Effects Of EtOH Research Articles

C-Jun N-Terminal Kinase-2 (JNK2) Plays a Crucial Role in Alcohol and Corticosterone-Induced Dysbiosis of Gut Microbiota and Tissue Injury at the Gut-Liver Axis

Introduction: About 15% of heavy drinkers develop liver disease, suggesting the “Multiple hit hypothesis” of Alcohol-Associated Liver Diseases (AALD). Chronic stress is a potential second hit in AALD pathogenesis. Our recent study showed that alcohol-induced gut and liver injury is exacerbated by chronic restraint stress or corticosterone. JNK2 plays an essential role in stress-induced cell injury. In this study, we investigated the role of JNK2 in ethanol (EtOH) and corticosterone-induced gut and liver injury and microbiota dysbiosis in mice. Methods: Wildtype (WT) and JNK2 knockout (JNK2-KO) mice (male and female; 8-10 wks) were fed a Lieber-DiCarli liquid diet with or without EtOH (0% 2d, 1% 2d, 2% 2d, 4% 1wk, 5% 1wk, & 6% 1wk). Control groups were pair-fed isocaloric EtOH-free diet. One group of pair-fed and EtOH-fed WT and JNK2-KO mice were administered corticosterone (CORT, 25 mg/kg/day; s.c.). Intestinal permeability was measured by vascular-to-luminal flux of FITC-inulin in vivo, tight junction (TJ) and adherens junction (AJ) integrity was assessed by confocal microscopy for occludin, ZO-1, E-cadherin, and β-catenin, and mucosal inflammation by RT-PCR for cytokines/chemokines. Plasma lipopolysaccharide (LPS) and cytokines were measured to evaluate endotoxemia and systemic inflammation. Liver damage was assessed by measuring plasma AST/ALT, liver triglyceride, and cytokine/chemokine mRNA. Gut microbiota was analyzed by 16S rRNA sequencing of colonic flushing followed by bioinformatics analyses. Results and conclusion: In WT mice, EtOH feeding increased intestinal permeability, disrupted epithelial TJ and AJ, and elevated mucosal cytokine expression, indicating epithelial barrier dysfunction and mucosal inflammation. EtOH increased plasma LPS and cytokine/chemokine levels, indicating endotoxemia and systemic inflammation. Plasma AST/ALT and liver triglyceride levels were increased, indicating EtOH-induced liver damage. CORT significantly elevated EtOH-induced intestinal permeability, TJ/AJ disruption, endotoxemia, systemic inflammation, and liver damage in WT mice. These effects of EtOH and CORT were absent in JNK2-KO mice. Microbiota α-diversity was higher in JNK2-KO mice than in WT mice, and β-diversity analysis showed that the microbiota composition in WT mice differed from that of KO mice. Microbiota α- and β-diversities were altered by EtOH and CORT in WT mice but not in JNK2-KO mice. The relative abundance of Nocardiaceae and Verrucomicrobiaceae was high, and Ruminococcaceae and Bacillaceae were low in KO mice. CORT decreased the abundance of Caulobacteriaceae and Ruminococcaceae and increased Verrucomicrobiaceae and Lactobacillaceae in WT but not KO mice. EtOH increased Ruminococcaceae and Bacillaceae and decreased Lactobacillaceae in WT mice but not KO mice. CORT-induced modulation of EtOH-induced microbiota dysbiosis was observed in WT mice but not KO mice. In conclusion, these data demonstrate that JNK2 regulates gut microbiota in mice, and JNK2 deficiency confers resistance to alcohol and corticosterone-induced tissue injury and microbiota dysbiosis. Acknowledgements: NIH/NIAAA - R01AA012307, AA029270; VA - IO1BX003014. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Recurrent Ethanol Treatment does not Produce a Cumulative Effect on Plasma Leptin Levels in Rats

