Excessive Ethanol Consumption Research Articles

Enhanced autophagy and phagocytosis of apoptotic lymphocytes in splenic macrophages of acute ethanol-treated rats: Light and electron microscopic studies.

Autophagy is a prosurvival mechanism for the clearance of damaged cellular components, specifically upon exposure to various stressors. In lymphoid organs, excessive ethanol consumption increases lymphocyte apoptosis, resulting in immunosuppression. However, ethanol-induced autophagy and related phagocytosis of apoptotic lymphocytes in the spleen have not been studied yet. Adult male Wistar rats were injected intraperitoneally either with 5 g/kg ethanol or phosphate-buffered saline (as a control group) and then sacrificed 0, 3, 6, and 24 hours after injection. Light and transmission electron microscopy (TEM) findings indicated enhanced T cell apoptosis in the white pulps of ethanol-treated rats (ETRs) compared with the control group, which peaked at 6 h and was associated with the accumulation of tingible body macrophages (TBMs). These macrophages exhibited an upregulated autophagic response, as evidenced by enhanced LC3-II (a specific marker of autophagosomes) expression, which peaked at 24h. In addition, double labeling immunofluorescence of LC3-II with lysosomal markers revealed the enhanced formation of autolysosomes in TBMs of ETRs, which was associated with suppression of p62 immunostaining, indicating the enhanced autophagic flux. Interestingly, this elevated autophagic response in ETR TBMs was accompanied by evidence of LC3-associated phagocytosis (LAP) of apoptotic splenocytes. This is based on TUNEL/LC3-II double labeling and TEM observations of phagosomes containing apoptotic bodies, enclosed within phagosomal membranes adjacent to the autophagic vacuoles. It can be concluded that enhanced prosurvival autophagy in splenic TBMs of ETRs and clearing of apoptotic lymphocytes via LAP may contribute to preventing secondary necrosis and autoimmune diseases.

Chemogenetic inhibition of locus coeruleus to rostromedial tegmental nucleus noradrenergic pathway increases light cycle ethanol drinking in male and female mice and blunts ethanol-induced CTA

We recently showed that chemogenetic activation of the locus coeruleus (LC) to the rostromedial tegmental nucleus (RMTg) noradrenergic (NE) pathway significantly blunted binge-like ethanol drinking and induced aversive-like behaviors in mice. The aim of the present study is to determine if silencing this TH + LC → RMTg noradrenergic pathway promotes increased levels of binge-like ethanol intake and reduced ethanol-induced conditioned taste aversion (CTA). To this end, both male and female TH-ires-cre mice on a C57BL/6 J background were cannulated in the RMTg and injected in the LC with rAVV viruses that encode cre-dependent Gi-expressing designer receptor exclusively activated by designer drugs (DREADDs), or its control, to directly control the activity of NE neurons. Inhibition of the LC to RMTg pathway had no effect on the binge-ethanol drinking in a “drinking-in-the-dark” (DID) paradigm. However, when using this paradigm during the light cycle, silencing of this circuit significantly increased ethanol intake without altering sucrose drinking. Moreover, we found that inhibition of this circuit significantly attenuated an ethanol-induced CTA. In addition, when compared to control animals, pairing RMTg-directed Clozapine N-oxide (CNO) with an i.p. injection of 1.5 g/kg ethanol reduced c-Fos activation in the LC, and increased c-Fos expression in the ventral tegmental area (VTA) in Gi-expressing mice. Our data show that inhibition of the TH + LC to the RMTg pathway significantly increased ethanol drinking as well as attenuated ethanol-induced CTA, supporting the involvement of the LC to RMTg noradrenergic circuit as an important protective mechanism against excessive ethanol consumption.

