EtOH Concentrations Research Articles

Vulnerable windows for developmental ethanol toxicity in the Japanese medaka fish ( Oryzias latipes)

Ethanol (EtOH) is a well-known developmental toxicant that produces a range of abnormal phenotypes in mammalian systems including craniofacial abnormalities, cognitive deficits and growth retardation. While the toxic potential of developmental EtOH exposure is well characterized clinically, the effect of timing on the extent of toxicity remains unknown. Fish models such as the Japanese medaka, Oryzias latipes, provide a convenient system for investigating the effects of developmental EtOH exposure in vivo. In this study, medaka embryo toxicity tests were used to assess temporal variations in developmental EtOH toxicity. Fertilized eggs were collected and incubated during early, middle or late egg development (e.g., 0–3, 3–6 or 6–9 days post-fertilization) with various sub-lethal concentrations of EtOH [0.1% (17.2 mM), 0.5% (86.0 mM) or 1% (172 mM)]. Uptake of EtOH by the embryo was 60–68% of the solution concentration across all windows. Time to hatch, head width, total body length and whole embryo caspase activity were used to assess toxicity. Hatching delays were noted only at the highest concentration of EtOH. Head width was affected at all ethanol levels, regardless of the window of exposure. EtOH-induced decreases in body length, however, appeared to be most pronounced when exposure occurred either during the first or last window. The effect on caspase-3/7 activity also depended on the window of exposure, with increases in caspase noted in embryos treated on days 1 or 2 (first window) and decreases seen in embryos treated on day 6 (second window) or day 8 (third window). In general, these data suggest that critical periods for heightened sensitivity to developmental EtOH exposure may vary according to the specific endpoint used to assess toxicity.

LOW-DOSE ALCOHOL REDIRECTS POSTPRANDIAL CHOLINERGIC-STIMULATED APICAL EXOCYTOSIS TO THE BASOLATERAL MEMBRANE IN PANCREATIC ACINAR CELLS, THE SUSCEPTIBILITY MECHANISM OF ALCOHOLIC PANCREATITIS

Background: We previously demonstrated that alcohol (EtOH) pretreatment of pancreatic acini caused the basal plasma membrane (BPM) to become receptive to aberrant BPM exocytosis upon stimulation with submaximal CCK-8. These actions of alcohol on the acinar cell were due to induction of PM-bound Munc18c to dissociate into the cytosol, rendering the PM Syntaxin4 to interact with PM-SNAP-23 and granule-VAMP to form a complex capable of mediating basolateral exocytosis into the interstitial space, which we had postulated to be a mechanism of alcoholic pancreatitis. Rat pancreas however lacks CCK receptors and instead responds to cholinergic stimulation. Aim: We therefore examined whether 1- low EtOH concentration (20 mM) is able to alter submaximal cholinergic stimulation of rat pancreatic acinar cell secretion and exocytosis; 2- alcohol diet-fed rats are susceptible to develop pancreatitis upon low postprandial cholinergic stimulation. Methods: 1- Dispersed rat pancreatic acini were pretreated with non-stimulatory EtOH (20 mM-1 h) followed by stimulation with postprandial carbachol (3 uM-1 h), a dose stimulating only half maximal amylase secretion; 2- rats were fed with either a control or ethanol (36% of total calories intake replacement) liquid diet for 6 weeks and then challenged with low carbachol (5 ip injections of 0.5 uM/kg/h) stimulation.We then examined: a) PM levels of Munc18c and SNARE proteins, and Munc18c displacement into the cytosol by confocal microscopy and cell subfractionation; b) amylase release; c) real-time epifluorescent imaging of exocytosis using FM1-43; d) electron microscopy to visualize the distribution of granules within the cell; e) tissue hematoxylin and eosin (H&E) staining to examine morphologic changes of pancreatitis; f) serum amylase, lipase and ethanol concentrations as clinical parameters of pancreatitis. Results: 1- a) Whereas 20 mM EtOH has no effect on amylase secretion, pretreatment with 20 mM EtOH inhibited 3 uM carbachol stimulated secretion. b) Whereas 3 uM carbachol evokes apical exocytosis, pretreatment with 20 mM EtOH redirected exocytosis to the basal (by FM1-43 imaging) and lateral plasma membrane (by E.M.). c) While neither 20 mM EtOH nor 3 uM carbachol affected PM-Munc18c (confocal microscopy and subcellular fractionation), pretreatment of acini with 20 mM ETOH induced 3 uM carbachol to displace Munc18c from the BPM into the cytosol. 2-Alcohol diet-fed rats (serum ETOH levels of 20-30 mM) were normal and did not exhibit major morphologic defects in the pancreas. However, subsequent exposure to low-dose carbachol induced mild pancreatitis, observed as altered morphology (E.M and H&E) showing vacuolization in acinar cells, leukocyte infiltrate, hemorrhage and necrotic spots; along with clinical parameters of elevated serum levels of amylase and lipase. These morphologic changes correlated to Munc18c displacement from the BPM and its degradation in the cytosol. Conclusion: Postprandial EtOH renders acini PM-Munc18c conducive for postprandial cholinergic stimulation to displace into the cytosol, relieving Syntaxin4 to bind SNAP-23 and ZG VAMP proteins to promote basolateral exocytosis. We propose this to be a 'susceptibility' mechanism by which alcohol predisposes to human pancreatitis. (Funded by: Alcohol Beverage Medical Research Foundation and NIH -R21 AA015579-01).

