Direct Effect Of Ethanol Research Articles

Acute alcoholic hepatitis – the role of modern hepatoprotectors

Alcoholic hepatitis is a progressive inflammatory-dystrophic lesion of the liver, the pathogenetic mechanism of which is based on alcoholic damage. Acute alcoholic hepatitis is defined primarily as an exacerbation of the chronic process of alcoholic liver disease. Two mechanisms are defined as the basis for the formation of alcohol-associated liver damage: primary (direct effect of ethanol on hepatocytes and oxidative stress provoked by it) and secondary (through changes in the gut-liver axis with dysbacteriosis and increased permeability of the intestinal wall). For the treatment of acute alcoholic hepatitis, mainly glucocorticosteroids are used, the action of which is directed at cytotoxic and inflammatory mechanisms of the pathogenesis of this disease. Also, phosphodiesterase inhibitors, broad-spectrum antibiotics (rifaximin), probiotics, prebiotics, synbiotics, enterosorbents and hepatoprotectors are actively used. Choosing a hepatoprotector that is effective and safe for patients is still a challenge. At the moment one of the most promising and optimal in terms of “price-quality” ratio drug from this group is a domestic drug from the group of combined hepatoprotectors – Remaxol (inosine + meglumine + methionine + nicotinamide + succinic acid). A clinical case of application of this drug in a patient diagnosed with acute alcoholic hepatitis combined with chronic alcoholic hepatopathy is presented. Not severe course (MELD: 16. Maddrey’s index: 14.04). Ademetionine was prescribed. On the background of the prescribed treatment slight improvements were noted, no significant changes in laboratory data were registered (MELD: 16, Maddrey index: 12.54). After replacement of the hepatoprotector by Remaxol, the following was observed: correction of the general condition, correction of the mental status, reduction of the severity of hepatosplenomegaly, normalization of laboratory parameters (MELD: 10. Maddrey’s index: 6.06). Based on the review of Russian and foreign literature, as well as personal experience in the use of Remaxol, we can conclude that this pharmacological agent contributes to a more favorable course of acute alcoholic hepatitis, a significant reduction in the risk of complications, as well as reducing the length of hospital stay and the cost of treatment.

Binge alcohol disrupts skeletal muscle core molecular clock independent of glucocorticoids.

Circadian rhythms are central to optimal physiological function, as disruption contributes to the development of several chronic diseases. Alcohol (EtOH) intoxication disrupts circadian rhythms within liver, brain, and intestines, but it is unknown whether alcohol also disrupts components of the core clock in skeletal muscle. Female C57BL/6Hsd mice were randomized to receive either saline (control) or alcohol (EtOH) (5 g/kg) via intraperitoneal injection at the start of the dark cycle [Zeitgeber time (ZT12)], and gastrocnemius was collected every 4 h from control and EtOH-treated mice for the next 48 h following isoflurane anesthetization. In addition, metyrapone was administered before alcohol intoxication in separate mice to determine whether the alcohol-induced increase in serum corticosterone contributed to circadian gene regulation. Finally, synchronized C2C12 myotubes were treated with alcohol (100 mM) to assess the influence of centrally or peripherally mediated effects of alcohol on the muscle clock. Alcohol significantly disrupted mRNA expression of Bmal1, Per1/2, and Cry1/2 in addition to perturbing the circadian pattern of clock-controlled genes, Myod1, Dbp, Tef, and Bhlhe40 (P < 0.05), in muscle. Alcohol increased serum corticosterone levels and glucocorticoid target gene, Redd1, in muscle. Metyrapone prevented the EtOH-mediated increase in serum corticosterone but did not normalize the EtOH-induced change in Per1, Cry1 and Cry2, and Myod1 mRNA expression. Core clock gene expression (Bmal, Per1/2, and Cry1/2) was not changed following 4, 8, or 12 h of alcohol treatment on synchronized C2C12 myotubes. Therefore, binge alcohol disrupted genes of the core molecular clock independently of elevated serum corticosterone or direct effects of EtOH on the muscle.NEW & NOTEWORTHY Alcohol is a myotoxin that impairs skeletal muscle metabolism and function following either chronic consumption or acute binge drinking; however, mechanisms underlying alcohol-related myotoxicity have not been fully elucidated. Herein, we demonstrate that alcohol acutely interrupts oscillation of skeletal muscle core clock genes, and this is neither a direct effect of ethanol on the skeletal muscle, nor an effect of elevated serum corticosterone, a major clock regulator.