Vascular injury associated with ethanol intake is driven by AT1 receptor and mitochondrial dysfunction

BackgroundRenin-angiotensin (Ang II)-aldosterone system (RAAS) is crucial for the cardiovascular risk associated with excessive ethanol consumption. Disturbs in mitochondria have been implicated in multiple cardiovascular diseases. However, if mitochondria dysfunction contributes to ethanol-induced vascular dysfunction is still unknown. We investigated whether ethanol leads to vascular dysfunction via RAAS activation, mitochondria dysfunction, and mitochondrial reactive oxygen species (mtROS). MethodsMale C57/BL6J or mt-keima mice (6–8-weeks old) were treated with ethanol (20% vol./vol.) for 12 weeks with or without Losartan (10 mg/kg/day). ResultsEthanol induced aortic hypercontractility in an endothelium-dependent manner. PGC1α (a marker of biogenesis), Mfn2, (an essential protein for mitochondria fusion), as well as Pink-1 and Parkin (markers of mitophagy), were reduced in aortas from ethanol-treated mice. Disturb in mitophagy flux was further confirmed in arteries from mt-keima mice. Additionally, ethanol increased mtROS and reduced SOD2 expression. Strikingly, losartan prevented vascular hypercontractility, mitochondrial dysfunction, mtROS, and restored SOD2 expression. Both MnTMPyP (SOD2 mimetic) and CCCP (a mitochondrial uncoupler) reverted ethanol-induced vascular dysfunction. Moreover, L-NAME (NOS inhibitor) and EUK 134 (superoxide dismutase/catalase mimetic) did not affect vascular response in ethanol group, suggesting that ethanol reduces aortic nitric oxide (NO) and H2O2 bioavailability. These responses were prevented by losartan. ConclusionAT1 receptor modulates ethanol-induced vascular hypercontractility by promoting mitochondrial dysfunction, mtROS, and reduction of NO and H2O2 bioavailability. Our findings shed a new light in our understanding of ethanol-induced vascular toxicity and open perspectives of new therapeutic approaches for patients with disorder associated with abusive ethanol drinking.

Nuclear miR-150 enhances hepatic lipid accumulation by targeting RNA transcripts overlapping the PLIN2 promoter

Alcohol-associated liver disease is a prevalent chronic liver disease caused by excessive ethanol consumption. This study aims to investigate the role of miR-150 in regulating hepatic lipid homeostasis in alcoholic fatty liver (AFL). miR-150 was mainly distributed in the nucleus of hepatocytes and correlated with the degree of liver injury. The decreased expression of miR-150 observed in AFL was a compensatory response to ethanol-induced hepatic steatosis. Overexpression of miR-150 facilitated hepatic lipid accumulation in cellulo and exacerbated ethanol-induced liver steatosis invivo. In silico analysis identified perilipin-2 (PLIN2) as a potential target gene of miR-150. miR-150 activated PLIN2 transcription by directly binding the RNA transcripts overlapping PLIN2 promoter and facilitating the recruitment of DNA helicase DHX9 and RNA polymeraseⅡ. Overall, our study provides fresh insights into the homeostasis regulation of hepatic steatosis induced by ethanol and identifies miR-150 as a pro-steatosis effector driving transcriptional PLIN2 gene activation.

Identification of hepatoprotective peptides from porcine liver and its interaction with ethanol metabolizing enzymes in vitro

Alcoholic liver disease (ALD) is a worldwide disease caused by excessive ethanol consumption. In current study, hepatoprotective peptides were isolated from the porcine liver while their activities and interactions with ethanol metabolizing enzymes (alcohol dehydrogenase: ADH/acetaldehyde dehydrogenase: ALDH) were analyzed. The alcalase digested porcine liver hydrolysate (PLH) was confirmed to possess ADH/ALDH activation ability in a dose-dependent manner. The peptide sequences of PLH were identified by mass spectrometry. Molecular docking and in silico analysis were federatively used for screening. NTLPHPTAP had the lowest binding energy with ADH (−0.88 kcal/mol) and low toxicity. Fluorescence quenching data showed a dynamic fluorescence quenching constant (Kq) for NTLPHPTAP interacting with ADH/ALDH (ADH: 1.57 × 10−10 M−1 s−1; ALDH:1.961 × 10−10 M−1 s−1). Circular dichroism (CD) spectra and infrared spectrometer (FTIR) revealed that the enzyme secondary structures became flexible and disordered after interaction with NTLPHPTAP. Meanwhile, the ADH/ALDH was speculated to bind NTLPHPTAP via amido and carbonyl groups, respectively. These results indicated that the formation of ADH/ALDH-NTLPHPTAP complexes expanded the overall structure of the original enzymes and further increased the possibility of enzyme-substrate binding. In conclusion, the study proposed a method for the extraction of porcine liver hepatoprotective peptides and suggested further explanation for the interaction between NTLPHPTAP and ADH/ALDH in vitro.