Background: Hypoleptinemia is a potent orexigenic signal. Chronic ethanol (EtOH) consumption is associated with weight gain in humans, and acute ethanol treatment leads to reductions in leptin concentrations in both fasted humans and rats. The objective of this study was to determine whether short-term recurrent ethanol treatment has a cumulative effect on plasma leptin levels in rats, and whether treatment in the fed or fasted state influences the leptin lowering effect of EtOH. We hypothesized that recurrent EtOH treatment would have a cumulative effect on leptin levels, and that these effects would be more pronounced in the fed state, when leptin levels are highest. Methods: Two separate groups of male Sprague Dawley rats were exposed to either oral or intravenous (IV) treatment with EtOH (2g/kg via oral gavage or 0.067 g/kg/min IV for 30 min; n=7/group) on three consecutive days. Each rat served as its own control and received saline treatment either 7 d prior to or 7 d following exposure to EtOH. The oral group was fasted 12-14 h (overnight) prior to treatment, while the IV group was not fasted. Plasma leptin and EtOH levels were measured each day at baseline and 3 h following EtOH treatment. Data are expressed as mean ± SEM. A two-way repeated measures ANOVA was used to test for differences in plasma leptin and EtOH levels due to treatment (saline vs. EtOH) or day (cumulative effect of treatment). Results: Post-treatment ethanol levels in the oral and IV groups (55 ± 6.9 and 45 ± 6.3 mM, respectively) were higher than saline-treated rats (p>0.001). Baseline leptin levels in the oral treatment (fasted) group were 3.4 ± 0.3 and 3.9 ± 0.2 ng/mL under saline and leptin treated conditions, respectively. There was a significant main effect of treatment but not of day in the oral group (p=0.04 and p=0.38, respectively). In the oral group, average daily change in leptin levels 3 h post treatment in saline and leptin treated rats were -0.18 ± 0.05 and -0.72 ± 0.1 ng/mL, respectively. Baseline leptin levels in the IV group (fed) were 7.8 ± 0.3 and 6.7 ± 0.1 ng/mL, in saline and leptin treated rats respectively. There was no significant main effect of treatment or day in the IV group (p=0.17 and p=0.41, respectively). In the IV group, average daily changes in leptin levels 3 h following treatment in saline and leptin treated rats were -1.7 ± 0.2 and -1.3 ± 0.3 ng/mL, respectively. Conclusion: Although we were able to replicate previous work indicating reduced leptin levels following acute EtOH treatment in fasted rats, our results do not support a cumulative leptin-lowering effect of recurrent EtOH treatment. Furthermore, IV EtOH treatment in the fed state did not have any impact on leptin levels. This suggests that the leptin-lowering effects of EtOH may either require the fasted state or may require ingestion and exposure of the GI system to EtOH. NIH R01 DK131165-01A1. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Untangling the Impacts of Ethanol on Myoblast Proliferation

Alcohol-mediated myopathy frequently accompanies chronic alcohol misuse. This decreased skeletal muscle mass and function increases risk for frailty and falls, decreases quality of life, and worsens prognosis for other comorbid conditions. After muscle injury or atrophy, satellite cells are activated as myoblasts, which proliferate, differentiate, and fuse with myofibers to aid in repairing and rebuilding skeletal muscle. While decreased myoblast differentiation with ethanol (EtOH) is well-documented, we have also observed fewer myoblasts with in vitro EtOH after the proliferative phase of myogenesis. The extent of this decrease and whether this is due primarily to increased cell death, decreased cell division, or a combination thereof remains unanswered. Therefore, the objective of this study is to quantify effects of EtOH on myoblast cell division and death during the proliferative phase. C2C12 myoblasts were proliferated in the presence or absence of 50 mM EtOH for 24, 48, and 72 hrs. Nuclei were stained with DAPI to quantify differences in cell count at each time point. Lactate dehydrogenase (LDH) activity was measured in cell culture supernatant at 48 and 72 hrs and normalized to cell count to assess overall cytotoxicity. T-tests were used to determine differences between conditions at each time point. There were no significant differences in cell counts between cells grown with 0 and 50 mM EtOH at 24 or 48 hrs. At 72 hrs, EtOH significantly decreased cell count (p=0.03). There were no significant differences for LDH activity at 48 hrs. At 72 hrs, LDH activity was significantly lower in the 50 mM group (p=0.05). These preliminary results suggest that EtOH decreases cell count in a time-dependent manner. Cytotoxicity appeared lower with EtOH, although this is likely due to lower intracellular LDH activity given our previously observed adverse impacts of EtOH on myoblast glycolytic function. Regardless, the present data do not support increased cytotoxicity with 50 mM EtOH. Next steps will be to assess alterations in myoblast apoptosis and cell division with EtOH. Future work based on the results of these experiments will examine possible nutraceutical supplements and exercise-like stimulation as interventions to rescue alcohol-mediated effects on myoblast proliferation, which may improve subsequent differentiation and therefore regenerative capacity. This work was supported by Texas Tech University (TTU) startup funds and a TTU College of Arts & Sciences Undergraduate Research Experiences grant (DL). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Altered reward processing following sucrose bingeing in male and female mice

ABSTRACT Objectives: Binge eating disorder (BED) is the most prevalent eating disorder associated with multiple adverse health effects, especially mental health issues, including substance use disorders and mood and anxiety disorders. Given these high comorbidities, the objective of our study was to examine whether bingeing behavior would lead to altered perception of reinforcing properties of EtOH and changes in well-being. Methods: We used a sucrose bingeing model based on an intermittent access paradigm with a two-bottle choice, without fasting, in male and female mice. We examined the effect of 2-week sucrose paradigm on ethanol-reinforcing properties using a conditioned place preference test (CPP). Well-being, anxiety- and depressive-like behavioral tests were performed to assess emotional state following 2 and 8-week sucrose bingeing paradigm. Results: Mice with intermittent access to sucrose developed a binge-like behavior assessed by higher sucrose intake and escalation rate during the 1st hour of access, in comparison with mice with a continuous sucrose access. We show for the first time that sucrose bingeing in mice modifies positive reinforcing effect of EtOH in a CPP paradigm without marked alteration of emotional state. Interestingly, prolonging sucrose access for 8 weeks revealed an exacerbated bingeing behavior in female mice, and some signs of emotional state alterations in female with continuous access. Discussion : In sum, our findings broaden the understanding of behavioral alterations associated with bingeing, highlighting the need to investigate addictive-like properties of palatable food both in male and female mice.