Ethanol-Induced Neuronal and Cognitive/Emotional Impairments are Accompanied by Down-Regulated NT3-TrkC-ERK in Hippocampus.

Ethanol ingestion affects cognition and emotion, which have been attributed to the dysfunction of specific brain structures. Studies of alcoholic patients and animal models consistently identify reduced hippocampal mass as a key ethanol-induced brain adaptation. This study evaluated how neuroadaptation in the hippocampus (Hip) produced by ethanol contributed to related behavioral deficits in male and female rats. Effects of acute, short-term and long-term ethanol exposure on the anxiety-like behavior and recognition memory on adult male and female Sprague-Dawley rats were assessed using elevated plus maze test and novel object recognition test, respectively. In addition, in order to investigate the direct effect of ethanol on hippocampal neurons, primary culture of hippocampal neurons was exposed to ethanol (10, 30 and 90mM; 1, 24 and 48h), and viability (CCK-8) and morphology (immunocytochemistry) were analyzed at structural levels. Western blot assays were used to assess protein levels of NT3-TrkC-ERK. Acute and short-term ethanol exposure exerted anxiolytic effects, whereas long-term ethanol exposure induced anxiogenic responses in both sexes. Short-term ethanol exposure impaired spatial memory only in female rats, whereas long-term ethanol exposure impaired spatial and recognition memory in both sexes. These behavioral impairments and ethanol-induced loss of hippocampal neurons and decreased cell viability were accompanied by downregulated NT3-TrkC-ERK pathway. These results indicate that NT3-TrkC-ERK signaling in the Hip may play an important role in ethanol-induced structural and behavioral impairments.

Direct effects of ethanol at relevant concentrations on conformation and ligand binding of Toll-like Receptor 3 (TLR3)

Abstract The purpose of this study was to seek direct physical evidence that ethanol at concentrations relevant to binge drinking alters the conformation of TLR3 and decreases ligand binding. We and others have reported that TLR3-mediated responses are significantly suppressed by concentrations of ethanol that occur in binge drinkers (J. Immunol., 2004, 173: 2715–2724). This suppression can be detected at all levels of signaling that have been tested (from MAP kinases to NF-kB), but it remains possible that ethanol acts directly on TLR3 and that its other effects are secondary to altered TLR3 conformation. Purified ectodomains of TLR3 were used in this study, because they comprise most of the TLR3 molecule, and dimerization of TLR3 ectodomains is necessary for signal transduction and activation of TLR3-mediated innate immunity. Using circular dichroism (CD) to measure changes in protein conformation, we demonstrated that ethanol concentrations from 20–100 mM substantially altered the conformation of human and mouse TLR3 ectodomains complexed with a defined dsRNA ligand. Binding of poly I:C, a dsRNA analog, was significantly inhibited by ethanol. Modeling with Autodock 4 software indicated that mouse and human TLR3 contain a putative ethanol binding site with a dissociation constant consistent with the concentration dependence of the conformational changes. These results support the hypothesis that ethanol inhibits TLR3-mediated functions by directly altering the conformation of TLR3. This work was supported by NIH R01AA09505 and SBP was supported by NIH grant P20GM103646.