Somatostatin signaling modulates binge drinking behavior via the central nucleus of the amygdala

Somatostatin (SST) is a neuropeptide widely expressed in the central nervous system with dense expression in limbic regions such as the extended amygdala. It has recently gained attention for playing a role in modulating alcohol use disorders and co-morbid neuropsychiatric disorders. However, the role of SST in the central nucleus of the amygdala (CeA), a key region for neuropeptide regulation of alcohol and anxiety related behaviors, in alcohol consumption has not been assessed. In this work we perform an initial examination of the interaction between the CeA SST system and binge ethanol intake. Binge intake is a dangerous pattern of excessive ethanol consumption associated with health complications and the transition into alcohol dependence. We use the Drinking in the Dark (DID) model of binge intake in C57BL/6J male and female mice to examine: 1) the impact of 3 DID cycles on CeA SST expression; 2) the effect of intra-CeA SST injection on binge-like ethanol consumption; and 3) if the SST receptor 2 or 4 (SST2R or SST4R) mediate any effect on consumption. Our results show binge-like ethanol intake decreases SST expression in the CeA, but not neighboring basolateral amygdala. We further found intra-SST CeA administration reduces binge ethanol intake. This decrease was replicated by the administration of an SST4R agonist. These effects were not sex-dependent. Overall, this work lends further support for SST playing a role in alcohol related behaviors and as a potential therapeutic target.

Glucagon-like peptide 1 receptor agonist, exendin-4, reduces alcohol-associated fatty liver disease

Fatty liver is the earliest response to excessive ethanol consumption, which increases the susceptibility of the liver to develop advanced stage of liver disease. Our previous studies have revealed that chronic alcohol administration alters metabolic hormone levels and their functions. Of current interest to our laboratory is glucagon-like peptide 1 (GLP-1), a widely studied hormone known to reduce insulin resistance and hepatic fat accumulation in patients with metabolic-associated fatty liver disease. In this study, we examined the beneficial effects of exendin-4 (a GLP-1 receptor agonist) in an experimental rat model of ALD. Male Wistar rats were pair-fed the Lieber-DeCarli control or ethanol diet. After 4 weeks of this feeding regimen, a subset of rats in each group were intraperitoneally injected every other day with either saline or exendin-4 at a dose of 3 nmol/kg/day (total 13 doses) while still being fed their respective diet. At the end of the treatment, rats were fasted for 6 h and glucose tolerance test was conducted. The following day, the rats were euthanized, and the blood and tissue samples collected for subsequent analysis. We found that exendin-4 treatment had no significant effect on body weight gain among the experimental groups. Exendin-4-treated ethanol rats exhibited improved alcohol-induced alterations in liver/body weight and adipose/body weight ratio, serum ALT, NEFA, insulin, adiponectin and hepatic triglyceride levels. Reduction in indices of hepatic steatosis in exendin-4 treated ethanol-fed rats was attributed to improved insulin signaling and fat metabolism. These results strongly suggest that exendin-4 mitigates alcohol-associated hepatic steatosis by regulating fat metabolism.

Astrocyte expression in the extended amygdala of C57BL/6J mice is sex-dependently affected by chronic intermittent and binge-like ethanol exposure.

Excessive ethanol drinking is a major problem within the United States, causing alterations in brain plasticity and neurocognitive function. Astrocytes are glial cells that regulate neurosynaptic plasticity, modulate neurochemicals, and monitor other homeostatic roles. Astrocytes have been found to play a part in modulating excessive ethanol consumption. The basolateral amygdala (BLA), central amygdala (CeA), and bed nucleus of the stria terminalis (BNST) are brain regions that process stress, anxiety, and reward; they are also implicated in modulating ethanol intake. Little is understood, however, about how astrocyte expression in each region is modulated by chronic and binge-like ethanol drinking patterns. In the present report, we utilized two separate animal models of excessive drinking: chronic intermittent ethanol (CIE) and "Drinking-in-the-dark" (DID). Following these paradigms, animal brains were processed through immunohistochemistry (IHC) and stained for glial fibrillary acidic protein (GFAP). Collected data illustrated a sex-dependent relationship between ethanol intake and GFAP immunoreactivity (IR) in the BLA and BNST, but not in the CeA. Specifically, CIE and DID ethanol drinking resulted in blunted GFAP-IR (specifically via GFAP-positive cell count) in the BLA and BNST, particularly in males. These findings may implicate sex-dependent ethanol-induced changes in BLA and BNST astrocytes, providing a potential therapeutic target for anxiety and stress disorders.

