Dose Of EtOH Research Articles

Dysregulated hepatic alcohol metabolism: a key factor involved in the pathogenesis of alcohol-associated liver disease.

Alcohol use disorder is a major risk factor for alcohol-associated liver disease (ALD), characterized by reduced hepatic alcohol dehydrogenase (ADH) activity, increased body burden of alcohol and its nonoxidative metabolism to fatty acid ethyl esters (FAEEs). However, the mechanism(s) underlying ALD remain unclear. This study investigated metabolic basis and mechanism(s) of ALD in chronic ethanol (EtOH) fed hepatic ADH1-deficient (ADH-) deer mice administered with a single dose of binge EtOH with/without FAEEs. Hepatic ADH- and ADH normal (ADH+) deer mice fed chronic EtOH daily for 3 months, followed by a single dose of binge EtOH [3g/kg body weight (BW)] with/without FAEEs (100 mg/kg BW), one week prior to euthanasia. Blood alcohol and acetaldehyde and liver injury markers in the plasma, hepatic FAEEs, lipids and inflammatory markers were analyzed. Hepatic histology, ultrastructure, protein/mRNA expression of genes involved in alcohol metabolism and lipogenesis, cAMP, phosphodiesterase (PDE) activity, and AMPKα signaling were assessed. Blood alcohol, hepatic lipids and FAEEs, inflammation, oxidative stress, and the expression of lipogenic proteins/genes were significantly increased in various chronic EtOH-fed groups of ADH- vs. ADH+ deer mice. Additionally, hepatic cAMP levels were reduced, while PDE activity and plasma transaminases were elevated. Binge EtOH with/without FAEEs did not significantly exacerbate the liver injury in chronic EtOH-fed ADH- as well as ADH+ deer mice. Overall, an increased body burden of EtOH and endogenously formed FAEEs due to hepatic ADH deficiency, along with dysregulated cAMP and AMPKα signaling, could be the determining factors for EtOH-induced liver injury leading to ALD.

Rosuvastatin repurposing for prophylaxis against ethanol-induced acute gastric ulceration in rats: a biochemical, histological, and ultrastructural perspective.

Ethanol (EtOH) consumption is frequently associated with acute and chronic gastrointestinal disorders. Rosuvastatin (RSV), a third-generation statin, has demonstrated certain biological functions beyond its lipid-lowering properties. This study is designed to explore the gastroprotective impact of RSV in a rat model of EtOH-induced gastric ulceration in a dose-dependent manner through the evaluation of oxidant/antioxidant biomarkers, inflammatory myeloperoxidase (MPO) enzyme activity, and prostaglandin E2 (PGE2) levels in gastric tissues, along with histopathological examination of the gastric tissues. Therefore, 40 adult male rats were randomly divided into five equal groups as control, EtOH (gastric ulcer), RSV-low dose plus EtOH and RSV-high dose plus EtOH. The EtOH rat model of gastric ulceration was achieved by intragastric administration of a single dose of EtOH. Seven days before EtOH administration, rats were orally administered either omeprazole (20mg/kg/day) or RSV (10mg/kg/day or 20mg/kg/day). RSV administration enhanced the antioxidant glutathione reduced, countered oxidative malondialdehyde, augmented cytoprotective PGE2, suppressed inflammatory MPO enzyme activity in gastric tissues, decreased ulcer index scoring, increased the percentage of ulcer inhibition, and reversed the associated histological and ultrastructural abnormalities, additionally, RSV treatment resulted in weak positive nuclear staining for the inflammatory nuclear factor kappa B in a dose-dependent manner. It is concluded that RSV demonstrates gastroprotective potential, attributable at least in part, to its antioxidant and anti-inflammatory properties, as well as its ability to promote ulcer protection through the maintenance of mucosal content and PGE2 levels. Thus, RSV therapy emerges as a safe option for patients with gastric ulcers.