Ethanol sensitizes skeletal muscle to ammonia-induced molecular perturbations

Ethanol causes dysregulated muscle protein homeostasis while simultaneously causing hepatocyte injury. Because hepatocytes are the primary site for physiological disposal of ammonia, a cytotoxic cellular metabolite generated during a number of metabolic processes, we determined whether hyperammonemia aggravates ethanol-induced muscle loss. Differentiated murine C2C12 myotubes, skeletal muscle from pair-fed or ethanol-treated mice, and human patients with alcoholic cirrhosis and healthy controls were used to quantify protein synthesis, mammalian target of rapamycin complex 1 (mTORC1) signaling, and autophagy markers. Alcohol-metabolizing enzyme expression and activity in mouse muscle and myotubes and ureagenesis in hepatocytes were quantified. Expression and regulation of the ammonia transporters, RhBG and RhCG, were quantified by real-time PCR, immunoblots, reporter assays, biotin-tagged promoter pulldown with proteomics, and loss-of-function studies. Alcohol and aldehyde dehydrogenases were expressed and active in myotubes. Ethanol exposure impaired hepatocyte ureagenesis, induced muscle RhBG expression, and elevated muscle ammonia concentrations. Simultaneous ethanol and ammonia treatment impaired protein synthesis and mTORC1 signaling and increased autophagy with a consequent decreased myotube diameter to a greater extent than either treatment alone. Ethanol treatment and withdrawal followed by ammonia exposure resulted in greater impairment in muscle signaling and protein synthesis than ammonia treatment in ethanol-naive myotubes. Of the three transcription factors that were bound to the RhBG promoter in response to ethanol and ammonia, DR1/NC2 indirectly regulated transcription of RhBG during ethanol and ammonia treatment. Direct effects of ethanol were synergistic with increased ammonia uptake in causing dysregulated skeletal muscle proteostasis and signaling perturbations with a more severe sarcopenic phenotype.

The Effect of Ethanol on Inflammation Markers and FGF-21 in Healthy Individuals

Introduction: The mechanisms behind the detrimental effects of ethanol on the liver remain uncertain. It has been suggested that the presence of ethanol in the gut lumen promotes a ’leaky gut’ allowing bacterial products to translocate to the bloodstream causing an inflammatory response. Furthermore, the ethanol-induced hormone fibroblast growth factor 21 (FGF-21) has emerged as a potential safeguard from ethanol-induced liver injury. Here, we aimed to investigate how ethanol administered intragastrically and intravenously, respectively, influences circulating markers of inflammation and FGF-21. Materials and Method: In a double-blinded, randomized cross-over design, we subjected 12 fasted healthy men (age 25.3±3.9 years; body mass index (BMI) 22.6±2.6 kg/m2 (mean±SD)) to intragastrically instilled ethanol (0.70 g ethanol per kg body weight in a 20% (v/w) solution infused over 5 minutes) and isoethanolaemic iv ethanol infusion, respectively, on two separate experimental days, and evaluated a range of circulating markers of inflammation and liver function as well as FGF-21. Results: No increment in plasma sCD163, or any other inflammation markers (lipopolysaccharide binding protein, tumor necrosis factor-α, interferon-γ, interleukin-10 (IL), IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-6, and IL-8), was observed. Plasma FGF-21 levels increased significantly (nine times baseline level), similarly and throughout both experimental days. Conclusion: Neither ’oral’ nor iv ethanol elicited significant changes in circulating markers of macrophage activation or inflammation, but increased plasma FGF-21 significantly regardless of the administration form suggesting a direct ethanol effect on FGF-21 secretion. Disclosure A.R. Lanng: None. L.S. Gasbjerg: Stock/Shareholder; Self; Antag Therapeutics. N.C. Bergmann: Employee; Self; Zealand Pharma A/S. H. Møller: Other Relationship; Self; IQ-products. M. Gillum: None. H. Gronbaek: Board Member; Self; Ipsen Biopharmaceuticals, Inc., Novartis AG. Research Support; Self; Intercept Pharmaceuticals, Inc., AbbVie Inc., Novo Nordisk A/S. T. Vilsbøll: Advisory Panel; Self; Amgen Inc., Boehringer Ingelheim GmbH, Eli Lilly and Company, AstraZeneca, MSD K.K., Sanofi, Novo Nordisk A/S. Consultant; Self; Amgen Inc., Boehringer Ingelheim GmbH, Eli Lilly and Company, AstraZeneca, MSD K.K., Sanofi, Novo Nordisk A/S. Speaker's Bureau; Self; Amgen Inc., Boehringer Ingelheim GmbH, Eli Lilly and Company, AstraZeneca, MSD K.K., Sanofi, Novo Nordisk A/S. Research Support; Self; Eli Lilly and Company, Novo Nordisk A/S. F.K. Knop: Advisory Panel; Self; AstraZeneca. Consultant; Self; AstraZeneca. Research Support; Self; AstraZeneca. Speaker's Bureau; Self; AstraZeneca. Advisory Panel; Self; Eli Lilly and Company. Speaker's Bureau; Self; Eli Lilly and Company. Advisory Panel; Self; Amgen Inc.. Speaker's Bureau; Self; Merck Sharp &amp; Dohme Corp.. Advisory Panel; Self; Novo Nordisk A/S. Consultant; Self; Novo Nordisk A/S. Speaker's Bureau; Self; Novo Nordisk A/S. Consultant; Self; Amgen Inc.. Advisory Panel; Self; MedImmune, Sanofi. Consultant; Self; Sanofi. Research Support; Self; Sanofi. Speaker's Bureau; Self; Sanofi.