Fractal analysis is a useful tool for evaluating prostate tissue remodeling caused by ethanol consumption and androgen therapy.

Alcohol has been widely consumed for centuries and is linked to the aggravation of diseases. Several studies have shown that excessive consumption of ethanol results in morphophysiological changes in the male reproductive system. One of the effects of ethanol is the decrease in testosterone concentration and hormonal therapies are an alternative to minimize the changes resulting from chronic alcoholism. Qualitative studies were commonly carried out to evaluate the male histopathological alterations resulting from ethanol consumption, being necessary quantitative and non-subjective techniques. This study analyzes the importance of fractal analysis as a useful tool to identify and quantify tissue remodeling in rats submitted to ethanol consumption and hormone therapy with testosterone. Prostate of animals submitted to chronic ethanol consumption showed tissue disorganization, which was confirmed by an increasing of fractal dimension. Regarding the prostatic stroma, collagen fractal dimension and quantification revealed lower values in animals that were only submitted to androgen therapy. Thus, we can conclude that the fractal analysis was a useful tool to quantify tissue changes caused by ethanol consumption and androgen therapy.

Chemogenetic inhibition of corticotropin-releasing factor neurons in the central amygdala alters binge-like ethanol consumption in male mice.

Repetitive bouts of binge drinking can lead to neuroplastic events that alter ethanol's pharmacologic effects and perpetuate excessive consumption. The corticotropin-releasing factor (CRF) system is an example of ethanol-induced neuroadaptations that drive excessive ethanol consumption. Our laboratory has previously shown that CRF antagonist, when infused into the central amygdala (CeA), reduces binge-like ethanol consumption. The present study extends this research by assessing the effects of silencing CRF-producing neurons in CeA on binge-like ethanol drinking stemming from "Drinking in the Dark" (DID) procedures. CRF-ires-Cre mice underwent surgery to infuse Gi/o-coupled Designer Receptors Exclusively Activated by Designer Drugs (DREADD) virus or a control virus into either the CeA or basolateral amygdala (BLA). Gi/o-DREADD-induced CRF-neuronal inhibition in the CeA resulted in a 33% decrease in binge-like ethanol consumption. However, no effect on ethanol consumption was seen after DREADD manipulation in the BLA. Moreover, CeA CRF-neuronal inhibition had no effect on sucrose consumption. The effects of silencing CRF neurons in the CeA on ethanol consumption are not secondary to changes in motor function or anxiety-like behaviors as assessed in the open-field test (OFT). Finally, the DREADD construct's functional ability to inhibit CRF-neuronal activity was demonstrated by reduced ethanol-induced c-Fos following DREADD activation. Together, these data suggest that the CRF neurons in the CeA play an important role in binge ethanol consumption and that inhibition of the CRF-signaling pathway remains a viable target for manipulating binge-like ethanol consumption. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Overexpression of wild type glycine alpha 1 subunit rescues ethanol sensitivity in accumbal receptors and reduces binge drinking in mice.

The nucleus accumbens (nAc) is a critical region in the brain reward system since it integrates abundant synaptic inputs contributing to the control of neuronal excitability in the circuit. The presence of inhibitory α1 glycine receptor (GlyRs) subunits, sensitive to ethanol, has been recently reported in accumbal neurons suggesting that they are protective against excessive binge consumption. In the present study, we used viral vectors (AAV) to overexpress mutant and WT α1 subunits in accumbal neurons in D1 Cre and α1 KI mice. Injection of a Cre-inducible AAV carrying an ethanol insensitive α1 subunit in D1 Cre neurons was unable to affect sensitivity to ethanol in GlyRs or affect ethanol drinking. On the other hand, using an AAV that transduced WT α1 GlyRs in GABAergic neurons in the nAc of high-ethanol consuming mice caused a reduction in ethanol intake as reflected by lowered drinking in the dark and reduced blood ethanol concentration. As expected, the AAV increased the glycine current density by 5-fold without changing the expression of GABAA receptors. Examination of the ethanol sensitivity in isolated accumbal neurons indicated that the GlyRs phenotype changed from an ethanol resistant to an ethanol sensitive type. These results support the conclusion that increased inhibition in the nAc can control excessive ethanol consumption and that selective targeting of GlyRs by pharmacotherapy might provide a mechanistic procedure to reduce ethanol binge.