The effect of STAT1, miR-99b, and MAP2K1 in alcoholic liver disease (ALD) mouse model and hepatocyte.

Alcoholic liver disease (ALD) serves as the leading cause of chronic liver diseases-related morbidity and mortality, which threatens the life of millions of patients in the world. However, the molecular mechanisms underlying ALD progression remain unclear. Here, we applied microarray analysis and experimental approaches to identify miRNAs and related regulatory signaling that associated with ALD. Microarray analysis identified that the expression of miR-99b was elevated in the ALD mouse model. The AML-12 cells were treated with EtOH and the expression of miR-99b was enhanced in the cells. The expression of miR-99b was positively correlated with ALT levels in the ALD mice. The microarray analysis identified the abnormally expressed mRNAs in ALD mice and the overlap analysis was performed with based on the differently expressed mRNAs and the transcriptional factors of miR-99b, in which STAT1 was identified. The elevated expression of STAT1 was validated in ALD mice. Meanwhile, the treatment of EtOH induced the expression of STAT1 in the AML-12 cells. The expression of STAT1 was positively correlated with ALT levels in the ALD mice. The positive correlation of STAT1 and miR-99b expression was identified in bioinformatics analysis and ALD mice. The expression of miR-99b and pri-miR-99b was promoted by the overexpression of STAT1 in AML-12 cells. ChIP analysis confirmed the enrichment of STAT1 on miR-99b promoter in AML-12 cells. Next, we found that the expression of mitogen-activated protein kinase kinase 1 (MAP2K1) was negatively associated with miR-99b. The expression of MAP2K1 was downregulated in ALD mice. Consistently, the expression of MAP2K1 was reduced by the treatment of EtOH in AML-12 cells. The expression of MAP2K1 was negative correlated with ALT levels in the ALD mice. We identified the binding site of MAP2K1 and miR-99b. Meanwhile, the treatment of miR-99b mimic repressed the luciferase activity of MAP2K1 in AML-12 cells. The expression of MAP2K1 was suppressed by miR-99b in the cells. We observed that the expression of MAP2K1 was inhibited by the overexpression of STAT1 in AML-12 cells. Meanwhile, the apoptosis of AML-12 cells was induced by the treatment of EtOH, while miR-99b mimic promoted but the overexpression of MAP2K1 attenuated the effect of EtOH in the cells. In conclusion, we identified the correlation and effect of STAT1, miR-99b, and MAP2K1 in ALD mouse model and hepatocyte. STAT1, miR-99b, and MAP2K1 may serve as potential therapeutic target of ALD.

Biomarkers of Affective Dysregulation Associated with In Utero Exposure to EtOH.

Children with fetal alcohol spectrum disorders (FASD) exhibit behavioral and affective dysregulation, including hyperactivity and depression. The mechanisms are not known, but they could conceivably be due to postnatal social or environmental factors. However, we postulate that, more likely, the affective dysregulation is associated with the effects of EtOH exposure on the development of fetal serotonergic (5-HT) and/or dopaminergic (DA) pathways, i.e., pathways that in postnatal life are believed to regulate mood. Many women who use alcohol (ethanol, EtOH) during pregnancy suffer from depression and take selective serotonin reuptake inhibitors (SSRIs), which might influence these monoaminergic pathways in the fetus. Alternatively, monoaminergic pathway abnormalities might reflect a direct effect of EtOH on the fetal brain. To distinguish between these possibilities, we measured their expressions in fetal brains and in fetal brain-derived exosomes (FB-Es) isolated from the mothers' blood. We hypothesized that maternal use of EtOH and/or SSRIs during pregnancy would be associated with impaired fetal neural development, detectable as abnormal levels of monoaminergic and apoptotic biomarkers in FB-Es. Fetal brain tissues and maternal blood were collected at 9-23 weeks of pregnancy. EtOH groups were compared with unexposed controls matched for gestational age (GA). The expression of 84 genes associated with the DA and 5-HT pathways was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) on microarrays. FB-Es also were assayed for serotonin transporter protein (SERT) and brain-derived neurotrophic factor (BDNF) by enzyme-linked immunosorbent assay (ELISA). Six EtOH-exposed human fetal brain samples were compared to SSRI- or polydrug-exposed samples and to unexposed controls. EtOH exposure was associated with significant upregulation of DA receptor D3 and 5-HT receptor HTR2C, while HTR3A was downregulated. Monoamine oxidase A (MAOA), MAOB, the serine/threonine kinase AKT3, and caspase-3 were upregulated, while mitogen-activated protein kinase 1 (MAPK1) and AKT2 were downregulated. ETOH was associated with significant upregulation of the DA transporter gene, while SERT was downregulated. There were significant correlations between EtOH exposure and (a) caspase-3 activation, (b) reduced SERT protein levels, and (c) reduced BDNF levels. SSRI exposure independently increased caspase-3 activity and downregulated SERT and BDNF. Early exposure to EtOH and SSRI together was associated synergistically with a significant upregulation of caspase-3 and a significant downregulation of SERT and BDNF. Reduced SERT and BDNF levels were strongly correlated with a reduction in eye diameter, a somatic manifestation of FASD. Maternal use of EtOH and SSRI during pregnancy each was associated with changes in fetal brain monoamine pathways, consistent with potential mechanisms for the affective dysregulation associated with FASD.