Sex Differences in Alcohol-related Cardiomyopathy

Alcohol-related Cardiomyopathy (ACM) manifests in humans with a significant history of alcohol use and is characterized by ventricular dilation and cardiac function impairment. Although the most common age group for ACM is males between the ages of 30-55, women require a lesser lifetime alcohol exposure to develop ACM than men. The objective of this study is to assess sexual differences in a mouse model of chronic plus binge alcohol consumption. We hypothesize that female mice exposed to chronic plus binge alcohol will not develop as severe cardiac dysfunction as their male counterparts. In this study, male (n=19) and female (n=14) C57BL/6J mice were provided chronic plus binge alcohol feeding. After 5 days of acclimation to the liquid diet, mice are fed ad libitum 5% ethanol (EtOH) liquid or isocaloric control liquid diet for 30 days. At 10 and 30 days, mice received an oral binge dose of EtOH (5 g/kg body weight), or isocaloric maltose dextrin solution (9 g/kg body weight). Noninvasive cardiac structural and functional data were obtained via echocardiography at baseline and following binges. After 30 days, cardiac hemodynamics was measured by open chest left ventricle catheterization. Two-way ANOVA with Tukey’s post hoc analysis was performed for each cardiac parameter, and significance was defined as p<0.05. Male EtOH mice exhibited a significant decrease in stroke work (1119 ± 297 mmHg/mL when compared to male controls, 1622 ± 365 mmHg/mL; p= 0.03). Female EtOH mice had a nonsignificant decrease in stroke work (1242 ± 374 mmHg/mL when compared to female controls, 1423 ± 469 mmHg/mL; p= 0.80). Maximum left ventricular pressure was decreased 20% in male EtOH mice (p=0.003) but increased 10% in female EtOH mice (p=0.46). The maximum rate of pressure change within the ventricle was 24% lower in the male EtOH mice (p=0.02) and 9% higher in female EtOH mice (p=0.87). The minimum rate of pressure change within the ventricle was 24% higher in male EtOH mice (p=0.02) and 17% lower in female ETOH mice (p=0.44). After 30 days of EtOH exposure, male mice showed significant systolic and diastolic dysfunction. Female mice on the other hand, did not show substantial cardiac dysfunction after chronic plus binge ethanol exposure. Further experiments involving qPCR of proinflammatory and profibrotic markers will elucidate the molecular mechanism of sex differences in ACM development. R21AA029747 (JG & CB) & T32AA007577. 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 Epigenetic Marks and Gene Expression in Fetal Brain, and Postnatal Behavioural Disorders, Following Prenatal Exposure of Ogg1 Knockout Mice to Saline or Ethanol.

Oxoguanine glycosylase 1 (OGG1) is widely known to repair the reactive oxygen species (ROS)-initiated DNA lesion 8-oxoguanine (8-oxoG), and more recently was shown to act as an epigenetic modifier. We have previously shown that saline-exposed Ogg1 -/- knockout progeny exhibited learning and memory deficits, which were enhanced by in utero exposure to a single low dose of ethanol (EtOH) in both Ogg1 +/+ and -/- progeny, but more so in Ogg1 -/- progeny. Herein, OGG1-deficient progeny exposed in utero to a single low dose of EtOH or its saline vehicle exhibited OGG1- and/or EtOH-dependent alterations in global histone methylation and acetylation, DNA methylation and gene expression (Tet1 (Tet Methylcytosine Dioxygenase 1), Nlgn3 (Neuroligin 3), Hdac2 (Histone Deacetylase 2), Reln (Reelin) and Esr1 (Estrogen Receptor 1)) in fetal brains, and behavioural changes in open field activity, social interaction and ultrasonic vocalization, but not prepulse inhibition. OGG1- and EtOH-dependent changes in Esr1 and Esr2 mRNA and protein levels were sex-dependent, as was the association of Esr1 gene expression with gene activation mark histone H3 lysine 4 trimethylation (H3K4me3) and gene repression mark histone H3 lysine 27 trimethylation (H3K27me3) measured via ChIP-qPCR. The OGG1-dependent changes in global epigenetic marks and gene/protein expression in fetal brains, and postnatal behavioural changes, observed in both saline- and EtOH-exposed progeny, suggest the involvement of epigenetic mechanisms in developmental disorders mediated by 8-oxoG and/or OGG1. Epigenetic effects of OGG1 may be involved in ESR1-mediated gene regulation, which may be altered by physiological and EtOH-enhanced levels of ROS formation, possibly contributing to sex-dependent developmental disorders observed in Ogg1 knockout mice. The OGG1- and EtOH-dependent associations provide a basis for more comprehensive mechanistic studies to determine the causal involvement of oxidative DNA damage and epigenetic changes in ROS-mediated neurodevelopmental disorders.