Assessment of prenatal exposure to alcohol and gestational consumption by EtG testing in neonatal meconium and maternal hair and neonatal: First Italian national survey

Use of alcohol during pregnancy and subsequent fetal exposure can cause multiple perinatal disorders (prematurity at birth, withdrawal syndromes, tremors, hyperreflexia) and impaired physical and mental development in the later stages of life, known as fetal alcohol spectrum disorders (FADS) including as major expression of impairment fetal alcohol syndrome (FAS). Gestational drinking is usually, worldwide reported by questionnaires filled during antenatal visits, but international studies demonstrated their inconsistency for under-reporting or misreporting of toxic habits. Information concerning the true prevalence of alcohol consumption during pregnancy and consequent prenatal exposure to this teratogen is essentially lacking in Italy. To assess objective gestational consumption to alcohol and fetal exposure to it, we measured one of the most specific ethyl alcohol metabolites, ethylglucuronide (EtG), in maternal hair and neonatal meconium involving delivering mothers and newborns along the whole National Italian territory. Non-paired twenty delivering women and twenty newborns per city from hospitals of the National Health system located in the North, Centre and South Italy (including islands) were enrolled in the project. Maternal hair, 8 cm as maximum length, was collected at the end of pregnancy and analyzed as entire strand and neonatal meconium was collected within the first 24 hours after birth. Ethylglucuronide was quantified in both biological matrices through a validated gas chromatography–tandem mass spectrometry methodology. Hair EtG concentrations below 5 pg/mg hair were attributed to abstinent women, those > 5 pg/mg and below 30 pg/mg were attributed to women consuming “some” alcohol during pregnancy and ≥ 30 pg/mg were attributed to excessive drinkers. Furthermore, according to previous studies from our research group, maternal hair EtG above 11 pg/mg was shown to correspond with high probability to a neonatal meconium with EtG > 30 ng/g. Hence, it was considered as a “cut-off” matching to fetal exposure to alcohol. Up to now, 781 maternal hair and 642 meconium samples have been analyzed. Only one woman out of 781(0.1%) presented chronic excessive ethanol consumption with EtG concentration > 30 pg/mg, while the 8.2% of maternal hair presented EtG concentrations > 5 pg/mg with 1.4% > 11 pg/mg. Four newborns (0.6%) resulted prenatally exposed to ethanol with meconium EtG concentration > 30 ng/g. The highest percentage of women involved in the study (mean age + SD 34 + 5.1 years) were Italian (88%) and employed (69%). With regard to educational level, 44% had a university degree and 39% high-school education level. The four exposed newborns did not present at birth any sign of evident disability or malformation. These preliminary findings, obtained in separate cohorts of pregnant and delivering mothers and newborns showed that, currently, a negligible amount of Italian women drink during pregnancy and that a minute percentage of newborns are prenatally exposed to alcohol. The policies applied by health Italian women have acquired awareness about risks associated with drinking during pregnancy.

Sex-specific effects of adolescent intermittent ethanol exposure-induced dysregulation of hippocampal glial cells in adulthood.