Alcohol-Induced Conformation Changes and Thermodynamic Signatures in the Binding of Polyphenols to Proline-Rich Salivary Proteins.

The first contact of polyphenols (tannins) with the human body occurs in the mouth, where they are known to interact with proline-rich proteins (PRPs). These interactions are important at a sensory level, especially for the development of astringency, but affect also various other biochemical processes. Employing thermodynamic measurements, fluorescence and CD spectroscopy, we investigated the binding process of the prototypical polyphenol ellagic acid (EA) to different IB-PRPs and BSA, also in the presence of ethanol, which is known to influence tannin-protein interactions. Binding of EA to BSA and the small peptide IB7-14 is weak, but very strong to IB9-37. The differences in binding strength and stoichiometry are due to differences in the binding motifs, which also lead to differences in the thermodynamic signatures of the binding process. EA binding to BSA is enthalpy-driven, whereas binding to both IB7-14 and IB9-37 is entropy-driven. The presence of 10 vol.% EtOH, as present in wines, increases the binding constant of EA with BSA and IB7-14 drastically, but not that with IB9-37; however, it changes the binding stoichiometry. These differences can be attributed to the effect of EtOH on the conformation dynamics of the proteins and to changes in hydration properties in alcoholic solution.

NAP-XPS study of surface chemistry of CO and ethanol sensing with WO3 nanowires-based gas sensor

The nanowires prepared on a commercial Al2O3-based sensor platform were studied by near ambient pressure X-ray photoelectron spectroscopy (NAP-XPS) upon detecting CO and ethanol. Our findings indicated that the ex-situ prepared WO3 NWs-based gas sensor might not be suitable for NAP-XPS studies, as the surfaces tend to accumulate significant carbon contamination, which remains on the surface even after long-term annealing in mbar range oxygen atmosphere at 593 K. To address this challenge, we developed a methodology involving exposure of the sensor to NOx, which effectively cleans the surface and enables the detection of subtle chemical changes upon exposure to various carbon-containing analytes. Using NAP-XPS analysis, we investigated the gas sensing mechanisms of WO3 NWs for CO and ethanol at different temperatures, revealing that it might combine several mechanisms commonly observed in metal-oxide chemiresistors. It is shown that the response to CO rather arises from the quick interaction of CO molecules with the ionosorbed oxygen. In the case of ethanol sensing, the main driving force for the sensor macroscopic electrical response at low temperatures is the adsorption of ethanol molecules. In contrast, at high temperatures, it combines the adsorption of ethoxy groups with the strong reducing effect of EtOH on the WO3 surface.

Psidium guajava L.: Chemical composition and protective effects of a leaf extract against ethanol-induced cardiotoxicity

It has been previously reported that a leaf extract of Psidium guajava L. (GV.E) can impart numerous health benefits that were attributed to its antioxidant and anti-inflammatory effects. Ethanol (EtOH, alcohol) misuse is a global health burden, with profound effects on the younger population. The toxic effects of EtOH, that are largely attributed to oxidative stress, target a variety of organs including the heart. The current study tested the protective effects of two different doses of GV.E (1 and 2 g/kg/d) on EtOH-induced cardiac injury in 8-10 (post-pubertal) weeks-old male rats. The plant metabolites of the extract were profiled using HPLC-PDA-ESI-MS, and fifty-one metabolites, mostly polyphenols, were tentatively identified. Importantly, the EtOH-induced increase in heart weight and heart coefficient (heart/body weight ratio) were mitigated by the GV.E at both tested doses. Additionally, the EtOH-induced increase in the selective heart injury marker; creatine kinase-m b, and the non-selective tissue injury marker, lactate dehydrogenase, were attenuated by the GV.E (1 g/kg/d). The EtOH-induced increase in serum aspartate aminotransferase and c-reactive protein were normalized by GV.E (2 g/kg/d). Cardiac lipid peroxidation, nitrates, and nitrites (markers of oxidative stress) were reduced by GV.E. However, cardiac reduced glutathione was not affected by EtOH, which could be due to upregulation of the body's defense mechanisms. GV.E also caused a suppression of the cardiac mRNA expression of two inflammatory cytokines; interleukin-6 and tumor necrosis factor (TNF)-α, which were upregulated by EtOH, with a normalization of TNF-α by GV.E (1 g/kg/d). Taken together, these results indicate that GV.E exerts cardio-protective effects against EtOH-induced cardiac damage in male rats.