Sarcopenia in Alcoholic Liver Disease: Clinical and Molecular Advances.

Despite advances in treatment of alcohol use disorders that focus on increasing abstinence and reducing recidivism, alcoholic liver disease (ALD) is projected to be the major cause of cirrhosis and its complications. Malnutrition is recognized as the most frequent complication in ALD, and despite the high clinical significance, there are no effective therapies to reverse malnutrition in ALD. Malnutrition is a relatively imprecise term, and sarcopenia or skeletal muscle loss, the major component of malnutrition, is primarily responsible for the adverse clinical consequences in patients with liver disease. It is, therefore, critical to define the specific abnormality (sarcopenia) rather than malnutrition in ALD, so that therapies targeting sarcopenia can be developed. Skeletal muscle mass is maintained by a balance between protein synthesis and proteolysis. Both direct effects of ethanol (EtOH) and its metabolites on the skeletal muscle and the consequences of liver disease result in disturbed proteostasis (protein homeostasis) and consequent sarcopenia. Once cirrhosis develops in patients with ALD, abstinence is unlikely to be effective in completely reversing sarcopenia, as other contributors including hyperammonemia, hormonal, and cytokine abnormalities aggravate sarcopenia and maintain a state of anabolic resistance initiated by EtOH. Cirrhosis is also a state of accelerated starvation, with increased gluconeogenesis that requires amino acid diversion from signaling and substrate functions. Novel therapeutic options are being recognized that are likely to supplant the current "deficiency replacement" approach and instead focus on specific molecular perturbations, given the increasing availability of small molecules that can target specific signaling components. Myostatin antagonists, leucine supplementation, and mitochondrial protective agents are currently in various stages of evaluation in preclinical studies to prevent and reverse sarcopenia, in cirrhosis in general, and ALD, specifically. Translation of these data to human studies and clinical application requires priority for allocation of resources.

Observation on the ultrastructure morphology of HeLa cells treated with ethanol: Statistical analysis

