100mM Ethanol Research Articles

MiR-92a-3p regulates ethanol-induced apoptosis in H9c2 cardiomyocytes.

The role of miR-92a-3p in the ethanol-induced apoptosis of H9c2 cardiomyocytes remains unclear. In this study, we explored the role of miR-92a-3p in the ethanol-induced apoptosis of H9c2 cardiomyocytes and identified its target genes and signaling pathways. H9c2 cells were cultured with or without 100mM ethanol for 24h. The differential expression of miR-92a-3p was verified in H9c2 cells through reverse transcription-quantitative polymerase chain reaction (RT-qPCR). To manipulate the expression of miR-92a-3p, both a mimic and an inhibitor were transfected into H9c2 cells. An Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit and apoptosis-related antibodies were used for apoptosis detection through flow cytometry and Western blotting, respectively. Target genes were verified through RT-qPCR, Western blotting, and double luciferase reporter gene assays. miR-92a-3p was significantly overexpressed in ethanol-stimulated H9c2 cardiomyocytes (P<0.001). After ethanol stimulation, H9c2 myocardial cells exhibited increased apoptosis. The apoptosis rate was higher in the miR-92a-3p mimic group than in the control group. However, the apoptosis rate was lower in the miR-92a-3p inhibitor group than in the control group, indicating that miR-92a-3p promotes the ethanol-induced apoptosis of H9c2 myocardial cells. RT-qPCR and Western blotting revealed that the miR-92a-3p mimic and inhibitor significantly regulated the mRNA and protein expression levels of mitogen- and stress-activated protein kinase 2 and cyclic AMP-responsive element-binding protein 3-like protein 2 (CREB3L2), suggesting that miR-92a-3p promotes the apoptosis of H9c2 cardiomyocytes by inhibiting the MSK2/CREB/Bcl-2 pathway. Therefore, the apoptosis of H9c2 cardiomyocytes increases after ethanol stimulation, and miR-92a-3p can directly target MSK2 and CREB3L2, thereby promoting the ethanol-induced apoptosis of H9c2 myocardial cells.

Chronic Ethanol Exposure of Basal Cells causes Barrier Dysfunction of Differentiated Mucocilliary Monolayers

Alcohol use disorder affects over 14 million adults and causes 88,000 deaths each year in the United States. Chronic alcohol use significantly increases people’s risk of developing lung infections and acute respiratory distress syndrome (ARDS). This increased sensitivity to injury is a condition known as alcoholic lung syndrome, and it is caused by dysfunction of the lung immune system and alveolar epithelial barrier. However, little is known about how alcohol impacts epithelial cells in the conducting airway, i.e. the trachea and bronchi, which is the first line of defense against infectious pathogens in the lungs. We hypothesize that in vitro ethanol exposure decreases barrier function of human primary bronchial epithelial cells. To test this, we dosed cells with ethanol at various timepoints: 1) during both the basal‐cell expansion and differentiation stages 2) the first 14 days of differentiation on Transwell permeable supports at air liquid interface (ALI); 3) beginning on day 14 of differentiation and continuing for 10 days. We treated cells with 10mM, 60mM or 100mM ethanol almost daily and measured barrier function by transepithelial electrical resistance (TER). We found that ethanol caused a dose‐dependent decrease in barrier function, but only in the group treated during both the expansion and differentiation stages. This suggests that chronically dosing basal cells with ethanol is required for barrier dysfunction of a fully differentiated cell monolayer. Additionally, we cultured primary airway epithelial cells isolated from healthy and alcoholic patients on Transwell permeable supports and differentiated them in vitro to determine if in vivo exposure caused long‐term changes to tight junction protein levels. It is known that the transmembrane protein junctional adhesion molecule A (JAM‐A) regulates barrier function by controlling the paracellular flow of small molecules as part of the tight junction barrier. Our results show in response to alcohol exposure, cells have lower JAM‐A protein expression compared to healthy cells. Together, these results indicate that chronic alcohol exposure detrimentally alters the conducting airway epithelial cell barrier potentially through the loss of JAM‐A. Future directions include determining if other tight junction proteins are altered by alcohol and whether the alcohol‐induced reduction in JAM‐A is sufficient to cause barrier dysfunction in airway cells.