Sexual dimorphic vulnerability of respiratory control in a neonatal rodent model of fetal alcohol spectrum disorder

Fetal alcohol spectrum disorder (FASD) has been linked to numerous poor neurological outcomes as well as impairments in respiratory neural control. Females are known to metabolize ethanol (EtOH) differently than males suggesting a sexual dimorphic sensitivity to EtOH exposure. We used a rodent model of FASD to investigate whether EtOH disrupts respiratory neural control. Rat pups received a single intraperitoneal injection of 2 different doses (0.8 mg/g or 4.4 mg/g) of EtOH. Whole-body plethysmography was used ∼24 h later to assess ventilatory responses to acute hypoxia (HVR) and hypercapnia (HCVR). Females treated with 4.4 mg/g of EtOH exhibited an attenuated HVR and HCVR, but there was no effect on males, and no effect of 0.8 mg/g on either sex. There was unexpected mortality of unknown causes, especially in females, that occurred 2–3 days after EtOH administration. These data suggest that important ventilatory defense responses in females are impaired following developmental EtOH exposure, and this may be associated with increased risk of later death.

Gastroprotective effect of dapagliflozin in ethanol-induced gastric lesions in rats: Crosstalk between HMGB1/RAGE/PTX3 and TLR4/MyD88/VEGF/PDGF signaling pathways.

Alcohol abuse may lead to the development of gastric mucosal lesions. Dapagliflozin (DAPA), a sodium-glucose cotransporter-2 inhibitor, is clinically used to treat type 2 diabetes mellitus. However, studies showed protective effect of DAPA under various experimental conditions by alleviating oxidative stress and inflammation. The effect of DAPA on experimental gastric ulcer has not been studied yet. Therefore, we attempted to investigate DAPA's protective effect against ethanol (EtOH)-induced gastric lesions. Fifty-six (8-week-old) male Wistar rats were divided into seven groups. DAPA (1, 5, and 10mg/kg/day; p.o.) was given for seven days, plus a single dose of absolute EtOH (5ml/kg) on day 8. According to hematoxylin and eosin, and Alcian blue staining of gastric tissue sections, titratable acidity, and macroscopic assessments, DAPA high dose (10mg/kg) was the most protective, with lesser ulcerations, and higher mucin, relative to the lower two doses and the standard treatment omeprazole (OME). In rats pre-treated with DAPA high dose, colorimetric and ELISA analyses revealed significantly decreased oxidative stress, pro-inflammatory, and apoptosis indices and increased levels of platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). Western blot analysis revealed reduced pentraxin-3 (PTX3), high-mobility group box 1 (HMGB1), receptor for advanced glycation end products (RAGE), toll-like receptor 4 (TLR4), and myeloid differentiation factor 88 (MyD88) expression. These results were comparable in DAPA (10mg/kg) and OME pre-treated groups. Overall, DAPA exerted a dose-dependent protective effect against EtOH-induced gastric injury. Gastroprotective effects of DAPA (10mg/kg) may be associated with influencing HMGB1/RAGE/PTX3 and TLR4/MyD88/VEGF/PDGF pathways.

Mechanical Stimulation Alters Chronic Ethanol-Induced Changes to VTA GABA Neurons, NAc DA Release and Measures of Withdrawal.

Therapeutic activation of mechanoreceptors (MStim) in osteopathy, chiropractic and acupuncture has been in use for hundreds of years with a myriad of positive outcomes. It has been previously shown to modulate the firing rate of neurons in the ventral tegmental area (VTA) and dopamine (DA) release in the nucleus accumbens (NAc), an area of interest in alcohol-use disorder (AUD). In this study, we examined the effects of MStim on VTA GABA neuron firing rate, DA release in the NAc, and behavior during withdrawal from chronic EtOH exposure in a rat model. We demonstrate that concurrent administration of MStim and EtOH significantly reduced adaptations in VTA GABA neurons and DA release in response to a reinstatement dose of EtOH (2.5 g/kg). Behavioral indices of EtOH withdrawal (rearing, open-field crosses, tail stiffness, gait, and anxiety) were substantively ameliorated with concurrent application of MStim. Additionally, MStim significantly increased the overall frequency of ultrasonic vocalizations, suggesting an increased positive affective state.