Adolescent alcohol abuse is a significant public health concern, with approximately 4.3 million U.S. adolescents reporting monthly binge drinking. Excessive ethanol consumption during adolescence has been linked to dysregulation of the neuroimmune system, particularly in the hippocampus. Because there are sex differences in both neuroimmune responses and ethanol's pharmacologic actions, this study tested whether there were disparate effects based on sex in glial cells and neurodegeneration in adulthood after the adolescent intermittent ethanol (AIE) model. Male and female adolescent Sprague-Dawley rats underwent AIE. In adulthood, immunohistochemical techniques were utilized to determine the effects of AIE on astrocytes and microglia, and Fluoro-Jade C (FJC) was used to assess neurodegeneration in the hippocampus. AIE exposure significantly increased astrocyte activation in the cornu ammonis 1 (CA1), CA2/3, and dentate gyrus (DG) in both male and female rats with no discernible sex differences in immunoreactivity. Likewise, the number of GFAP+cells was significantly increased by AIE across the hippocampus. In our microglial assessment, AIE only led to increased Iba1 immunoreactivity in the CA1 but not CA2/3 or DG regions. However, the number of Iba1+ cells was increased by AIE in both the CA1 and DG subregions. In the DG, the ethanol effect was observed in both sexes, but in the CA1, AIE-induced increased Iba1 cells were only observed in females. In regard to neurodegeneration, there were no persisting AIE effects on FJC+cells. These findings indicate that AIE alters hippocampal glial cells in adulthood, in the absence of active neurodegeneration. However, while AIE induced long-term elevation of astroglial measures in both males and females, persisting AIE-induced microglial activation was more sparse and sex-dependent. While the majority of these findings suggest that AIE has similar effects on glial morphology and number between males and females, additional work should determine whether there are molecular differences as well as innate sex differences in glial interaction with AIE's influence on glial functions in behavior.

Protective effect and mechanism of Lycium barbarum L. polyphenol on cognitive impairment induced by ethanol in mice

BackgroundChronic excessive ethanol consumption damages the central nervous system and causes neurobehavioral changes, such as cognitive impairment, which is related to oxidative stress and inhibition of neurogenesis in the hippocampus. It is known that promoting neurogenesis improves learning memory, anxiety and depression. Lycium barbarum L. polyphenol (LBP) is the main active ingredient of Lycium barbarum L., which has excellent neuroprotective effects. However, the effects and mechanisms of LBP on ethanol-induced cognitive impairment are unclear. PurposeTo assess the effects and mechanisms of LBP on ethanol-induced cognitive impairment in mice. MethodsEight-weeks-old adult C57BL/6J mice were allowed to drink ethanol (10%) to establish a model of ethanol-induced cognitive impairment. From the 29th day of LBP (25, 50, 100, 200, 400 mg/kg, intragastric administration), the locomotor activity, novel object recognition (NOR), Y maze and Morris water maze (MWM) were sequentially performed to investigate the effect of LBP on ethanol-induced cognitive impairment in mice. Next, enzyme-linked immunosorbent assay, immunofluorescence, and western blotting were used to study the underlying mechanism of LBP on ethanol-induced cognitive impairment. ResultsLBP significantly decreased the escape latency and increased the number of crossings of the original platform in MWM, increased the spontaneous alteration behavior in the Y maze, and increased the preference index in the NOR in ethanol-induced mice. Notably, LBP significantly promoted the proliferation of neural stem cells, neural progenitor cells and neuroblasts, and increased the proportion of activated NSCs in mice with ethanol-induced cognitive impairment. Similarly, LBP significantly increased the number of newborn immature neurons and mature neurons. Moreover, LBP increased the levels of nuclear factor erythroid2-related factor 2 (Nrf2) and the downstream heme oxygenase-1(HO-1) protein expression, which led to a decrease of oxidative stress levels. ConclusionLBP significantly improves cognitive impairment in ethanol-induced mice, which is attributed to the promotion of hippocampal neurogenesis and reduction of oxidative stress.

Methylsulfonylmethane protects against ethanol-induced brain injury in mice through the inhibition of oxidative stress, proinflammatory mediators and apoptotic cell death

Excessive ethanol consumption causes brain injury through oxidative stress, inflammation and apoptotic cell death. Methylsulfonylmethane (MSM) is a natural compound that has therapeutic effects on oxidative and inflammatory disorders. The aim of this study was to investigate the protective effect and underlying mechanisms of MSM on ethanol-induced brain injury in an experimental model. Male C57BL/6 mice were exposed to binge ethanol (5 g/kg/day, orally) and treated with MSM (200 and 400 mg/kg/day) concomitantly for 12 days. At the end of the experiment brain tissues were removed for histological and biochemical analysis. The results showed that MSM reduced ethanol-mediated oxidative stress by decreasing the levels of malondialdehyde (MDA) and carbonyl protein. The Nrf2/HO-1 pathway and the levels of cytoprotective antioxidants superoxide dismutase (SOD), catalase and glutathione (GSH) were increased by MSM in the brain tissue. MSM treatment reduced the ethanol-induced inflammatory factors including myeloperoxidase (MPO), iNOS/NO, cyclooxygenase (COX)-2, nuclear factor kappa B (NF-κB), NLRP3 inflammasome and proinflammatory cytokines including TNF-α, IL-1β, IL-6 and MCP-1. MSM also decreased the levels of pro-apoptotic caspase-3 and TUNEL positive cells while increased the level of anti-apoptotic Bcl-2 in the brain tissue. Our findings demonstrated that MSM protects against ethanol-induced brain injury by improving anti-oxidant defense mechanism and reducing ethanol-mediated inflammation and apoptosis. Therefore, MSM may be a potential protective approach for brain damage caused by high levels of alcohol.