Alcohol Decreases the Barrier Function of Airway Epithelial cells through the TGFß1 Pathway

Alcohol use disorder affects over 14 million adults and causes 88,000 deaths each year in the United States. Chronic alcohol use significantly increases people's risk of developing lung infections and acute respiratory distress syndrome (ARDS). This increased sensitivity to injury is a condition known as alcoholic lung syndrome, and it is caused by dysfunction of the lung immune system and alveolar epithelial barrier. However, little is known about how alcohol impacts epithelial cells in the conducting airway, i.e. the trachea and bronchi, which are the first line of defense against infectious pathogens in the lung. We hypothesize that alcohol exposure causes a decrease in barrier function of airway epithelial cells through a TGFß1-dependent mechanism. To test this, we cultured primary airway epithelial cells isolated from rats and humans on Transwell permeable supports and differentiated them in vitro at air liquid interface (ALI). We treated cells in vitro with 60mM or 100mM ethanol changed daily for 2 weeks and measured barrier function by transepithelial electrical resistance (TER). We found that ethanol caused a decrease in barrier function compared to control cells. However, cells treated with ethanol and a TGFß1 inhibitor had barrier function similar to control cells. Additionally, we cultured primary airway epithelial cells isolated from healthy and alcoholic patients on Transwell permeable supports and differentiated them in vitro to determine if in vivo exposure caused long-term changes to tight junction protein levels. It is known that the transmembrane protein junctional adhesion molecule A (JAM-A) regulates barrier function by controlling the paracellular flow of small molecules as part of the tight junction barrier. Our results show in response to alcohol exposure, cells have lower JAM-A protein expression compared to healthy cells. Rat airway cells exposed to ethanol in vitro also show a decrease in JAM-A at both the RNA and protein level. Together, these results indicate that chronic alcohol exposure detrimentally alters the conducting airway epithelial cell barrier potentially through the loss of JAM-A. Future directions include determining if other tight junction proteins are altered by alcohol and whether the alcohol-induced reduction in JAM-A is sufficient to cause barrier dysfunction in airway cells.

Human ESC-derived Expandable Hepatic Organoids Enable Therapeutic Liver Repopulation and Pathophysiological Modeling of Alcoholic Liver Injury

Introduction: Here, we report the generation of human ESC-derived, expandable hepatic organoids (hEHOs) enabling therapeutic liver repopulation and pathophysiological modeling of alcoholic liver injury. Methods: The culture system including a mix of A83-01, Forskolin, R-spondin 1, Wnt3a and EGF along with the extracellular matrix was used to generate hEHOs. The liver repopulation potential of hEHOs was assayed in FRG mouse model of inherited metabolic liver disorders. The tumorigenesis and lineage restriction of hEHOs were evaluated by transplanting the hEHOs into epididymal fat pads of NOD/SCID mice. To model alcoholic liver disease, a derivative model by incorporating human fetal liver mesenchymal cells (hFLMCs) into the hEHOs, referred to as hFLMC/hEHO was generated and treated with 100mM ethanol for 7days. Results: The hEHOs stably maintain phenotypic features of bipotential liver stem/progenitor cells that can differentiate into functional hepatocytes or cholangiocytes. The hEHOs can expand for 20 passages enabling large scale expansion to cell numbers requisite for industry or clinical programs. The hEHOs display remarkable repopulation capacity in injured livers of FRG mice following transplantation. If implanted into the epididymal fat pads of immune-deficient mice, they do not generate non-hepatic lineages and have no tendency to form teratomas. The hFLMC/hEHO can model alcoholic liver disease-associated pathophysiologic changes, including oxidative stress generation, steatosis, inflammatory mediators release and fibrosis, under ethanol treatment. Conclusion: The hEHOs have considerable potential to be a novel, ex vivo pathophysiological model for studying alcoholic liver disease as well as a promising cellular source for treating human liver diseases.

MiR-155-5p alleviates ethanol-induced myocardial insulin resistance in H9C2 cells via regulating the mTOR signalling pathway.