ABSTRACTIt is estimated that 5.9% of all human deaths are attributable to alcohol consumption and that the harmful use of ethanol ranks among the top five risk factors for causing disease, disability, and death worldwide. Ethanol is known to disrupt phospholipid packing and promote membrane hemifusion at lipid bilayers. With the exception of mitochondria involved in hormone synthesis, the sterol content of mitochondrial membranes is low. As membranes that are low in cholesterol have increased membrane fluidity and are the most easily disordered by ethanol, we hypothesize that mitochondria are sensitive targets for ethanol damage. HeLa cells were exposed to 50 mM ethanol and the direct effects of ethanol on cellular ultrastructure were examined utilizing transmission electron microscopy. Our ultramicroscopic analysis revealed that cells exposed to ethanol harbor fewer incidence of apoptotic morphology; however, significant alterations to mitochondria and to nuclei occurred. We observed statistical increases in the amount of irregular cells and cells with multiple nuclei, nuclei harboring indentations, and nuclei with multiple nucleolus-like bodies. Indeed, our analysis revealed that mitochondrial damage is the most extensive type of cellular damage. Rupturing of cristae was the most prominent damage followed by mitochondrial swelling. Ethanol exposure also resulted in increased amounts of mitochondrial rupturing, organelles with linked membranes, and mitochondria localizing to indentations of nuclear membranes. We theorize that these alterations could contribute to cellular defects in oxidative phosphorylation and, by extension, the inability to generate regular levels of cellular adenosine triphosphate.

Direct effects of ethanol on neuronal differentiation: An in vitro analysis of viability and morphology.

The deleterious effects of ethanol (EtOH) on the brain have been widely described, but its effects on the neuronal cytoskeleton during differentiation have not yet been firmly established. In this context, our aim was to investigate the direct effect of EtOH on cortical neurons during the period of differentiation. Primary cultures of cortical neurons obtained from 1-day-old rats were exposed to EtOH after 7days of culture, and viability and morphology were analyzed at structural and ultrastructural levels after 24-h EtOH exposure. EtOH caused a significant reduction of 73±7% in the viability of cultured cortical neurons, by preferentially inducing apoptotic cellular death. This effect was accompanied by an increase in caspase 3 and 9 expression. Furthermore, EtOH induced a reduction in total dendrite length and in the number of dendrites per cell. Ultrastructural studies showed that EtOH increased the number of lipidic vacuoles, lysosomes and multilamellar vesicles and induced a dilated endoplasmatic reticulum lumen and a disorganized Golgi apparatus with a ring-shape appearance. Microtubules showed a disorganized distribution. Apposition between pre- and postsynaptic membranes without a defined synaptic cleft and a delay in presynaptic vesicle organization were also observed. Synaptophysin and PSD95 expression, proteins pre- and postsynaptically located, were reduced in EtOH-exposed cultures. Overall, our study shows that EtOH induces neuronal apoptosis and changes in the cytoskeleton and membrane proteins related with the establishment of mature synapses. These direct effects of EtOH on neurons may partially explain its effects on brain development.

Anxiolytic, Psychostimulant, and Analgesic Effects of Various Volumes of Ethanol Solution in Different Concentrations, but in the Same Dose.

We studied the effect of ethanol (dose 2 g/kg) in various concentrations (5, 13, and 40%) and different volumes (40, 15.5, and 5 ml/kg) on the level of anxiety, locomotor activity, and pain sensitivity in rats. Administration of 40 ml/kg water to animals was followed by a significant increase in the time spent in the open arms of the elevated plus maze. Administration of water in a volume of 5 or 15.5 ml/kg had little effect on the level of anxiety. The greater was the volume of intragastrically administered ethanol, the stronger was the anxiolytic effect. The psychostimulant and analgesic effects of ethanol were more pronounced after administration of medium volumes and intermediate concentrations of ethanol-containing solutions. Since administration of these solutions cannot produce maximum blood concentration of ethanol, we believe that the observed effects are mainly related to the direct effect of ethanol on the stomach tissue.

Bone changes in alcoholic liver disease.