Changes in cholinergic function in the frontal cortex and hippocampus of rat exposed to ethanol and acetaldehyde

Our previous microdialysis study demonstrated that both ethanol (EtOH) and acetaldehyde (ACe) decrease in vivo acetylcholine (ACh) release in the medial frontal cortex of freely moving rats. To better understand the mechanisms of EtOH and ACe’s effects on the cholinergic system in the brain, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) expression was examined at 40 and 240 min after a dose of EtOH (1 g/kg) in the rat frontal cortex and hippocampus. The control group was treated with 0.9% saline, and other groups received EtOH or cyanamide (CY, 50 mg/kg, a potent aldehyde dehydrogenase inhibitor) and 60 min later by EtOH intraperitoneally. Reverse-transcription polymerase chain reaction (RT-PCR) analysis revealed that ChAT mRNA levels were decreased by 72.8% and 71.6% in the EtOH and CY+EtOH groups, respectively, at 40 min after EtOH injection compared with saline in the frontal cortex. The hippocampal ChAT levels were reduced by 76.5% and 53.0% in the EtOH and CY+EtOH groups, respectively, at this time. CY+EtOH-induced depletion in ChAT mRNA levels was markedly higher than EtOH in the hippocampus. A similar decrease pattern of ChAT was observed at protein levels as determined by Western blot, but the reduced ChAT levels were significantly higher in the CY+EtOH group as compared with the EtOH group both in the frontal cortex and hippocampus. At 240 min after EtOH injection, the EtOH group had no effect on ChAT at mRNA levels, as compared with saline, whereas CY+EtOH group induced a significant decrease in ChAT mRNA expression to 62.0% and 65.5% in the frontal cortex and hippocampus, respectively. These data were consistent with the results of the Western blot analysis. AChE expression at mRNA levels was not changed at either 40 or 240 min after EtOH dosing in either of these groups in the frontal cortex and hippocampus. Within 40 and 240 min, a statistically significant difference in ChAT expression at mRNA and protein levels was found in the EtOH and CY+EtOH groups both in the frontal cortex and hippocampus. The data obtained from this study demonstrate that EtOH and ACe concentrations decreased ChAT expression at 40 and 240 min after EtOH administration in the frontal cortex and hippocampus, and this result suggests that reduced ChAT expression is strongly related to a decrease in ACh release in the rat brain.

Immature hippocampal neuronal networks do not develop tolerance to the excitatory actions of ethanol

Ethanol (EtOH) damages the hippocampus, a brain region that is involved in learning and memory processes. The mechanisms responsible for this effect of EtOH are not fully understood. We recently demonstrated that acute EtOH exposure potently stimulates oscillatory activity driven by the excitatory actions of GABA in the CA3 region of the neonatal rat hippocampus. This activity can be recorded during the growth spurt period as giant depolarizing potentials (GDPs). Here, we characterized the effects of prolonged EtOH exposure on GDPs. In the first study, we prepared hippocampal coronal slices from neonatal rats and exposed these to control artificial cerebrospinal fluid (ACSF) or ACSF plus 50 mM EtOH for 3–4 h. We then performed whole-cell patch-clamp electrophysiological recordings from CA3 pyramidal neurons, which revealed that tolerance to the GDP stimulating effects of EtOH did not occur after continuous exposure. In the second study, we exposed neonatal rats to air or air plus 1.9 g/dl EtOH in vapor chambers for 4 h/day for 1 or 3 days (neonatal peak blood EtOH concentration = 40–45 mM). We then performed slice electrophysiological studies 24 h after the end of EtOH exposure and found that there was no statistically significant difference in the acute effect of 50 mM EtOH on GDP frequency in samples from neonates exposed to air or air plus EtOH. These findings indicate that EtOH persistently stimulates network-driven oscillatory activity in the developing hippocampus. We propose that the lack of adaptive response to continuous EtOH exposure could make immature neuronal networks particularly vulnerable to the actions of this agent.