Alcohol exposure impairs myocardium insulin sensitivity, which links to heart dysfunction. miR-155 regulates mTOR signaling pathway and is involved in multiple functions. However, the underlying mechanism of miR-155 in ethanol-induced myocardial insulin resistance remains unclear. Here, in this study we aimed to identify the role of miR-155 in myocardial insulin sensitivity and the involvement of mTOR pathway. H9C2 cells were cultured with or without 100mM ethanol for 24h. miR-155-5p inhibitor, miR-155-5p mimics or their respective negative control (inhibitor NC and mimic NC) were transfected to regulate miR-155-5p expression. mTOR signaling, including Ras homolog enriched in brain (Rheb), rapamycin insensitive companion of mTOR (Rictor) and ribosomal protein S6 kinase B2 (S6K2), was investigated by western blotting and qPCR, and insulin responsiveness was evaluated by glucose uptake and phosphorylation of insulin receptor substrate-1 (p-IRS1). The miR-155-5p level increased under ethanol exposure, accompanied by a decrease in glucose uptake, an increase in p-IRS1(ser 307) and activation of the mTOR signaling pathway in H9C2 cells. In addition, miR-155-5p downregulation decreased the glucose uptake, increased the p-IRS1(ser 307) level and activated the mTOR signaling pathway. miR-155-5p upregulation increased the glucose uptake, decreased the p-IRS1(ser 307) level and suppressed the mTOR signaling pathway. Collectively, these findings suggest miR-155-5p upregulation ameliorates myocardial insulin resistance via mTOR signaling in vitro, and miR-155-5p downregulation attenuates myocardial insulin resistance, which might become a potential therapeutic target for alcohol-induced cardiomyopathy.

Region-dependent regulation of acute ethanol on γ oscillation in the rat hippocampal slices.

Ethanol use disorders are a serious medical and public health problem in the world today. Acute ethanol intoxication can lead to cognitive dysfunction such as learning and memory impairment. Gamma oscillations (γ, 30-80Hz) are synchronized rhythmic activity generated by population of neurons within local network, and closely related to learning and memory function. The hippocampus is a critical anatomic structure that supports learning and memory. On the grounds of structure and function, hippocampus can be divided into the intermediate (IH), the dorsal (DH), and ventral hippocampus (VH). The current study is the first to investigate the effects of acute ethanol on γ oscillations in these sub-regions of rat hippocampal slices. The sustained γ oscillations were induced by 200nM kainate (KA) in the CA3c of IH, DH, and VH. When KA-induced γ oscillation reached the steady state, ethanol (50mM or 100mM) was applied and the effects of ethanol on γ oscillation power was measured in the slices sequentially sectioned from ventral to dorsal hippocampus of adult rats. In the intermediate hippocampal slices, compared with control (KA only), ethanol (50mM) caused 36.1 ± 3.9% decrease in γ power (p < 0.05, n = 10), while ethanol (100mM) caused 55.3 ± 5.5% decrease in γ power (p < 0.001, n = 14). In the dorsal hippocampus, only ethanol (100mM) caused 18.1 ± 8.6% decrease in γ power (p < 0.05, n = 12). However, in the ventral hippocampus, neither 50mM nor 100mM ethanol affected γ oscillation. Our results demonstrate that ethanol may produce the differential suppression of γ oscillations in a dose-dependent manner in different sub-regions of hippocampus, suggesting that the modulation of ethanol on hippocampal γ oscillation is region-dependent.

Ethanol Stimulation of Microglia Release Increases ERK1/2-Dependent Neuronal cPLA2 Activity in Immature Cultured Cortical Preparations.

Ethanol consumption typically begins during adolescence and is associated with age-dependent responses and maladaptive neuronal consequences. Our previous work established the role of a putative signaling cascade involving cytoplasmic phospholipase A2 (cPLA2), arachidonic acid (AA) and novel protein kinase C isoforms in adolescent hypnotic sensitivity. The current study aimed to further delineate this pathway by ascertaining the cellular specificity as well as the upstream activators of cPLA2 using an immature cultured cortical preparation. A threefold increase in cPLA2 was detected within 2min of 100mM ethanol exposure as measured by phosphorylation of serine 505 (Ser505). Increases in cPLA2 activity were further observed to be primarily confined to neuronal cells. Increases in the number of neurons co-expressing cPLA2 Ser505 phosphorylation were prevented by preincubation with an ERK1/2 inhibitor, but not P38 MAPK inhibition. Finally, conditioned media studies were used to determine whether glial cells were involved in the ethanol-induced neuronal cPLA2 activity. Rapid increases in neuronal cPLA2 activity appears to be initiated through ethanol stimulated microglial, but not astrocytic releasable factors. Taken together, these data extend the proposed signaling cascade involved in developmental ethanol responding.