Alcoholism has been associated with growth impairment, osteomalacia, delayed fracture healing, and aseptic necrosis (primarily necrosis of the femoral head), but the main alterations observed in the bones of alcoholic patients are osteoporosis and an increased risk of fractures. Decreased bone mass is a hallmark of osteoporosis, and it may be due either to decreased bone synthesis and/or to increased bone breakdown. Ethanol may affect both mechanisms. It is generally accepted that ethanol decreases bone synthesis, and most authors have reported decreased osteocalcin levels (a "marker" of bone synthesis), but some controversy exists regarding the effect of alcohol on bone breakdown, and, indeed, disparate results have been reported for telopeptide and other biochemical markers of bone resorption. In addition to the direct effect of ethanol, systemic alterations such as malnutrition, malabsorption, liver disease, increased levels of proinflammatory cytokines, alcoholic myopathy and neuropathy, low testosterone levels, and an increased risk of trauma, play contributory roles. The treatment of alcoholic bone disease should be aimed towards increasing bone formation and decreasing bone degradation. In this sense, vitamin D and calcium supplementation, together with biphosphonates are essential, but alcohol abstinence and nutritional improvement are equally important. In this review we study the pathogenesis of bone changes in alcoholic liver disease and discuss potential therapies.

Covering the Cover

Covering the Cover

Correcting direct effects of ethanol on translation and transcription machinery confers ethanol tolerance in bacteria

The molecular mechanisms of ethanol toxicity and tolerance in bacteria, although important for biotechnology and bioenergy applications, remain incompletely understood. Genetic studies have identified potential cellular targets for ethanol and have revealed multiple mechanisms of tolerance, but it remains difficult to separate the direct and indirect effects of ethanol. We used adaptive evolution to generate spontaneous ethanol-tolerant strains of Escherichia coli, and then characterized mechanisms of toxicity and resistance using genome-scale DNAseq, RNAseq, and ribosome profiling coupled with specific assays of ribosome and RNA polymerase function. Evolved alleles of metJ, rho, and rpsQ recapitulated most of the observed ethanol tolerance, implicating translation and transcription as key processes affected by ethanol. Ethanol induced miscoding errors during protein synthesis, from which the evolved rpsQ allele protected cells by increasing ribosome accuracy. Ribosome profiling and RNAseq analyses established that ethanol negatively affects transcriptional and translational processivity. Ethanol-stressed cells exhibited ribosomal stalling at internal AUG codons, which may be ameliorated by the adaptive inactivation of the MetJ repressor of methionine biosynthesis genes. Ethanol also caused aberrant intragenic transcription termination for mRNAs with low ribosome density, which was reduced in a strain with the adaptive rho mutation. Furthermore, ethanol inhibited transcript elongation by RNA polymerase in vitro. We propose that ethanol-induced inhibition and uncoupling of mRNA and protein synthesis through direct effects on ribosomes and RNA polymerase conformations are major contributors to ethanol toxicity in E. coli, and that adaptive mutations in metJ, rho, and rpsQ help protect these central dogma processes in the presence of ethanol.

Modulation of proopiomelanocortin gene expression by ethanol in mouse anterior pituitary corticotrope tumor cell AtT20

In humans, alcoholism is associated with a decrease in basal ACTH and cortisol levels, and blunted pituitary ACTH responses to administered corticotropin-releasing hormone (CRH) during active drinking and after long-term abstinence. Preclinical studies indicate that a persistent decrease in pituitary activation after chronic exposure to ethanol is due to a direct effect of ethanol on the corticotrope of the anterior pituitary. The present studies were undertaken to determine if ethanol has effects on proopiomelanocortin (POMC) gene transcription activity in mouse anterior pituitary corticotrope tumor cell AtT20. We measured the levels of the POMC primary nuclear RNA transcript (PT), processing intermediate, and mature mRNA in the nucleus and the levels of the POMC mRNA in the cytoplasm after treatment of AtT20 cells with 5–15mM concentrations of ethanol. After 15mM ethanol for 60 to 120min, the POMC PT levels were significantly decreased. This decreased POMC gene transcription activity was coupled with a significant reduction of the POMC cytoplasmic mRNA levels. After ethanol for 4h, however, both the decreases were no longer observed. After 8h, a decrease in the ACTH secretion in the medium was found. We further investigated if CRH or glutamate modulates the effects of ethanol on the POMC gene transcription activity. CRH at 10nM after 60min increased the POMC PT levels, and 15mM ethanol attenuated the effect of CRH on the nuclear transcription activity. Glutamate receptor proteins, including NMDA receptor subtype NR1 (but not NR2A or NR2B) and GluR2, were identified by Western immunoblot analysis in AtT20 cells, with similar sizes to those in mouse hypothalamus. The inhibitory effect of 60min ethanol at 5 to 15mM on the POMC PT levels was attenuated by 50μML-glutamate. Together, our data showed that: (1) ethanol treatment in intoxicate doses significantly inhibited POMC gene transcription activity in a dose- and time-dependent manner in AtT20 cells, and (2) the POMC gene transcription activity in response to CRH or glutamate was altered by ethanol. Our results suggest that ethanol has an inhibitory effect on the POMC gene transcription activity in the anterior pituitary corticotrope, which may contribute to the persistent decrease in pituitary activation after chronic ethanol exposure.