Physiologic and Genomic Analyses of Nutrition-Ethanol Interactions During Gestation: Implications for Fetal Ethanol Toxicity

Nutrition-ethanol (EtOH) interactions during gestation remain unclear primarily due to the lack of appropriate rodent models. In the present report we utilize total enteral nutrition (TEN) to specifically understand the roles of nutrition and caloric intake in EtOH-induced fetal toxicity. Time-impregnated rats were intragastrically fed either control or diets containing EtOH (8-14 g/kg/day) at a recommended caloric intake for pregnant rats or rats 30% undernourished, from gestation day (GD) 6-20. Decreased fetal weight and litter size (P < 0.05) and increased full litter resorptions (33% vs. 0%), were observed in undernourished dams compared to adequately fed rats given the same dose of EtOH, while undernutrition alone did not produce any fetal toxicity. Undernutrition led to impairment of EtOH metabolism, increased blood EtOH concentrations (160%), and decreased maternal hepatic ADH1 mRNA, protein, and activity. Microarray analyses of maternal hepatic gene expression on GD15 revealed that 369 genes were altered by EtOH in the presence of undernutrition, as compared to only 37 genes by EtOH per se (+/-2-fold, P < 0.05). Hierarchical clustering and gene ontology analysis revealed that stress and external stimulus responses, transcriptional regulation, cellular homeostasis, and protein metabolism were affected uniquely in the EtOH-under-nutrition group, but not by EtOH alone. Microarray data were confirmed using real-time RT-PCR. Undernourished EtOH-fed animals had 2-fold lower IGF-1 mRNA and 10-fold lower serum IGF-1 protein levels compared to undernourished controls (P < 0.0005). Examination of maternal GH signaling via STAT5a and -5b revealed significant reduction in both gene and protein expression produced by both EtOH and undernutrition. However, despite significantly elevated fetal BECs, fetal IGF-1 mRNA and protein were not affected by EtOH or EtOH-undernutrition combinations. Our data suggest that undernutrition potentiates the fetal toxicity of EtOH in part by disrupting maternal GH-IGF-1, signaling thereby decreasing maternal uterine capacity and placental growth.

Synthesis of nanoparticulate yttrium aluminum garnet in supercritical water–ethanol mixtures

Single-phase YAG nanoparticles have been produced by the rapid hydrolysis and dehydration of Al 3+ and Y 3+ salts in EtOH–H 2O mixtures using continuous supercritical water hydrothermal synthesis. The effects of the nature of the starting solution, the metal concentration, the EtOH content, the flow rate and the temperature on the particle size were studied. EtOH promotes crystallization and highly crystalline materials are obtained at relatively low temperatures. Furthermore the critical temperature of EtOH–H 2O mixtures is lower than that of pure H 2O, reducing the operating temperature. From the variables studied, the most important parameter seems to be the temperature and much smaller particles have been obtained when experiments are conducted under supercritical conditions (average 50 nm), in comparison to near critical or subcritical conditions (average 150 nm). The method is simple and clean and requires neither addition of a base to the system nor the use of very high reaction temperatures and potentially allows the continuous production of large quantities of material.

Vapor deposited ethanol–H 2O ice mixtures investigated by micro-Raman scattering

Solid deposits have been formed at 88 K and 10 −1 Torr from ethanol–water gas collected above aqueous solutions of ethanol (EtOH) (0.6, 2, 4.5, 9 and 17 mol%). The composition of different gas mixtures varying between 1:16 and 1:0.8 EtOH:H 2O are determined at 295 K using our experimental vapor–liquid equilibrium (VLE) data in combination with the Wilson model [28]. The Wilson constants derived at this temperature are Λ 12 = 0.37(4) and Λ 21 = 0.58(5). The concentration of EtOH in the ice mixture can be calculated using these data and a kinetic model of condensation. It is found to vary between 9 and 65 mol% EtOH. The ice mixtures are analyzed in situ in a modified cryostage by micro-Raman spectroscopy. The distinct vibrational signatures of pure EtOH, EtOH aqueous solutions and EtOH–ice mixtures are identified in the 400–3800 cm −1 spectral range. Internal vibrational motions of EtOH molecules are affected by temperature and concentration. The presence of amorphous EtOH–ice phases at 88 K is demonstrated by the characteristic vibrational signatures of the ν OH stretching modes. The crystallization of an EtOH hydrate is proposed during annealing at ∼140 K of a 65 mol% EtOH–ice mixture. According to our preliminary X-ray diffraction work, this phase has apparently a distinct structure from that of solid EtOH or from EtOH–clathtrate structures usually found in frozen aqueous solutions. For ice mixtures of lower EtOH content, a distinct hydrate phase crystallizes at ∼170 K. These results suggest that ice mixtures obtained by vapor deposition reflect the existence of EtOH clusters of a distinctive structural nature with respect to those encountered in frozen aqueous mixtures.