The Prolyl Isomerase PIN1 Impairs Methionine Adenosyltransferase α1 Mitochondrial Targeting in Alcoholic Liver Disease

BackgroundMethionine adenosyltransferase alpha1 (MATα1) is responsible for hepatic biosynthesis of S‐adenosylmethionine (SAMe). Lower MATα1 activity and SAMe levels occur in alcoholic liver disease (ALD), a condition characterized by mitochondrial dysfunction. We recently showed MATα1 is in the mitochondrial matrix of hepatocytes and is important for mitochondrial function. PIN1 is a peptidyl‐prolyl cis‐trans isomerase that can alter the activity, stability, interactions and subcellular localization of its targets by binding to a specific phosphorylated motif pSer/pThr‐Pro. PIN1’s role in ALD is unknown and MATα1 has a PIN1 binding motif (Ser114‐Pro115). The aim of this study was to examine whether PIN1 can regulate MATα1 function in ALD.MethodsMATα1 phosphorylation was examined using immunoprecipitation (IP) with anti‐phospho‐Ser antibody followed by immunoblotting, phosphoproteomics and mass spectrometry (MS). IP using recombinant proteins, liver and cell lysates studied MATα1‐PIN1 interaction. Mitochondria were isolated from AML12 cells treated with 100mM ethanol for 48 hours (in vitro model), NIAAA livers (in vivo model), liver samples from patients with alcoholic hepatitis, and HepG2 cells where PIN1 expression was varied to evaluate MATα1 mitochondrial content. MATα1 S114A mutation investigated its impact on PIN1 interaction.ResultsPIN1 expression was unchanged in ALD models and in the livers of ALD patients. We found that MATα1 and PIN1 interact directly, in liver and cell lysates, and that their interaction is enhanced by ethanol in experimental models and in the livers of patients with alcoholic cirrhosis. Ethanol treatment in AML12 cells and in the NIAAA model increased MATα1 phosphorylation. Phosphoproteomics/MS revealed MATα1 is phosphorylated at Ser114 and mutation of Ser114 to alanine significantly reduced its interaction with PIN1. Increased PIN1‐MATα1 interaction reduced MATα1 mitochondrial targeting, as silencing PIN1 raised mitochondrial MATα1 content in AML12 and HepG2 cells, and attenuated ethanol‐induced triglyceride accumulation. Consistently, mitochondrial MATα1 content was preferentially reduced (by 60–80%) as compared to total MATα1 content (by 20–50%) in both in vitro and in vivo models and in livers from alcoholic hepatitis patients.ConclusionWe unveiled a novel mechanism of mitochondrial injury in ALD that involves PIN1. In ALD, increased MATα1 phosphorylation and interaction with PIN1 lead to impaired MATα1 mitochondrial targeting. Inhibiting PIN1‐MATα1 interaction may serve as a new therapeutic strategy in ALD.Support or Funding InformationThis work was supported by NIH grants R01AA026759 (SC Lu and J Van Eyk) and R24AA025017 (Sun Z).In ALD, increased MATα1 phosphorylation and interaction with PIN1 impaired MATα1 mitochondrial targeting.Figure 1

Ethanol Augments Monosodium Urate-Induced NLRP3 Inflammasome Activation via Regulation of AhR and TXNIP in Human Macrophages.

PurposeEthanol elicits several inflammatory responses and affects the innate immune response. The aim of this study was to identify the mechanism by which ethanol affects uric acid-induced NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation by regulation of aryl hydrocarbon receptor (AhR) and thioredoxin-interacting protein (TXNIP).Materials and MethodsHuman myeloid leukemia cells (U937 cells) were used to assess the role of ethanol in NLRP3 inflammasome activation induced by monosodium urate (MSU) crystals. Expression of target molecules, such as NLRP3 inflammasome components, AhR, and TXNIP, were measured using quantitative real-time PCR and Western blot analyses. The effect of ethanol-induced TXNIP on the NLRP3 inflammasome was assessed in human macrophages transfected with TXNIP siRNA.ResultsU937 cells treated with 100 mM ethanol for 24 h induced NLRP3 and interleukin (IL)-1β expression. Ethanol increased reactive oxygen species generation in a time- and dose-dependent manner. AhR mRNA expression was downregulated in U937 cells treated with 100 mM ethanol, whereas CYP1A1 mRNA expression increased. Treatment with ethanol increased NLRP3 and IL-1β mRNA and protein expression in U937 cells exposed to 1.0 mg/mL of MSU crystals for 24 h. TXNIP expression in U937 cells incubated with both 100 mM ethanol and 1.0 mg/mL of MSU crystals was significantly higher than in cells incubated with MSU crystals alone. Treatment with 100mM ethanol for 24 h downregulated NLRP3 and IL-1β expression in MSU crystal-activated U937 cells transfected with TXNIP siRNA, compared to those with scramble siRNA.ConclusionEthanol stimulates uric acid-induced NLRP3 inflammasome activation through regression of AhR and upregulation of TXNIP.