Astrocyte ethanol exposure reveals persistent and defined calcium response subtypes and associated gene signatures

Astrocytes play a critical role in brain function, but their contribution during ethanol (EtOH) consumption remains largely understudied. In light of recent findings on the heterogeneity of astrocyte physiology and gene expression, an approach with the ability to identify subtypes and capture this heterogeneity is necessary. Here, we combined measurements of calcium signaling and gene expression to define EtOH-induced astrocyte subtypes. In the absence of a demonstrated EtOH receptor, EtOH is believed to have effects on the function of many receptors and downstream biological cascades that underlie calcium responsiveness. This mechanism of EtOH-induced calcium signaling is unknown and this study provides the first step in understanding the characteristics of cells displaying these observed responses. To characterize underlying astrocyte subtypes, we assessed the correlation between calcium signaling and astrocyte gene expression signature in response to EtOH. We found that various EtOH doses increased intracellular calcium levels in a subset of astrocytes, distinguishing three cellular response types and one nonresponsive subtype as categorized by response waveform properties. Furthermore, single-cell RNA-seq analysis of astrocytes from the different response types identified type-enriched discriminatory gene expression signatures. Combining single-cell calcium responses and gene expression analysis identified specific astrocyte subgroups among astrocyte populations defined by their response to EtOH. This result provides a basis for identifying the relationship between astrocyte susceptibility to EtOH and corresponding measurable markers of calcium signaling and gene expression, which will be useful to investigate potential subgroup-specific influences of astrocytes on the physiology and pathology of EtOH exposure in the brain.

Gastroprotective and Antioxidant Properties of Trigonella foenum graecum Seeds Aqueous Extract (Fenugreek) and Omeprazole Against Ethanol-Induced Peptic Ulcer.

Trigonella foenum graecum (Fenugreek) is used in traditional phytomedicine for its anti-inflammatory, antiseptic, antidiabetic, and several other therapeutic virtues. The current study was intended to investigate the protecting effects of fenugreek seeds' aqueous extract (FSAE) using experimentally ethanol (EtOH)-induced gastric peptic ulcer in rats, as immense alcohol consumption can lead to gastric ulcer. Sixty adult male Wistar rats were divided into 6 groups of 10 each: control, EtOH (4 g/kg body weight [b.w.]), EtOH + several doses of FSAE (50, 100, and 200 mg/kg b.w.), and EtOH + Omeprazole (OM, 20 mg/kg orally [p.o.]). Animals were p.o. pretreated with FSAE for 21 days and exposed to a single oral administration of EtOH (4 g/kg b.w.) for 2 h. Gastric ulcer in rats was induced with a single dose of EtOH. Ulcer index, malondialdehyde (MDA), hydrogen peroxide (H2O2), and thiol groups (-SH) content in stomach, and antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were measured. Our recorded results showed that EtOH induced gastric damage, evidenced by the level of oxidative stress markers such as MDA and H2O2 in rats exposed to EtOH. However, significant increases in the activities of antioxidant enzymes were recorded, such as SOD, CAT, and GPx, and a decrease in nonenzymatic antioxidants, such as (-SH). Moreover, histopathological examinations showed the presence of lesions associated with severe tissue damage in the untreated rats. Interestingly, FSAE meaningfully protects against all gastric damages caused by EtOH. We propose that FSAE exhibits protective effects in EtOH-induced peptic ulcer in rats. This protection might be related to its antioxidant and anti-inflammatory properties as well as its opposite effects on some studied intracellular mediators.

The peripheral dopamine 2 receptor antagonist domperidone attenuates ethanol enhancement of dopamine levels in the nucleus accumbens.

Dopamine neuron firing in the ventral tegmental area (VTA) and dopamine release in the nucleus accumbens have been implicated in reward learning. Ethanol is known to increase both dopamine neuron firing in the VTA and dopamine levels in the nucleus accumbens. Despite this, some discrepancies exist between the dose of ethanol required to enhance firing in vivo and ex vivo. In the present study we investigated the effects of peripheral dopamine 2 subtype receptor antagonism on ethanol's effects on dopamine neurotransmission. Plasma catecholamine levels were assessed following ethanol administration across four different doses of EtOH. Microdialysis and voltammetry were used to assess the effects of domperidone pretreatment on ethanol-mediated increases in dopamine release in the nucleus accumbens. A place conditioning paradigm was used to assess conditioned preference for ethanol and whether domperidone pretreatment altered this preference. Open-field and loss-of-righting reflex paradigms were used to assess the effects of domperidone on ethanol-induced sedation. A rotarod apparatus was used to assess the effects of domperidone on ethanol-induced motor impairment. Domperidone attenuated ethanol's enhancement of mesolimbic dopamine release under non-physiological conditions at intermediate (1.0 and 2.0g/kg) doses of ethanol. Domperidone also decreased EtOH-induced sedation at 2.0g/kg. Domperidone did not alter ethanol conditioned place preference nor did it affect ethanol-induced motor impairment. These results show that peripheral dopamine 2 receptors mediate some of the effects of ethanol on nonphysiological dopamine neurotransmission, although these effects are not related to the rewarding properties of ethanol.