Resveratrol can only partially attenuate ethanol-induced oxidative stress in embryonic chick brains

Ethanol (EtOH) exposure promotes increased levels of reactive oxygen species that degrade unsaturated long-chain membrane fatty acids within embryonic chick brains and is associated with apoptosis and reduced embryo viability. In vitro studies have demonstrated that resveratrol, a known antioxidant, attenuated EtOH-induced damage. In order to test whether or not resveratrol can attenuate EtOH-induced embryonic damage, fertile chicken eggs were injected daily with EtOH (6.05 mmol/kg egg) and various concentrations of trans-resveratrol (0–29.5 mmol/kg egg) during the first three days of embryonic development. At 11 days of embryonic development, viable embryos were collected, brains isolated, and brain membrane fatty acid composition analyzed. Embryonic EtOH exposure promoted fewer viable embryos at 11 days of development as compared to controls. Embryonic EtOH exposure also promoted reduced levels of unsaturated long-chain membrane fatty acids, increased levels of saturated short-chain membrane fatty acids, and elevated brain lipid hydroperoxides (LPO) levels. Embryonic exposure to moderate (2.95 nmol/kg egg) and high (29.5 nmol/kg egg) levels of trans-resveratrol attenuated EtOH-induced changes in brain membrane fatty acid composition but failed to attenuate EtOH-induced increases in brain LPO levels and increased brain Casp-3 activities.

Ethanol potently and competitively inhibits binding of the alcohol antagonist Ro15-4513 to α 4/6 β 3 δ GABA A receptors

Although GABA(A) receptors have long been implicated in mediating ethanol (EtOH) actions, receptors containing the "nonsynaptic" delta subunit only recently have been shown to be uniquely sensitive to EtOH. Here, we show that delta subunit-containing receptors bind the imidazo-benzodiazepines (BZs) flumazenil and Ro15-4513 with high affinity (K(d) < 10 nM), contrary to the widely held belief that these receptors are insensitive to BZs. In immunopurified native cerebellar and recombinant delta subunit-containing receptors, binding of the alcohol antagonist [(3)H]Ro15-4513 is inhibited by low concentrations of EtOH (K(i) approximately 8 mM). Also, Ro15-4513 binding is inhibited by BZ-site ligands that have been shown to reverse the behavioral alcohol antagonism of Ro15-4513 (i.e., flumazenil, beta-carbolinecarboxylate ethyl ester (beta-CCE), and N-methyl-beta-carboline-3-carboxamide (FG7142), but not including any classical BZ agonists like diazepam). Experiments that were designed to distinguish between a competitive and allosteric mechanism suggest that EtOH and Ro15-4513 occupy a mutually exclusive binding site. The fact that only Ro15-4513, but not flumazenil, can inhibit the EtOH effect, and that Ro15-4513 differs from flumazenil by only a single group in the molecule (an azido group at the C7 position of the BZ ring) suggest that this azido group in Ro15-4513 might be the area that overlaps with the alcohol-binding site. Our findings, combined with previous observations that Ro15-4513 is a behavioral alcohol antagonist, suggest that many of the behavioral effects of EtOH at relevant physiological concentrations are mediated by EtOH/Ro15-4513-sensitive GABA(A) receptors.

Michaelis-Menten elimination kinetics of acetaldehyde during ethanol oxidation.

Acetaldehyde (AcH) is a toxic metabolite of ethanol (EtOH). The pharmacokinetics of blood AcH during EtOH oxidation was studied with or without the administration of aldehyde dehydrogenase 2 inhibitor (cyanamide) in rabbits. An bolus of EtOH saline solution (0.25, 0.5, 1.0, 1.5, and 2.0 g/kg) was injected intravenously. Cyanamide was administered intraperitoneally (25 mg/kg body weight) to the cyanamide-treated group. Blood EtOH and AcH concentrations were measured by using head-space gas chromatography. In the control group, the first peak of the blood AcH appeared immediately and the second elevation appeared 1 to 4 hr after administration at a high EtOH dose. The blood AcH levels other than the second elevation part were significantly correlated to the blood EtOH levels. In the cyanamide-treated group, a peak and a plateau formed at the time corresponding to the second peak in the control group. The peak and plateau concentration of AcH increased markedly. We attempted simultaneous curve fitting, using the five blood EtOH and AcH concentration-time curves, to determine the pharmacokinetic model. Consequently, the AcH elimination was best described by a Michaelis-Menten kinetic model in both groups. The blood AcH profile was suggested to consist of the first and second components that are related to the blood EtOH concentration itself and the metabolic formation of AcH, respectively. With higher EtOH doses or aldehyde dehydrogenase 2 inhibition, the second component becomes prominent as a result of the capacity-limited property of the metabolism of AcH, which is described by Michaelis-Menten elimination kinetics.

