p < 0.0001), and with presentation of the alcohol (2 beers) (p < 0.0001) prior to the ATT, as well as with alcohol amount consumed during the ATT (p < 0.003). CONCLUSIONS: These data establish significant association between binge or heavy levels of alcohol drinking and elevated levels of methylation and reduced levels of expression of POMC and PER2 genes. Furthermore, elevated methylation of POMC and PER2 genes is associated with greater subjective and behavioral motivation for alcohol.

Prenatal alcohol exposure increases the susceptibility to develop aggressive prolactinomas in the pituitary gland.

Excess alcohol use is known to promote development of aggressive tumors in various tissues in human patients, but the cause of alcohol promotion of tumor aggressiveness is not clearly understood. We used an animals model of fetal alcohol exposure that is known to promote tumor development and determined if alcohol programs the pituitary to acquire aggressive prolactin-secreting tumors. Our results show that pituitaries of fetal alcohol-exposed rats produced increased levels of intra-pituitary aromatase protein and plasma estrogen, enhanced pituitary tissue growth, and upon estrogen challenge developed prolactin-secreting tumors (prolactinomas) that were hemorrhagic and often penetrated into the surrounding tissue. Pituitary tumors of fetal alcohol-exposed rats produced higher levels of hemorrhage-associated genes and proteins and multipotency genes and proteins. Cells of pituitary tumor of fetal alcohol exposed rat grew into tumor spheres in ultra-low attachment plate, expressed multipotency genes, formed an increased number of colonies, showed enhanced cell migration, and induced solid tumors following inoculation in immunodeficient mice. These data suggest that fetal alcohol exposure programs the pituitary to develop aggressive prolactinoma after estrogen treatment possibly due to increase in stem cell niche within the tumor microenvironment.

Hypothalamic beta-endorphin neurons suppress preneoplastic and neoplastic lesions development in 1,2-dimethylhydrazine induced rat colon cancer model.

In recent years, experimental studies demonstrated negative impacts of impaired body stress response on colonic pathologies. In this study, we tested if reducing body stress response by the use of ß-endorphin (BEP) neuronal transplants in the hypothalamus suppresses pre-neoplastic and neoplastic lesions. Colon cancer was induced by injecting 1,2-dimethylhydrazine (DMH) for sixteen weeks in Sprague Dawley rats with BEP neuron transplants or control neuron transplants, and their colonic histopathologies, colon tissue levels of pro-inflammatory cytokines and epithelial-mesenchymal transition (EMT) proteins and splenic levels of cytotoxic proteins were measured. Our results revealed that DMH induced tumors in colon at 100% incidence in control rats but failed to induce colonic tumors in 70% of animal with BEP neuronal transplants. The mean volume of tumor at the colon was smaller in BEP neurons transplanted rats than those in controls. Histopathologies of colon tissues revealed that BEP neurons transplanted animals had lesser tissue lesions such as aberrant crypt foci (ACF) and adenocarcinoma development in the colon than those in control groups. Immunohistochemical and western blot analyses identified reduced expression of Ki-67, TNF-α and NF-κB nuclear translocation in colonic tissues of BEP neurons transplanted rats than those in controls. BEP neurons transplanted rats also showed reduced expressions of transcription factors linked to EMT like Snail, Twist, and N-cadherin, but increased the levels of an epithelial cell marker E-cadherin in colon tissue. Furthermore, splenic NK cells cytolytic proteins such as perforin, granzyme B and IFN-γ levels in BEP neurons transplanted rats were higher than those in control rats. These data suggest that BEP neuron transplants suppress the growth and progression of colonic tumors possibly by decreasing inflammatory mileu and EMT via activation of innate immune responses.

Pathophysiology of the Effects of Alcohol Abuse on the Endocrine System.

Alcohol can permeate virtually every organ and tissue in the body, resulting in tissue injury and organ dysfunction. Considerable evidence indicates that alcohol abuse results in clinical abnormalities of one of the body's most important systems, the endocrine system. This system ensures proper communication between various organs, also interfacing with the immune and nervous systems, and is essential for maintaining a constant internal environment. The endocrine system includes the hypothalamic-pituitary-adrenal axis, the hypothalamic-pituitary-gonadal axis, the hypothalamic-pituitary-thyroid axis, the hypothalamic-pituitary-growth hormone/insulin-like growth factor-1 axis, and the hypothalamic-posterior pituitary axis, as well as other sources of hormones, such as the endocrine pancreas and endocrine adipose tissue. Alcohol abuse disrupts all of these systems and causes hormonal disturbances that may result in various disorders, such as stress intolerance, reproductive dysfunction, thyroid problems, immune abnormalities, and psychological and behavioral disorders. Studies in both humans and animal models have helped shed light on alcohol's effects on various components of the endocrine system and their consequences.