GAD65 deficient mice are susceptible to ethanol-induced impairment of motor coordination and facilitation of cerebellar neuronal firing.

γ-aminobutyric acid (GABA) is a major inhibitory neurotransmitter and its concentrations in the brain could be associated with EtOH-induced impairment of motor coordination. GABA is synthesized by two isoforms of glutamate decarboxylase (GAD): GAD65 and GAD67. Mice deficient in GAD65 (GAD65-KO) can grow up to adulthood, and show that GABA concentration in their adult brains was 50-75% that of wild-type C57BL/6 mice (WT). Although a previous study showed that there was no difference in recovery from the motor-incoordination effect of acute intraperitoneally administered injections of 2.0 g/kg EtOH between WT and GAD65-KO, the sensitivity of GAD65-KO to acute EtOH-induced ataxia has not been fully understood. Here, we sought to determine whether motor coordination and spontaneous firing of cerebellar Purkinje cells (PCs) in GAD65-KO are more sensitive to the effect of EtOH than in WT. Motor performance in WT and GAD65-KO was examined by rotarod and open-field tests following acute administration of EtOH at lower-doses, 0.8, 1.2 and 1.6 g/kg. In a rotarod test, there was no significant difference between WT and GAD65-KO in terms of baseline motor coordination. However, only the KO mice showed a significant decrease in rotarod performance of 1.2 g/kg EtOH. In the open-field test, GAD65-KO showed a significant increase in locomotor activity after 1.2 and 1.6 g/kg EtOH injections, but not WT. In in vitro studies of cerebellar slices, the firing rate of PCs was increased by 50 mM EtOH in GAD65-KO compared with WT, whereas no difference was observed in the effect of EtOH at more than 100 mM between the genotypes. Taken together, GAD65-KO are more susceptible to the effect of acute EtOH exposure on motor coordination and PC firing than WT. This different sensitivity could be attributed to the basal low GABA concentration in the brain of GAD65-KO.

Abstract 4247: Impact of 70% ethanol intraductal injections in MNU rat models for breast cancer prevention

Abstract Breast cancer (BC) is the leading cancer diagnosis in women. While many BC treatment options exist, only two methods are approved for prevention. Prophylactic mastectomy is an invasive surgery that removes the breast, thereby removing the targeted epithelial ductal tree cells, where most BC arises. Despite a 90% risk reduction, recovery time, risk of infection, pain management, and mental health toll on women deter them from this surgery. Hormonal preventatives, tamoxifen and raloxifene, reduce BC risk by up to 50%. However, increased risk of uterine cancer and intolerable sides effect deter women from this option. Therefore, new preventative methods are needed to maintain BC risk reduction while minimizing current BC prevention deterrents. Intraductal (ID) injection is a technique that can circumvent systemic toxicity by inserting a needle directly into the ductal tree opening. We study BC prevention using 70% ethanol (EtOH) as a cell killing, ablative solution for ID injections and have shown its effectiveness of ablation in an aggressive genetically engineered mouse model with minimum collateral tissue damage. EtOH is a safe, inexpensive reagent clinically used as a sclerosing and ablative agent. We introduced ethyl cellulose (EC), a gelling agent which further minimized damage outside the ductal tree. To confirm successful ductal tree filling without animal sacrifice, we introduced Tantalum Oxide (TaOx), a nanoparticle-based contrast agent for real time and long-term visualization of ductal tree filling. TaOx showed remarkable local retention, without impeding 70% EtOH ablative effects or EC compared to other contrast agents. Despite our refined ablative solution, ID injection is a difficult technique to perform with limited ability to confirm success of a fully filled ductal tree. To elucidate any impact of partial ductal tree filling, we utilized a Methyl-N-Nitrosourea (MNU) rat model. Rat models improve scalability toward clinical trials and increase ductal tree filling volume and control to study the impact of partial ductal tree filling. Our MNU rat model provides a time frame between carcinogen exposure and tumor formation optimal for prevention studies. Two weeks after intraperitoneal MNU injections, rats received full or half volume (partial) injections, fat pad (partial), or no injection into individual mammary glands. Rats were monitored for tumor formation from 2 weeks post injection until reaching euthanasia criteria. Fully injected glands had the highest tumor latency increase by 1 month (130 days, p < 0.0001) compared to non-injected controls (95 days). Partial and fat pad tumor latency also improved compared to control (122 days, p <0.01 and 110 days, p <0.05 respectively). No iatrogenic effects of EtOH were observed. Here, we show that ID injection of 70% EtOH is a safe, effective, and scalable technique for BC prevention, which can be enhanced with TaOx and EC for visualization and retention. Citation Format: Erin K. Zaluzec, Mohamed Ashry, Elizabeth Kenyon, Elizabeth G. Phelps, Legend Kenney, Katherine Powell, Maximilian Volk, Shatadru Chakravarty, Jeremy M.L. Hix, Matti Kiupel, Erik Shapiro, Lorenzo F. Sempere. Impact of 70% ethanol intraductal injections in MNU rat models for breast cancer prevention. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4247.