Ethanol contributes to neurogenic pancreatitis by activation of TRPV1

Alcohol abuse is a major cause of pancreatitis in people, but the mechanism is unknown. It has been recently demonstrated that transient receptor potential vanilloid 1 (TRPV1) activation causes neurogenic inflammation and plays an important role in acute pancreatitis. Moreover, TRPV1 is activated by ethanol. We examined the direct effects of ethanol on acute pancreatitis. Acute inflammation of the pancreas was produced by injection of ethanol and palmitoleic acid (POA), a nonoxidative metabolite of ethanol, in wild-type C57BL/6J mice and Trpv1-knockout C57BL/6J mice. Inflammatory indexes were analyzed 24 h later. Injection of ethanol + POA produced acute pancreatitis indicated by significant increases in histopathological damage, serum amylase levels, and pancreatic MPO concentrations (P<0.05-0.001). All parameters of pancreatitis were blocked by pretreatment with the TRPV1 antagonist drug AMG9810. In addition, ethanol + POA administration to Trpv1knockout mice did not produce pancreatic inflammation. Treatment with vehicle, ethanol alone, or POA alone had no inflammatory effects. TRPV1 partially mediates inflammation induced by ethanol + POA in the mouse pancreas, consistent with the ability of ethanol to activate TRPV1. We propose that ethanol may contribute to alcohol-induced pancreatitis by a neurogenic mechanism.

English

Abstract Introduction Alcoholism may damage the skeleton. In addition to a direct effect of ethanol, many other mechanisms are involved which should be considered in the clinical evaluation of alcoholic patients. We will review the mechanism and clinical consequences of bone alterations observed in alcoholics. Conclusion Osteoporosis is the main metabolic disease observed in alcoholic patients. The main factors involved in its pathogenesis are: a direct effect of ethanol, associated malnutrition, liver disease, altered hormone profile and cytokine pattern, and alcoholic myopathy. Osteoporosis may lead to bone fracture. It improves with ethanol withdrawal.

Elucidating the Roles of Ethanol Fermentation Metabolism in Causing Off-Flavors in Mandarins

To elucidate the roles of ethanol fermentation metabolism in causing off-flavors, 'Mor' mandarins were exposed to anaerobic atmospheres for 0, 2, 4, 7, and 10 days to gradually increase juice ethanol and acetaldehyde levels through enhanced fermentation. Exposure to anaerobic atmosphere caused progressive decline in fruit sensory quality, from nearly "good" to "very bad", because of decreased typical mandarin flavor and increased sensation of 'musty' and 'ethanol' off-flavors. GC-MS analysis revealed significant (p ≤ 0.05) increases in the contents of 12 aroma volatiles, including the ethanol fermentation metabolites ethanol and acetaldehyde, and several fatty acid and amino acid catabolism derivates, 7 of which were ethyl esters, which suggests that they were esterification products of ethanol and acyl-CoA's derived from fatty acid and amino acid catabolism. These de novo synthesized anaerobiosis-regulated ethyl esters impart 'pungent', 'ethereal', 'waxy', 'musty', and 'fruity' notes. Overall, these results suggest that besides the direct effects of ethanol and acetaldehyde, downstream ethanol esterification products may also be involved in causing off-flavor sensation in mandarins.