Withaferin A alleviates ethanol-induced liver injury by inhibiting hepatic lipogenesis

Withaferin A (WA) is a natural steroidal compound with reported hepatoprotective activities against various liver diseases. Whether WA has therapeutic effects on alcoholic liver disease has not been explored. A binge alcoholic liver injury model was employed by feeding C57BL/6J mice an ethanol (EtOH) diet for 10 days followed by an acute dose of EtOH to mimic clinical acute-upon-chronic liver injury. In this binge model, WA significantly reduced the binge EtOH-induced increase of serum aminotransaminase levels and decreased hepatic lipid accumulation. Mechanistically, WA decreased levels of hepatic lipogenesis gene mRNAs in vivo, including Srebp1c, Fasn, Acc1 and Fabp1. In EtOH-treated primary hepatocytes in vitro, WA decreased lipid accumulation by lowering the expression of the lipogenesis gene mRNAs Fasn and Acc1 as well as decreasing hepatocyte death. In the established binge alcoholic liver injury model, WA therapeutically reduced the EtOH-induced increase of serum aminotransaminase levels as well as hepatic lipid accumulation. These results demonstrate that WA reduces EtOH-induced liver injury by inhibiting hepatic lipogenesis, suggesting a potential therapeutic option for treating alcoholic liver injury.

Hepatoprotective potential of kirenol on ethanol-induced liver toxicity in albino rats and acetaminophen-induced oxidative stress-mediated apoptosis in hepatic HepG2 cells.

Liver diseases are amajor health issue in both men and women and cause significant mortalityworldwide. The hepatoprotective effects of kirenol were evaluated in acetaminophen (APAP)-induced toxicity in HepG2 cells and ethanol (EtOH)- induced hepatotoxicity in rats. The cytotoxicity of kirenol (IC50 , 25 µM/ml) and APAP (20 µg/ml) with sylimarin (IC50 , 15 µg/ml) wasobserved in HepG2 cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Furthermore, reactive oxygen species formation, mitochondrial membrane potential, and oxidative stress markers such as thiobarbituric acid-reactive substance, suproxide dismutase, and catalase were assayed. Rats were administered a different dose(10, 20, and 30 mg/kg/day) for a period of 4 weeks before a single dose of EtOH (40% vol/vol) 3 g/kg/day. EtOH administered rats appeared to have lower body weight gain, severe hepatic and kidney damage as proved by elevated aspartate transaminase, alanine transaminase, alkaline phosphatase, uric acid, increased malondialdehyde (MDA), and inflammatory markers, and reduced glutathione (GSH) levels. Results showed that the kirenol treatment enhanced the GSH and reducedMDA in the liver and renal tissues and restored TNF-α and IL-6. Histoanalysis proved the protective effects of kirenol. In conclusion, it was proved that the kirenol demonstrated a hepato-protective effect in APAP- and EtOH-induced liver toxicity in HepG2 cells and rats, respectively.

Rapid tolerance to behavioral effects of ethanol in rats: Prevention by R-(−)-ketamine