Sex chromosome and gonadal hormone contributions to binge-like and aversion-resistant ethanol drinking behaviors in Four Core Genotypes mice.

While substantial research has focused on the contribution of sex hormones to driving elevated levels of alcohol drinking in female rodents, fewer studies have investigated how genetic influences may underlie sex differences in this behavior. We used the Four Core Genotypes (FCG) mouse model to explore the contribution of sex chromosome complement (XX/XY) and gonad type [ovaries (Sry-)/testes (Sry+)] to ethanol (EtOH) consumption and quinine-resistant drinking across two voluntary self-administration tasks: limited access consumption in the home cage and an operant response task. For limited access drinking in the dark, XY/Sry + (vs. XX/Sry +) mice consumed more 15% EtOH across sessions while preference for 15% EtOH vs. water was higher in XY vs. XX mice regardless of gonad type. XY chromosomes promoted quinine-resistant drinking in mice with ovaries (Sry-) and the estrous cycle did not affect the results. In the operant response task, responding for EtOH was concentration dependent in all genotypes except XX/Sry + mice, which maintained consistent response levels across all concentrations (5-20%) of EtOH. When increasing concentrations of quinine (100-500 μM) were added to the solution, FCG mice were insensitive to quinine-punished EtOH responding, regardless of sex chromosome complement. Sry + mice were further found to be insensitive to quinine when presented in water. Importantly, these effects were not influenced by sensitivity to EtOH's sedative effect, as no differences were observed in the time to lose the righting reflex or the time to regain the righting reflex between genotypes. Additionally, no differences in EtOH concentration in the blood were observed between any of the genotypes once the righting reflex was regained. These results provide evidence that sex chromosome complement regulates EtOH consumption, preference, and aversion resistance and add to a growing body of literature suggesting that chromosomal sex may be an important contributor to alcohol drinking behaviors. Examination of sex-specific genetic differences may uncover promising new therapeutic targets for high-risk drinking.

Role of α6-Nicotinic Receptors in Alcohol-Induced GABAergic Synaptic Transmission and Plasticity to Cholinergic Interneurons in the Nucleus Accumbens.

The prevailing view is that enhancement of dopamine (DA) transmission in the mesolimbic system, consisting of DA neurons in the ventral tegmental area (VTA) that project to the nucleus accumbens (NAc), underlies the reward properties of ethanol (EtOH) and nicotine (NIC). We have shown previously that EtOH and NIC modulation of DA release in the NAc is mediated by α6-containing nicotinic acetylcholine receptors (α6*-nAChRs), that α6*-nAChRs mediate low-dose EtOH effects on VTA GABA neurons and EtOH preference, and that α6*-nAChRs may be a molecular target for low-dose EtOH. However, the most sensitive target for reward-relevant EtOH modulation of mesolimbic DA transmission and the involvement of α6*-nAChRs in the mesolimbic DA reward system remains to be elucidated. The aim of this study was to evaluate EtOH effects on GABAergic modulation of VTA GABA neurons and VTA GABAergic input to cholinergic interneurons (CINs) in the NAc. Low-dose EtOH enhanced GABAergic input to VTA GABA neurons that was blocked by knockdown of α6*-nAChRs. Knockdown was achieved either by α6-miRNA injected into the VTA of VGAT-Cre/GAD67-GFP mice or by superfusion of the α-conotoxin MII[H9A;L15A] (MII). Superfusion of MII blocked EtOH inhibition of mIPSCs in NAc CINs. Concomitantly, EtOH enhanced CIN firing rate, which was blocked by knockdown of α6*-nAChRs with α6-miRNA injected into the VTA of VGAT-Cre/GAD67-GFP mice. The firing rate of CINs was not enhanced by EtOH in EtOH-dependent mice, and low-frequency stimulation (LFS; 1Hz, 240 pulses) caused inhibitory long-term depression at this synapse (VTA-NAc CIN-iLTD) which was blocked by knockdown of α6*-nAChR and MII. Ethanol inhibition of CIN-mediated evoked DA release in the NAc was blocked by MII. Taken together, these findings suggest that α6*-nAChRs in the VTA-NAc pathway are sensitive to low-dose EtOH and play a role in plasticity associated with chronic EtOH.

Alcohol and energy drinks: individual contribution of common ingredients on ethanol-induced behaviour.

Since energy drinks (EDs) were sold to the general public as soft drinks and recreational beverages, mixing EDs with ethanol has grown in popularity, particularly among younger people. Given the research that links these drinks with higher risk behaviors and increased ethanol intake, ethanol combined with EDs (AmEDs) is a particularly worrying combination. EDs generally commonly include a variety of ingredients. Sugar, caffeine, taurine, and B-group vitamins are almost always present. Studies on the combined effect of ethanol and sugar and caffeine on ethanol-induced behaviors are extensive. Not so much in regards to taurine and vitamins. This review briefly summarises available information from research on the isolated compounds on EtOH-induced behaviors first, and secondly, the combination of AmEDs on EtOH effects. The conclusion is that additional research is needed to fully comprehend the characteristics and consequences of AmEDs on EtOH-induced behaviors.