Structural Basis for Alcohol Modulation of a Ligand-Gated Ion Channel Homolog

The molecular mechanisms underlying direct effects of ethanol and other n-alcohols on the nervous system, including intoxication and anesthesia, are poorly understood. Although physiological and behavioral evidence implicates ligand-gated ion channels in the action of alcohols and other general anesthetics, a lack of high-resolution structural information for these receptors has limited structural characterization of alcohol modulation. Recent structural and functional studies of the prokaryotic ligand-gated ion channel homolog GLIC show it to be a valuable model for structure, function, and modulation of these receptors. We are investigating the effects of alcohols on GLIC, and identifying specific protein sites underlying alcohol modulation. Our results demonstrate that GLIC exhibits differential modulation by short- and long-chain n-alcohols, similar to homologous cys-loop receptors. Mutations at putative alcohol binding sites modify alcohol modulation, while labeling with MTS reagents mimics alcohol effects. Our work supports a role for specific residues in the GLIC transmembrane domain in mediating alcohol binding and modulation.

Cytokines Reduce Toxic Effects of Ethanol on Oligodendroglia

To characterize immunomodulatory mechanisms that affect oligodendroglia (OL) and white matter following ethanol exposure during early CNS development, we investigated the direct effects of ethanol and cytokines on glia. Mixed glial cultures from newborn rat brain were exposed to 6.5-130 mM ethanol for 1-3 days. OL were sensitive to ethanol, with death ranging from 32 to 88% with increasing time and ethanol concentrations. Little cell death occurred in astroglia or microglia. Mixtures of cytokines representative of those produced by pro-inflammatory Th1 and monocyte/macrophage (M/M) cells as well as those produced by anti-inflammatory Th2 cells were all protective. Three of the cytokines in the Th1 mixture, IL-2, TNF-α and IFN-γ, were protective individually, although no single cytokine was as effective as the mixture. The protective effects of the Th1 mixture and of IL-2 were reversed by inhibition of both MAP kinase and PI-3 kinase signaling pathways. We conclude that cytokines can act either directly on OL or indirectly through effects on astroglia or microglia to protect OL from ethanol toxicity.

Acetaldehyde-Mediated Neurotoxicity: Relevance to Fetal Alcohol Spectrum Disorders

Ethanol-induced neuro-developmental abnormalities are associated with impaired insulin and IGF signaling, and increased oxidative stress in CNS neurons. We examined the roles of ethanol and its principal toxic metabolite, acetaldehyde, as mediators of impaired insulin/IGF signaling and oxidative injury in immature cerebellar neurons. Cultures were exposed to 3.5 mM acetaldehyde or 50 mM ethanol ± 4-methylpyrazole (4-MP), an inhibitor of ethanol metabolism, and viability, mitochondrial function, oxidative stress, DNA damage, and insulin responsiveness were measured 48 hours later. Acetaldehyde or ethanol increased neuronal death and levels of 8-OHdG and 4-HNE, and reduced mitochondrial function. Ethanol inhibited insulin responsiveness, whereas acetaldehyde did not. 4-MP abated ethanol-induced oxidative stress and mitochondrial dysfunction, but failed to restore insulin responsiveness. Furthermore, alcohol and aldehyde metabolizing enzyme genes were inhibited by prenatal ethanol exposure; this effect was mediated by acetaldehyde and not ethanol + 4MP. These findings suggest that brain insulin resistance in prenatal alcohol exposure is caused by direct effects of ethanol, whereas oxidative stress induced neuronal injury is likely mediated by ethanol and its toxic metabolites. Moreover, the adverse effects of prenatal ethanol exposure on brain development may be exacerbated by down-regulation of genes needed for metabolism and detoxification of alcohol in the brain.