Effects of ethanol on the anticancer function of doxorubicin in JJ012 cells.

Chondrosarcoma is difficult to treat because of resistance to conventional chemotherapy and radiotherapy. This study evaluated the effects of ethanol in combination with doxorubicin in chondrosarcoma cells. JJ012, was treated with doxorubicin alone or in combination with ethanol. Effects on cellular proliferation, migration, invasion, apoptosis, and the cell cycle were evaluated. Treatment of JJ012 cells with 100mM ethanol and doxorubicin resulted in reduced cell growth, invasion, and migration. In addition, doxorubicin uptake into the nucleus was enhanced and p53 mRNA expression was upregulated in JJ012 cells. Ethanol combined with doxorubicin increased doxorubicin uptake in the nucleus and enhanced the effects of doxorubicin in JJ012 cells.

Transcriptional profiling of human neural precursors post alcohol exposure reveals impaired neurogenesis via dysregulation of ERK signaling and miR-145.

Gestational alcohol exposure causes a range of neuropsychological disorders by modulating neurodevelopmental genes and proteins. The extent of damage depends on the stage of the embryo as well as dosage, duration and frequency of exposure. Here, we investigated the neurotoxic effects of alcohol using human embryonic stem cells. Multiple read-outs were engaged to assess the proliferation and differentiation capacity of neural precursor cells upon exposure to 100mM ethanol for 48h corresponding to the blood alcohol levels for binge drinkers. Whole-genome analysis revealed a spatiotemporal dysregulation of neuronal- and glial-specific gene expression that play critical roles in central nervous system (CNS) development. Alterations observed in the transcriptome may be attributed to epigenetic constitution witnessed by differential histone H3 Lys-4/Lys-27 modifications and acetylation status. In-depth mRNA and protein expression studies revealed abrogated extracellular signal-regulated kinases signaling in alcohol-treated cells. Consistent with this finding, ingenuity pathway analysis and micro-RNA profiling demonstrated up-regulation of miR-145 by targeting the neural specifier Sox-2. We also show that the neurite branching complexity of tubulin, beta 3 class III+ neurons was greatly reduced in response to alcohol. Finally, invivo studies using zebrafish embryos reconfirmed the invitro findings. Employing molecular endpoints in a human model, this report indicates for the first time that acute alcohol exposure could lead to impaired brain development via perturbation of extracellular signal-regulated kinases pathway and miR-145. However, it still needs to be addressed whether these modulations sustain throughout development, compromising the ability of the individual during adulthood and aging.

Increased synthesis of folate transporters regulates folate transport in conditions of ethanol exposure and folate deficiency.

Excessive alcohol consumption and dietary folate inadequacy are the main contributors leading to folate deficiency (FD). The present study was planned to study regulation of folate transport in conditions of FD and ethanol exposure in human embryonic kidney cell line. Also, the reversible nature of effects mediated by ethanol exposure and FD was determined by folate repletion and ethanol removal. For ethanol treatment, HEK293 cells were grown in medium containing 100mM ethanol, and after treatment, one group of cells was shifted on medium that was free from ethanol. For FD treatment, cells were grown in folate-deficient medium followed by shifting of one group of cells on folate containing medium. FD as well as ethanol exposure resulted in an increase in folate uptake which was due to an increase in expression of folate transporters, i.e., reduced folate carrier, proton-coupled folate transporter, and folate receptor, both at the mRNA and protein level. The effects mediated by ethanol exposure and FD were reversible on removal of treatment. Promoter region methylation of folate transporters remained unaffected after FD and ethanol exposure. As far as transcription rate of folate transporters is concerned, an increase in rate of synthesis was observed in both ethanol exposure and FD conditions. Additionally, mRNA life of folate transporters was observed to be reduced by FD. An increased expression of folate transporters under ethanol exposure and FD conditions can be attributed to enhanced rate of synthesis of folate transporters.

Chronic Alcohol Exposure Decreases 53BP1 Protein Levels Leading to a Defective DNA Repair in Cultured Primary Cortical Neurons.