R-(−)-ketamine has emerged as a potentially improved medication over that of the (S)-isomer (marketed as Spravato for depression). Recent data have suggested (R)-ketamine could have value in the treatment of substance use disorder. The present set of experiments was undertaken to examine whether (R)-ketamine might prevent tolerance development. Rapid ethanol (ETOH) tolerance was studied since racemic ketamine had previously been shown to block this tolerance development in rats. In the present study, male Sprague-Dawley rats were given two large doses of ETOH on Day 1 (2.3 + 1.7 g/kg) and 2.3 g/kg ETOH on Day 2. Animals were tested for effects of 2.3 g/kg ETOH on grip strength, inclined screen performance and rotarod performance on Day 1 with or without (R)-ketamine as a pretreatment. (R)-ketamine alone was tested at the highest dose studied (10 mg/kg) and did not significantly influence any dependent measure. (R)-ketamine (1–10 mg/kg) did not alter the acute effects of ETOH except for enhancing the effects of ETOH on the inclined screen test at 3 mg/kg. Between-subjects analysis documented that tolerance developed to the effects of ETOH only on the measure of grip strength. (R)-ketamine (3 mg/kg) given prior to ETOH on Day 1 exhibited a strong trend toward preventing tolerance development (p = 0.062). The present results extend prior findings on the potential value of (R)-ketamine in substance abuse disorder therapeutics and add to the literature on NMDA receptor blockade as a tolerance-regulating mechanism.

Recovery of Hippocampal-Dependent Learning Despite Blunting Reactive Adult Neurogenesis After Alcohol Dependence.

Background:The excessive alcohol drinking that occurs in alcohol use disorder (AUD) causes neurodegeneration in regions such as the hippocampus, though recovery may occur after a period of abstinence. Mechanisms of recovery are not clear, though reactive neurogenesis has been observed in the hippocampal dentate gyrus following alcohol dependence and correlates to recovery of granule cell number.Objective:We investigated the role of neurons born during reactive neurogenesis in the recovery of hippocampal learning behavior after 4-day binge alcohol exposure, a model of an AUD. We hypothesized that reducing reactive neurogenesis would impair functional recovery.Methods:Adult male rats were subjected to 4-day binge alcohol exposure and two approaches were tested to blunt reactive adult neurogenesis, acute doses of alcohol or the chemotherapy drug, temozolomide (TMZ).Results:Acute 5 g/kg doses of EtOH gavaged T6 and T7 days post binge did not inhibit significantly the number of Bromodeoxyuridine-positive (BrdU+) proliferating cells in EtOH animals receiving 5 g/kg EtOH versus controls. A single cycle of TMZ inhibited reactive proliferation (BrdU+ cells) and neurogenesis (NeuroD+ cells) to that of controls. However, despite this blunting of reactive neurogenesis to basal levels, EtOH-TMZ rats were not impaired in their recovery of acquisition of the Morris water maze (MWM), learning similarly to all other groups 35 days after 4-day binge exposure.Conclusions:These studies show that TMZ is effective in decreasing reactive proliferation/neurogenesis following 4-day binge EtOH exposure, and baseline levels of adult neurogenesis are sufficient to allow recovery of hippocampal function.

Development of a Physiologically Based Pharmacokinetic Model for Prediction of Ethanol Concentration-Time Profile in Different Organs.

A physiologically based pharmacokinetic (PBPK) modeling approach was used to simulate the concentration-time profile of ethanol (EtOH) in stomach, duodenum, plasma and other tissues upon consumption of beer and whiskey under fasted and fed conditions. A full PBPK model was developed for EtOH using the advanced dissolution, absorption and metabolism (ADAM) model fully integrated into the Simcyp Simulator® 15 (Simcyp Ltd., Sheffield, UK). The prediction performance of the developed model was verified and the EtOH concentration-time profile in different organs was predicted. Simcyp simulation showed ≤ 2-fold difference in values of EtOH area under the concentration-time curve (AUC) in stomach and duodenum as compared to the observed values. Moreover, the simulated EtOH maximum concentration (Cmax), time to reach Cmax (Tmax) and AUC in plasma were comparable to the observed values. We showed that liver is exposed to the highest EtOH concentration, faster than other organs (Cmax=839.50mg/L and Tmax=0.53h), while brain exposure of EtOH (AUC=1139.43mg·h/L) is the highest among all other organs. Sensitivity analyses (SAs) showed direct proportion of EtOH rate and extent of absorption with administered EtOH dose and inverse relationship with gastric emptying time (GE) and steady-state volume of distribution (Vss). The current PBPK model approach might help with designing in vitro experiments in the area of alcohol organ damage or alcohol-drug interaction studies.

High ethanol and acetaldehyde decrease extracellular glutamate in the frontal cortex of freely moving mice.