Polymorphisms of acetaldehyde dehydrogenase 2 and alcohol dehydrogenase 1B on the malignant transformation of vocal cord leukoplakia: A Chinese cohort

Severe dysplasia of vocal cord leukoplakia (VCL) is more likely to occur in laryngeal carcinoma. Alcohol dehydrogenase and acetaldehyde dehydrogenase are both important enzymes in alcohol metabolism. This study aimed to investigate the incidence rate of malignant transformation in patients with VCL and the role of drinking habits and ALDH2 and ADH1B genetic polymorphisms in the malignant transformation of VCL. From January 2007 to January 2017, 136 cases of VCL were included in this retrospective analysis. Information on medical history, alcohol and tobacco consumption habits, ALDH2 and ADH1B genotypes, gastroesophageal reflux, and clinical pathological characteristics of VCL was collected. As a result, patients had a median follow-up of 9.6 years (interquartile range: 7.5-12.5 years). Twenty-three of 136 VCL patients finally developed laryngeal carcinoma, resulting in a cumulative malignant transformation rate of 16.9%. Cox regression analysis demonstrated that the independent risk factors for the malignant transformation of VCL included age over 60 years (hazard ratio [HR]: 13.872, p < 0.001), ALDH2 *2 allele status (HR: 9.694, p < 0.001), alcohol (HR: 10.011, p < 0.001) and tobacco (HR: 8.869, p < 0.001) exposure after operation, and drinking frequency (HR: 2.178, p = 0.016). Therefore, among patients over 60 years old, an ALDH2-inactivating mutation and excessive ethanol and tobacco consumption are potential contributors to the malignant transformation of VCL.

Binge Ethanol Exposure in Mice Represses Expression of Genes Involved in Osteoblast Function and Induces Expression of Genes Involved in Osteoclast Differentiation Independently of Endogenous Catalase.

Excessive ethanol consumption is a risk factor for osteopenia. Since a previous study showed that transgenic female mice with overexpression of catalase are partially protected from ethanol-mediated trabecular bone loss, we investigated the role of endogenous catalase in skeletal ethanol toxicity comparing catalase knockout to wild-type mice. We hypothesized that catalase depletion would exacerbate ethanol effects. The mice were tested in a newly designed binge ethanol model, in which 12-week-old mice were exposed to 4 consecutive days of gavage with ethanol at 3, 3, 4, and 4.5 g ethanol/kg body weight. Binge ethanol decreased the concentration of serum osteocalcin, a marker of bone formation. The catalase genotype did not affect the osteocalcin levels. RNA sequencing of femoral shaft RNA from males was conducted. Ethanol exposure led to significant downregulation of genes expressed in cells of the osteoblastic lineage with a role in osteoblastic function and collagen synthesis, including the genes encoding major structural bone proteins. Binge ethanol further induced a smaller set of genes with a role in osteoclastic differentiation. Catalase depletion affected genes with expression in erythroblasts and erythrocytes. There was no clear interaction between binge ethanol and the catalase genotype. In an independent experiment, we confirmed that the binge ethanol effects on gene expression were reproducible and occurred throughout the skeleton in males. In conclusion, the binge ethanol exposure, independently of endogenous catalase, reduces expression of genes involved in osteoblastic function and induces expression of genes involved in osteoclast differentiation throughout the skeleton in males.

On the Use of Heterogeneous Stock Mice to Map Transcriptomes Associated With Excessive Ethanol Consumption.