Chronic alcohol consumption may cause neurodevelopmental and neurodegenerative disorders. Alcohol neurotoxicity is associated with the production of acetaldehyde and reactive oxygen species that induce oxidative DNA damage. However, the molecular mechanisms by which ethanol disturbs the DNA damage response (DDR), resulting in a defective DNA repair, remain unknown. Here, we have used cultured primary cortical neurons exposed to 50 or 100mM ethanol for 7days to analyze the ethanol-induced DDR. Ethanol exposure produced a dose-dependent generation of double strand breaks and the formation of DNA damage foci immunoreactive for the histone γH2AX, a DNA damage marker, and for the ubiquitylated H2A, which is involved in chromatin remodeling at DNA damage sites. Importantly, these DNA damage foci failed to recruit the protein 53BP1, a crucial DNA repair factor. This effect was associated with a drop in 53BP1 mRNA and protein levels and with an inhibition of global transcription. Moreover, ethanol-exposed neurons treated with ionizing radiation (2Gy) also failed to recruit 53BP1 at DNA damage foci and exhibited a greater vulnerability to DNA lesions than irradiated control neurons. Our results support that defective DNA repair, mediated by the deficient expression and recruitment of 53BP1 to DNA damage sites, represents a novel mechanism involved in ethanol neurotoxicity. The design of therapeutic strategies that increase or stabilize 53BP1 levels might potentially promote DNA repair and partially compensate alcohol neurotoxicity.

Blockade of store-operated calcium entry alleviates ethanol-induced hepatotoxicity via inhibiting apoptosis

Extracellular Ca2+ influx has been suggested to play a role in ethanol-induced hepatocyte apoptosis and necrosis. Previous studies indicated that store-operated Ca2+ entry (SOCE) was involved in liver injury induced by ethanol in HepG2 cells. However, the mechanisms underlying liver injury caused by SOCE remain unclear. We aimed to investigate the effects and mechanism of SOCE inhibition on liver injury induced by ethanol in BRL cells and Sprague–Dawley rats. Our data demonstrated that ethanol (0–400mM) dose-dependently increased hepatocyte injury and 100mM ethanol significantly upregulated the mRNA and protein expression of SOC for at least 72h in BRL cells. Blockade of SOCE by pharmacological inhibitors and sh-RNA knockdown of STIM1 and Orai1 attenuated intracellular Ca2+ overload, restored the mitochondrial membrane potential (MMP), decreased cytochrome C release and inhibited ethanol-induced apoptosis. STIM1 and Orai1 expression was greater in ethanol-treated than control rats, and the SOCE inhibitor corosolic acid ameliorated the histopathological findings and alanine transaminase and aspartate transaminase activity as well as decreased cytochrome C release and inhibited alcohol-induced cell apoptosis. These findings suggest that SOCE blockade could alleviate alcohol-induced hepatotoxicity via inhibiting apoptosis. SOCE might be a useful therapeutic target in alcoholic liver diseases.

PDE5 Inhibition with Sildenafil Blocks Induction of Carboxylesteras3 and Reduces Cell Necrosis and Autophagy in Acute Alcohol‐Induced Injury in Heart

Objective:Fatty Acid Ethyl Esters (FAEE) are esterified products of alcohol that can cause severe damage to tissue organs and are mediators of ethanol‐induced cytotoxic effects. The mechanism that underlie the onset, progression and severity of alcohol toxicity are poorly understood. Carboxylesterase3 (CES3) is an enzyme responsible for esterification of fatty acids and drugs. We hypothesized that CES3 may be involved in metabolizing alcohol and lead to cell injury.Methods and Results:Rat cardiac myoblast cells (H9C2) were treated with ethanol (100mM) or ethanol with sildenafil (1 µM) for 24hrs. Adult male 12‐14 weeks old FVB mice were randomized to receive saline (0.2 ml; i.p, n =3) or Alcohol (1.5 g/ BID; i.p, n=3) or alcohol with sildenafil (0.7mg/kg/day; i.p, n=3) for 3 days.Treatment of H9C2 cells with 100mM ethanol significantly increased the necrosis of cells (41.1 % vs 3.5 % control), whereas sildenafil treatment lowered cell death (17.9 % vs 41.1 % alcohol). CES‐3 protein level was increased 2 fold both in H9c2 cells and in acute ethanol treated mice. Moreover Beclin‐1, an inducer of autophagy was also significantly up‐ regulated upon alcohol treatment in H9C2 cells. Sildenafil treatment prevented the induction of CES3 both in H9C2 cells and in mice.ConclusionTherapeutic intervention using sildenafil can prevent the formation of esters by decreasing CES3 in heart and thus can decrease necrosis and autophagy. Our data reports a novel role of CES3 and a new mechanism in which sildenafil can protect heart from acute alcohol injury. This study was supported in part by grants from National Institutes of Health (HL51045, HL79424, HL118808 to RCK).