Revisiting the Solubility–Permeability Relationship with Hydrophobic Drug Umifenovir in Pluronic Solutions: Impact of pH and Co-Solvent

This study describes the influence of pluronic F-127 (F-127) and ethanol (EtOH) on the solubility of umifenovir (UMF) in buffer solutions of pH 2.0 and pH 7.4, and its permeability through cellulose membranes. A 44.4-fold greater UMF solubility in acidic medium as compared to an alkaline one was estimated at 310.15 K. The concentration of UMF in the saturated solution was enhanced by the interaction with F-127 micelles. The combined positive effect of EtOH and F-127 on the solubility was estimated. The aggregation number of F-127 micelles in the presence of 10% and 20% ethanol appeared to be reduced by 2.1-fold and 4.1-fold, respectively, as compared to buffer pH 7.4. The presence of ethanol in buffer pH 7.4 solution provided better solvent conditions but inhibited the formation of F-127 micelles. The impact of UMF on the aggregation number of F-127 was not pronounced and was expressed only by a slight increase of 1 and 3 units in 10% and 20% EtOH, respectively. According to the values of zeta potential, addition of EtOH reduced the stability of the system. The permeation of UMF in buffer pH 7.4 measured through the cellulose membrane MWCO 12-14 kDa was increased 1.4-fold by 10% EtOH. An increase in EtOH content to 20% reduced this effect to 1.2-fold. Decreasing effect of 1.5% F-127 on the permeability was inhibited by using 10% EtOH. The solution containing 1.5% F-127 and 10% EtOH was shown to be an advantageous system for UMF in view of the solubility-permeability balance. The authors suppose the findings of the study to be useful for the design of pharmaceutical formulations based on UMF antiviral drugs.

Bi-directional modulation of hyperpolarization-activated cation currents (Ih) by ethanol in rat hippocampal CA3 pyramidal neurons

It is widely acknowledged that ethanol (EtOH) can alter many neuronal functions, including synaptic signaling, firing discharge, and membrane excitability, through its interaction with multiple membrane proteins and intracellular pathways. Previous work has demonstrated that EtOH enhances the firing rate of hippocampal GABAergic interneurons and thus the presynaptic GABA release at CA1 and CA3 inhibitory synapses through a positive modulation of the hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels. Activation of HCN channels produce an inward current, commonly called Ih, which plays an essential role in generating/regulating specific neuronal activities in GABAergic interneurons and principal glutamatergic pyramidal neurons such as those in the CA3 subregion. Since the direct effect of EtOH on HCN channels expressed in CA3 pyramidal neurons was not thoroughly elucidated, we investigated the possible interaction between EtOH and HCN channels and the impact on excitability and postsynaptic integration of these neurons. Patch-clamp recordings were performed in single CA3 pyramidal neurons from acute male rat coronal hippocampal slices. Our results show that EtOH modulates HCN-mediated Ih in a concentration-dependent and bi-directional manner, with a positive modulation at lower (20 mM) and an inhibitory action at higher (60–80 mM) concentrations. The modulation of Ih by EtOH was mimicked by forskolin, antagonized by different drugs that selectively interfere with the AC/cAMP/PKA intracellular pathway, as well as by the selective HCN inhibitor ZD7288. Altogether, these data further support the evidence that HCN channels may represent an important molecular target through which EtOH may regulate neuronal activity.

Caveolin-1 inhibition mediates the opposing effects of alcohol on γ-secretase activity in arterial endothelial and smooth muscle cells.

Notch is important to vessel homeostasis. We investigated the mechanistic role of caveolin-1 (Cav-1) in mediating the effects of alcohol (Ethanol/EtOH) on the γ-secretase proteolytic activity necessary for Notch signaling in vascular cells. Human coronary artery endothelial cells (HCAEC) were treated with EtOH (0-50 mM), Notch ligand delta-like ligand 4 (Dll4), and the γ-secretase inhibitor DAPT. EtOH stimulated Notch signaling in HCAEC as evidenced by increased Notch receptor (N1, N4) and target gene (hrt2, hrt3) mRNA levels with the most robust response achieved at 25 mM EtOH. Ethanol (25 mM) stimulated γ-secretase proteolytic activity, to the same extent as Dll4, in HCAEC membranes. Ethanol inhibited Cav-1 mRNA and protein levels in HCAEC. Caveolin-1 negatively regulated γ-secretase activity in HCAEC as Cav-1 knockdown stimulated it, while Cav-1 overexpression inhibited it. Moreover, Cav-1 overexpression blocked the stimulatory effect of EtOH on γ-secretase activity in HCAEC. Although EtOH also inhibited Cav-1 expression in human coronary artery smooth muscle cells (HCASMC), EtOH inhibited γ-secretase activity in HCASMC in contrast to its effect in HCAEC. The inhibitory effect of EtOH on γ-secretase in HCASMC was mimicked by Cav-1 knockdown and prevented by Cav-1 overexpression, suggesting that in these cells Cav-1 positively regulates γ-secretase activity. In conclusion, EtOH differentially regulates γ-secretase activity in arterial EC and SMC, being stimulatory and inhibitory, respectively. These effects are both mediated by caveolin-1 inhibition which itself has opposite effects on γ-secretase in the two cell types. This mechanism may underlie, in part, the effects of moderate drinking on atherosclerosis.