We and many others have noted the advantages of using heterogeneous (HS) animals to map genes and gene networks associated with both behavioral and non-behavioral phenotypes. Importantly, genetically complex Mus musculus crosses provide substantially increased resolution to examine old and new relationships between gene expression and behavior. Here we report on data obtained from two HS populations: the HS/NPT derived from eight inbred laboratory mouse strains and the HS-CC derived from the eight collaborative cross inbred mouse strains that includes three wild-derived strains. Our work has focused on the genes and gene networks associated with risk for excessive ethanol consumption, individual variation in ethanol consumption and the consequences, including escalation, of long-term ethanol consumption. Background data on the development of HS mice is provided, including advantages for the detection of expression quantitative trait loci. Examples are also provided of using HS animals to probe the genes associated with ethanol preference and binge ethanol consumption.

Effect of chronic ethanol consumption in rhesus macaques on the nucleus accumbens core transcriptome.

The nucleus accumbens core (NAcc) has been repeatedly demonstrated to be a key component of the circuitry associated with excessive ethanol consumption. Previous studies have illustrated that in a nonhuman primate (NHP) model of chronic ethanol consumption, there is significant epigenetic remodeling of the NAcc. In the current study, RNA-Seq was used to examine genome-wide gene expression in eight each of control, low/binge (LD*), and high/very high (HD*) rhesus macaque drinkers. Using an FDR < 0.05, zero genes were significantly differentially expressed (DE) between LD* and controls, six genes between HD* and LD*, and 734 genes between HD* and controls. Focusing on HD* versus control DE genes, the upregulated genes (N = 366) were enriched in genes with annotations associated with signal recognition particle (SRP)-dependent co-translational protein targeting to membrane (FDR < 3 × 10−59), structural constituent of ribosome (FDR < 3 × 10−47), and ribosomal subunit (FDR < 5 × 10−48). Downregulated genes (N = 363) were enriched in annotations associated with behavior (FDR < 2 × 10−4), membrane organization (FDR < 1 × 10−4), inorganic cation transmembrane transporter activity (FDR < 2 × 10−3), synapse part (FDR < 4 × 10−10), glutamatergic synapse (FDR < 1 × 10−6), and GABAergic synapse (FDR < 6 × 10−4). Ingenuity Pathway Analysis (IPA) revealed that EIF2 signaling and mTOR pathways were significantly upregulated in HD* animals (FDR < 3 × 10−33 and <2 × 10−16, respectively). Overall, the data supported our working hypothesis; excessive consumption would be associated with transcriptional differences in GABA/glutamate-related genes.

Astaxanthin attenuates alcoholic cardiomyopathy via inhibition of endoplasmic reticulum stress-mediated cardiac apoptosis

Chronic excessive ethanol consumption is associated with a high incidence of mortality due to ethanol-induced dilated cardiomyopathy, known as alcoholic cardiomyopathy (ACM). Mechanistic studies have demonstrated that apoptosis is key to the pathogenesis of ACM, and endoplasmic reticulum (ER) stress-associated apoptosis contributes to various ethanol-related diseases. Astaxanthin (AST) is a natural carotenoid that exerts an anti-ER stress effect. Importantly, strong evidence has shown that AST induces beneficial effects in various cardiovascular diseases. The present study aimed to investigate whether AST induces beneficial effects on ACM by suppressing cardiac apoptosis mediated by ER stress. We showed that after 2 months of chronic excessive ethanol consumption, mice displayed obvious cardiac dysfunction and morphological changes associated with increased fibrosis, oxidative stress, ER stress and apoptosis. However, cardiac damage above was attenuated in response to AST treatment. The cardioprotective effect of AST against ethanol toxicity was also confirmed in both H9c2 cells and primary cardiomyocytes, indicating that AST-induced protection directly targets cardiomyocytes. Both in vivo and in vitro studies showed that AST inhibited all three ER stress signaling pathways activated by ethanol. Furthermore, administration of the ER stress inhibitor sodium 4-phenylbutyrate (4-PBA) strongly suppressed ethanol-induced cardiomyocyte damage. Interestingly, AST induced further anti-apoptotic effects once co-treated with 4-PBA, indicating that AST protects the heart from ACM partially by attenuating ER stress, but other mechanisms still exist. This study highlights that administration of AST ablated chronic excessive ethanol consumption-induced cardiomyopathy by suppressing cardiac ER stress and subsequent apoptosis.