Our recent study demonstrated that local perfusion of ethanol (EtOH) and acetaldehyde (AcH) into the hippocampus via microdialysis decreased extracellular glutamate; however, it is not clear whether this effect occurs in the frontal cortex. To address this issue, we investigated the effects of local perfusion of EtOH and AcH on extracellular glutamate in the frontal cortex of Aldh2-knockout (Aldh2-KO) and C57BL/6 N [wild-type (WT)] mice. Dialysates were collected every 20 minutes, and extracellular glutamate was measured using HPLC coupled with electrochemical detector. We found local perfusion of 200 and 500 mM EtOH into the frontal cortex of WT and Aldh2-KO mice produced significant decreases in extracellular glutamate levels (P < 0.05). A dose of 500 mM EtOH induced a greater decrease in Aldh2-KO mice (P < 0.05) than in WT mice, indicating the action of AcH. Similarly, perfusion of 200 and 500 µM AcH decreased glutamate in the frontal cortex of Aldh2-KO mice (P < 0.05), but this decrease was not seen in WT mice at any AcH dose, due to the subsequent oxidation of AcH by mitochondrial aldehyde dehydrogenase 2. A low dose of EtOH (100 mM) or AcH (100 µM) had no effect on glutamate. These results showed that high doses of EtOH and AcH induces a significant decrease in extracellular glutamate in the frontal cortex of mice, replicating previous findings and providing further evidence that reduced glutamate is likely to be involved in the depressant effects of EtOH.

High Ethanol and Acetaldehyde Inhibit Glutamatergic Transmission in the Hippocampus of Aldh2-Knockout and C57BL/6N Mice: an In Vivo and Ex Vivo Analysis.

We aimed to investigate whether ethanol (EtOH) and acetaldehyde (AcH) can affect glutamate and its receptors GluN1 and GluA1 in the hippocampus of Aldh2-knockout (Aldh2-KO) and C57BL/6N (wild-type (WT)) mice. To do this, we first examined the effect of local administration of EtOH (100mM, 200mM, and 500mM) and AcH (100μM, 200μM, and 500μM) on extracellular glutamate levels in freely moving mice. Retrodialysis of 200mM and 500mM EtOH into the hippocampus of WT and Aldh2-KO mice produced significant decreases in extracellular glutamate levels (p < 0.05). A dose of 500mM EtOH induced a greater decrease in Aldh2-KO mice (p < 0.05) than in WT mice, indicating the action of AcH. Similarly, perfusion of 200μM and 500μM AcH decreased glutamate in Aldh2-KO mice (p < 0.05), but this decrease was not seen in WT mice at any AcH dose. Second, we tested whether the EtOH- and AcH-induced decrease in glutamate was associated with decreases in GluN1 and GluA1 expression, as measured by real-time PCR and Western blot. We found a significant decrease in GluN1 (p < 0.05) and GluA1 (p < 0.05) subunits after a high dose of EtOH (4.0g/kg) and AcH (200mg/kg) in WT mice. However, a 2.0g/kg dose of EtOH did not produce a consistent decrease in GluN1 or GluA1 between messenger RNA and protein. In Aldh2-KO mice, all three doses of EtOH (1.0g/kg, 2.0g/kg, and 4.0g/kg) and AcH (50mg/kg, 100mg/kg, and 200mg/kg) decreased GluN1 expression (p < 0.05), while moderate-to-high doses of EtOH (2.0g/kg and 4.0g/kg) and AcH (100mg/kg and 200mg/kg) decreased GluA1 expression (p < 0.05). Together, these in vivo and ex vivo data suggest that EtOH and AcH decrease extracellular glutamate in the hippocampus of mice with a concomitant decrease in GluN1 and GluA1 subunits, but these effects require relatively high concentrations and may, therefore, explain the consequences of EtOH intoxication.

Linking Dysregulated AMPK Signaling and ER Stress in Ethanol-Induced Liver Injury in Hepatic Alcohol Dehydrogenase Deficient Deer Mice.