Synaptic Effects Induced by Alcohol.

Ethanol (EtOH) has effects on numerous cellular molecular targets, and alterations in synaptic function are prominent among these effects. Acute exposure to EtOH activates or inhibits the function of proteins involved in synaptic transmission, while chronic exposure often produces opposing and/or compensatory/homeostatic effects on the expression, localization, and function of these proteins. Interactions between different neurotransmitters (e.g., neuropeptide effects on release of small molecule transmitters) can also influence both acute and chronic EtOH actions. Studies in intact animals indicate that the proteins affected by EtOH also play roles in the neural actions of the drug, including acute intoxication, tolerance, dependence, and the seeking and drinking of EtOH. The present chapter is an update of our previous Lovinger and Roberto (Curr Top Behav Neurosci 13:31-86, 2013) chapter and reviews the literature describing these acute and chronic synaptic effects of EtOH with a focus on adult animals and their relevance for synaptic transmission, plasticity, and behavior.

Impact of alcohol exposure on neural development and network formation in human cortical organoids

Prenatal alcohol exposure is the foremost preventable etiology of intellectual disability and leads to a collection of diagnoses known as Fetal Alcohol Spectrum Disorders (FASD). Alcohol (EtOH) impacts diverse neural cell types and activity, but the precise functional pathophysiological effects on the human fetal cerebral cortex are unclear. Here, we used human cortical organoids to study the effects of EtOH on neurogenesis and validated our findings in primary human fetal neurons. EtOH exposure produced temporally dependent cellular effects on proliferation, cell cycle, and apoptosis. In addition, we identified EtOH-induced alterations in post-translational histone modifications and chromatin accessibility, leading to impairment of cAMP and calcium signaling, glutamatergic synaptic development, and astrocytic function. Proteomic spatial profiling of cortical organoids showed region-specific, EtOH-induced alterations linked to changes in cytoskeleton, gliogenesis, and impaired synaptogenesis. Finally, multi-electrode array electrophysiology recordings confirmed the deleterious impact of EtOH on neural network formation and activity in cortical organoids, which was validated in primary human fetal tissues. Our findings demonstrate progress in defining the human molecular and cellular phenotypic signatures of prenatal alcohol exposure on functional neurodevelopment, increasing our knowledge for potential therapeutic interventions targeting FASD symptoms.

Preparation of hydrophobic Ti-beta catalyst derived from Al-rich zeolite beta synthesized without using organic structure-directing agent and its enhanced catalytic performance for phenol oxidation

Crystalline microporous titanosilicates with multi-dimensional 12-ring channels are potentially useful as catalysts for the oxidation of phenol with H2O2. Among them, titanosilicate version of zeolite beta (Ti-beta), which has three-dimensional, 12-ring micropores, is one of the most promising catalytic materials. The Ti-beta catalysts are preferably with Al-free and Na-free chemical compositions, and they can be conventionally crystallized via direct hydrothermal synthesis with the aid of a dipiperidinium-type organic structure-directing agent (OSDA) or via the so-called fluoride method using tetraethylammonium in a fluoride-containing medium. Another route involves a post-synthesis treatment such as the isomorphous substitution of Ti for Al starting from Al-rich zeolite beta. One of the Al-rich beta was synthesized in the absence of OSDA. We succeeded in converting it to Ti-beta, denoted as (Ti)-beta_OSDAF, for the first time via deep dealumination along with framework stabilization followed by Ti-incorporation using TiCl4. The (Ti)-beta_OSDAF was much more effective catalyst for the phenol oxidation with H2O2 than the directly crystallized Ti-beta materials prepared in the conventional techniques. The high efficiency of phenol oxidation was ascribed to an appropriate balance between hydrophilicity and hydrophobicity realized by increasing hydrophobicity of titanosilicate catalysts. The bulk hydrophobicity reflects that of the vicinity of active Ti sites. The enhanced affinity of hydrophobic moiety of phenol molecules with the hydrophobic surface around the vicinity of active Ti sites should facilitate the diffusion of phenol molecules to the active sites inside the micropores. The hydrophobicity of Ti-beta samples prepared by the various techniques in this work differed substantially, depending on the amount of silanol groups within the zeolitic framework. Positive effect of EtOH as an additive in the reaction mixture on enhancing the shape-selectivity of the (Ti)-beta_OSDAF catalyst was obvious, probably due to the increase in steric restriction by the EtOH molecules inside the pores of the catalyst. Moreover, the phenylsilylation of (Ti)-beta_OSDAF catalyst drastically increased its hydrophobicity, which was found to be effective for enhancing para-selectivity and turnover number.