Involvement of seven in absentia homolog-1 in ethanol-induced apoptosis in neural crest cells

Ethanol-induced apoptosis in selected cell populations is a major component of pathogenesis underlying ethanol-induced teratogenesis. However, there is a fundamental gap in understanding how ethanol leads to apoptosis in embryos. In this study, we investigate the role of seven in absentia homolog-1 (Siah1) protein, an E3 ubiquitin ligase, in ethanol-induced apoptosis. Using an in vitro model of neural crest cell (NCC), JoMa1.3 cells, we found that exposure to 100mM ethanol resulted in a significant increase in Siah1 mRNA expression in NCCs, an ethanol-sensitive cell population implicated in Fetal Alcohol Spectrum Disorders (FASD). Treatment with 100mM ethanol for 24h also significantly increased the protein expression of Siah1 in JoMa1.3 cells. The nuclear translocation and accumulation of Siah1 was evidenced in the cells exposed to ethanol. In addition, we have found that the inhibition of Siah1 function with siRNA prevents ethanol-induced increase in Siah1 protein expression and nuclear translocation in NCCs. Down-regulation of Siah1 by siRNA also greatly diminished ethanol-induced cell death and caspase-3 activation, indicating that inhibition of Siah1 can attenuate ethanol-induced apoptosis. These results strongly suggest that Siah1 plays an important role in ethanol-induced apoptosis in NCCs.

Innate immune factors modulate ethanol interaction with GABAergic transmission in mouse central amygdala

Excessive ethanol drinking in rodent models may involve activation of the innate immune system, especially toll-like receptor 4 (TLR4) signaling pathways. We used intracellular recording of evoked GABAergic inhibitory postsynaptic potentials (eIPSPs) in central amygdala (CeA) neurons to examine the role of TLR4 activation by lipopolysaccharide (LPS) and deletion of its adapter protein CD14 in acute ethanol effects on the GABAergic system. Ethanol (44, 66 or 100mM) and LPS (25 and 50μg/ml) both augmented eIPSPs in CeA of wild type (WT) mice. Ethanol (44mM) decreased paired-pulse facilitation (PPF), suggesting a presynaptic mechanism of action. Acute LPS (25μg/ml) had no effect on PPF and significantly increased the mean miniature IPSC amplitude, indicating a postsynaptic mechanism of action. Acute LPS pre-treatment potentiated ethanol (44mM) effects on eIPSPs in WT mice and restored ethanol's augmenting effects on the eIPSP amplitude in CD14 knockout (CD14 KO) mice. Both the LPS and ethanol (44-66mM) augmentation of eIPSPs was diminished significantly in most CeA neurons of CD14 KO mice; however, ethanol at the highest concentration tested (100mM) still increased eIPSP amplitudes. By contrast, ethanol pre-treatment occluded LPS augmentation of eIPSPs in WT mice and had no significant effect in CD14 KO mice. Furthermore, (+)-naloxone, a TLR4-MD-2 complex inhibitor, blocked LPS effects on eIPSPs in WT mice and delayed the ethanol-induced potentiation of GABAergic transmission. In CeA neurons of CD14 KO mice, (+)-naloxone alone diminished eIPSPs, and subsequent co-application of 100mM ethanol restored the eIPSPs to baseline levels. In summary, our results indicate that TLR4 and CD14 signaling play an important role in the acute ethanol effects on GABAergic transmission in the CeA and support the idea that CD14 and TLR4 may be therapeutic targets for treatment of alcohol abuse.

Ethanol induces skeletal muscle autophagy and sarcopenia by an AMPK independent, PI3K dependent mechanism

Loss of skeletal muscle mass or sarcopenia is the most common but understudied complication of alcoholic liver disease. Skeletal muscle proteolysis due to ethanol has not been systematically evaluated.A comprehensive approach was used to study the proteolytic pathways in muscle of patients with alcoholic cirrhosis and alcohol fed rodent along with in vitro studies in C2C12 myotubes exposed to 100mM ethanol for 24 hrs. Ubiquitin proteasome activity and autophagy were studied using immunoblots, real time PCR, proteasome activity, standard autophagy readouts including LC3 lipidation, p62 degradation and Beclin overexpression. C2C12 cells transfected with GFP‐LC3 and GFP‐mCherry‐LC3 reporter were also studied. Pulse chase experiments were performed with and without 3‐methyladenine. Immunoblots for components of the PI3K pathway‐Akt, mTOR, p70s6k and 4EBP activation, pAMPK as well as protein phosphatase 2A and PI3Kγ activity were quantified by thin layer chromatography. Ubiquitin proteasomal activity was unaltered and autophagy was increased in the myotubes and in vivo with ethanol. mTOR and its target protein phosphorylation was lower with ethanol exposure. Increased PP2A activity resulted in an AMPK independent mTOR dependent increase in autophagy.