Ethanol (EtOH) metabolism itself can be a predisposing factor for initiation of alcoholic liver disease (ALD). Therefore, a dose dependent study to evaluate liver injury was conducted in hepatic alcohol dehydrogenase (ADH) deficient (ADH−) and ADH normal (ADH+) deer mice fed 1%, 2% or 3.5% EtOH in the liquid diet daily for 2 months. Blood alcohol concentration (BAC), liver injury marker (alanine amino transferase (ALT)), hepatic lipids and cytochrome P450 2E1 (CYP2E1) activity were measured. Liver histology, endoplasmic reticulum (ER) stress, AMP-activated protein kinase (AMPK) signaling and cell death proteins were evaluated. Significantly increased BAC, plasma ALT, hepatic lipids and steatosis were found only in ADH− deer mice fed 3.5% EtOH. Further, a significant ER stress and increased un-spliced X-box binding protein 1 were evident only in ADH− deer mice fed 3.5% EtOH. Both strains fed 3.5% EtOH showed deactivation of AMPK, but increased acetyl Co-A carboxylase 1 and decreased carnitine palmitoyltransferase 1A favoring lipogenesis were found only in ADH− deer mice fed 3.5% EtOH. Therefore, irrespective of CYP2E1 overexpression; EtOH dose and hepatic ADH deficiency contribute to EtOH-induced steatosis and liver injury, suggesting a linkage between ER stress, dysregulated hepatic lipid metabolism and AMPK signaling.

Discriminative Stimulus Effects and Metabolism of Ethanol in Rhesus Monkeys.

Animal models are an essential feature of drug and pharmacotherapy development for treating alcohol use disorders (AUDs). The rhesus macaque is a robust animal model for many aspects of AUDs particularly in exploiting individual differences in oral self-administration of ethanol (EtOH), endocrine orchestration of stress response, and menstrual cycle characteristics. However, the clearance rates of EtOH have not been reported in this species, and the GABAA and N-methyl-D-aspartate (NMDA) receptor involvement in EtOH's discriminative stimulus effects has not been fully characterized. EtOH clearance rates following 2 doses of EtOH on separate days (0.5 and 1.0g/kg, i.g.) were determined in 8 young adult male rhesus macaques. The EtOH was given by nasogastric gavage, and repeated blood samples were taken over 5hours without sedation. Next, all subjects were trained on a 2-choice 1.0g/kg EtOH (i.g.) versus water discrimination with a 60-minutes pretreatment period to capture peak blood EtOH concentration (BEC). Substitution testing was conducted with GABAA ligands pentobarbital (i.g. and i.m.) and midazolam (i.g.), as well as NMDA antagonist MK-801 (i.m.). Peak BECs were 34 and 87mg/dl for 0.5 and 1.0g/kg doses, respectively, and occurred at 66 and 87minutes following gavage. All GABAA and NMDA ligands tested resulted in responding on the EtOH-appropriate lever with the potency ranking of MK-801 (ED50 : 0.017mg/kg)>midazolam (ED50 : 1.6mg/kg)>pentobarbital (ED50 : 3.7mg/kg)>EtOH (ED50 : 700mg/kg, or 0.7g/kg) in these subjects. These results suggest that the compound discriminative stimulus effects of EtOH are highly consistent across species, providing further support for the rhesus macaque as strong model for pharmacotherapy development for AUD.

Effects of Acute Ethanol Administration on Brain Oxidative Status: The Role of Acetaldehyde.

Ethanol (EtOH), one of the most widely consumed substances of abuse, can induce brain damage and neurodegeneration. EtOH is centrally metabolized into acetaldehyde, which has been shown to be responsible for some of the neurophysiological and cellular effects of EtOH. Although some of the consequences of chronic EtOH administration on cell oxidative status have been described, the mechanisms by which acute EtOH administration affects the brain's cellular oxidative status and the role of acetaldehyde remain to be elucidated in detail. Swiss CD-I mice were pretreated with the acetaldehyde-sequestering agent d-penicillamine (DP; 75mg/kg, i.p.) or the antioxidant lipoic acid (LA; 50mg/kg, i.p.) 30minutes before EtOH (2.5g/kg, i.p.) administration. Animals were sacrificed 30minutes after EtOH injection. Glutathione peroxidase (GPx) mRNA levels; GPx and glutathione reductase (GR) enzymatic activities; reduced glutathione (GSH), glutathione disulfide (GSSG), glutamate, g-L-glutamyl-L-cysteine (Glut-Cys), and malondialdehyde (MDA) concentrations; and protein carbonyl group (CG) content were determined in whole-brain samples. Acute EtOH administration enhanced GPx activity and the GSH/GSSG ratio, while it decreased GR activity and GSSG concentration. Pretreatment with DP or LA only prevented GPx activity changes induced by EtOH. Altogether, these results show the capacity of a single dose of EtOH to unbalance cellular oxidative homeostasis.