Hepatic mitochondrial dysfunction induced by fatty acids and ethanol

Understanding the key aspects of the pathogenesis of alcoholic fatty liver disease particularly alterations to mitochondrial function remains to be resolved. The role of fatty acids in this regard requires further investigation due to their involvement in fatty liver disease and obesity. This study aimed to characterize the early effects of saturated and unsaturated fatty acids alone on liver mitochondrial function and during concomitant ethanol exposure using isolated liver mitochondria and VA-13 cells (Hep G2 cells that efficiently express alcohol dehydrogenase). Liver mitochondria or VA-13 cells were treated with increasing concentrations of palmitic or arachidonic acid (1 to 160μM) for 24 h with or without 100mM ethanol. The results showed that in isolated liver mitochondria both palmitic and arachidonic acids significantly reduced state 3 respiration in a concentration-dependent manner (P<0.001), implicating their ionophoric activities. Increased ROS production occurred in a dose-dependent manner especially in the presence of rotenone (complex I inhibitor), which was significantly more prominent in arachidonic acid at 80μM (+970%, P<0.001) than palmitic acid (+40%, P<0.01). In VA-13 cells, ethanol alone and both fatty acids (40μM) were able to decrease the mitochondrial membrane potential and cellular ATP levels and increase lipid formation. ROS production was significantly increased with arachidonic acid (+110%, P<0.001) exhibiting a greater effect than palmitic acid (+39%, P<0.05). While in the presence of ethanol, the drop in the mitochondrial membrane potential, cellular ATP levels, and increased lipid formation were further enhanced by both fatty acids, but with greater effect in the case of arachidonic acid, which also correlated with significant cytotoxicity (P<0.001). This study confirms the ability of fatty acids to promote mitochondrial injury in the development of alcoholic fatty liver disease.

Targeted mutagenesis of the Clostridium acetobutylicum acetone–butanol–ethanol fermentation pathway

The production of the chemical solvents acetone and butanol by the bacterium Clostridium acetobutylicum was one of the first large-scale industrial processes to be developed, and in the first part of the last century ranked second in importance only to ethanol production. After a steep decline in its industrial use, there has been a recent resurgence of interest in the acetone–butanol–ethanol (ABE) fermentation process, with a particular emphasis on butanol production. In order to generate strains suitable for efficient use on an industrial scale, metabolic engineering is required to alter the AB ratio in favour of butanol, and eradicate the production of unwanted products of fermentation. Using ClosTron technology, a large-scale targeted mutagenesis in C. acetobutylicum ATCC 824 was carried out, generating a set of 10 mutants, defective in alcohol/aldehyde dehydrogenases 1 and 2 (adhE1, adhE2), butanol dehydrogenases A and B (bdhA, bdhB), phosphotransbutyrylase (ptb), acetate kinase (ack), acetoacetate decarboxylase (adc), CoA transferase (ctfA/ctfB), and a previously uncharacterised putative alcohol dehydrogenase (CAP0059). However, inactivation of the main hydrogenase (hydA) and thiolase (thl) could not be achieved. Constructing such a series of mutants is paramount for the acquisition of information on the mechanism of solvent production in this organism, and the subsequent development of industrial solvent producing strains. Unexpectedly, bdhA and bdhB mutants did not affect solvent production, whereas inactivation of the previously uncharacterised gene CAP0059 resulted in increased acetone, butanol, and ethanol formation. Other mutants showed predicted phenotypes, including a lack of acetone formation (adc, ctfA, and ctfB mutants), an inability to take up acids (ctfA and ctfB mutants), and a much reduced acetate formation (ack mutant). The adhE1 mutant in particular produced very little solvents, demonstrating that this gene was indeed the main contributor to ethanol and butanol formation under the standard batch culture conditions employed in this study. All phenotypic changes observed could be reversed by genetic complementation, with exception of those seen for the ptb mutant. This mutant produced around 100mM ethanol, no acetone and very little (7mM) butanol. The genome of the ptb mutant was therefore re-sequenced, together with its parent strain (ATCC 824 wild type), and shown to possess a frameshift mutation in the thl gene, which perfectly explained the observed phenotype. This finding reinforces the need for mutant complementation and Southern Blot analysis (to confirm single ClosTron insertions), which should be obligatory in all further ClosTron applications.