Complexation of nicotinamide with Ag+ ions in water-organic solvents

The effect of the water-ethanol and water-DMSO solvent composition on the stability of nicotinamide (NicNH2) complexes with Ag+ ions at an ionic strength of 0.25 (NaClO4) and a temperature of 25 ± 0.1°C is studied by potentiometric titration. An increase in the EtOH concentration in the mixed solvent is found to result in a higher stability of the nicotinamide complex with silver(I) ions; as the DMSO concentration increases, the complex becomes less stable. The results of the study are analyzed in terms of the solvation thermodynamic approach taking into account the electronic structure of the ligand and the solvation contributions of the fragments of the molecule.

Ethanol affects the generation, cosignaling molecule expression, and function of plasmacytoid and myeloid dendritic cell subsets in vitro and in vivo

The influence of ethanol (EtOH) on multiple dendritic cell (DC) subsets, in the steady state or following their mobilization in vivo, has not been characterized. Herein, generation of mouse bone marrow-derived DC (BMDC) in response to fms-like tyrosine kinase 3 ligand was inhibited by physiologically relevant concentrations of EtOH with selective suppression of plasmacytoid (p)DC. EtOH reduced surface expression of costimulatory molecules (CD40, CD80, CD86) but not that of coinhibitory CD274 (B7-H1) on resting or CpG-stimulated DC subsets. Interleukin (IL)-12p70 production by activated DC was impaired. Consistent with these findings, EtOH-exposed BMDC exhibited a reduced capacity to induce naïve, allogeneic T cell proliferation and impaired ability to prime T cells in vivo. DC subsets freshly isolated from EtOH-fed mice were also examined. Liver DC, inherently immature and resistant to maturation, exhibited little change in their low surface cosignaling molecule expression, whereas splenic DC showed reduced expression of surface costimulatory molecules in response to CpG stimulation in vivo. These splenic DC elicited reduced naïve, allogeneic T cell proliferation in vitro, and the stimulatory capacity of resting but not CpG-activated liver DC was reduced by chronic EtOH administration. T cells from animals primed with EtOH-exposed DC produced elevated levels of IL-10 following ex vivo challenge with donor alloantigen. Thus, EtOH impairs cytokine-driven differentiation and function of myeloid DC and pDC in vitro. Hepatic DC from chronic EtOH-fed mice are less affected than splenic DC, which exhibit impaired functional maturation following CpG stimulation. These results indicate a potential mechanism by which alcohol consumption is associated with immunosuppression.

Técnica histoquímica aplicada ao tecido ósseo desmineralizado e parafinado para o estudo do osteócito e suas conexões

The osteocyte has been the subject of recent research; however the in situ evaluation of its morphology, activity and connection characteristics is difficult and has been performed solely through advanced techniques. Due to the importance of this type of cell in the maintenance of the bone matrix, this study proposes a technique of silver staining as an alternative for the study of the osteocyte and its connections in demineralized and paraffined bone tissue. Cuts of 4µm from the femur of female rats were de-mineralized, deparaffined in xylol and hydrated in decreasing concentrations of ETOH and miliQ water. For the impregnation, solutions of silver nitrate at 50% and formic acid at 1% with 2% microbiological gel were used and incubated at 40oC. This technique allows the easy visualizing of the osteocytes lacunae edges and its connections, offering a simple and efficient alternative to the study of this type of cellular morphology, so far not proposed with this purpose.

Removal of PCB-DNAPL from a rough-walled fracture using alcohol/polymer flooding

Phase behaviour experiments employing PCB (Aroclor 1242)/alcohol/water systems were conducted with ethanol (EtOH) and n-propanol (nPA). Both exhibited an affinity for the aqueous phase within the entire two-phase region. As much as 88% by volume (88% vol.) EtOH and 80% vol. nPA were necessary to achieve full miscibility of the PCB in the aqueous phase. DNAPL–water interfacial tension (IFT) was reduced from 38.9 dyn/cm to 4.7 dyn/cm and 2.4 dyn/cm with 80% vol. EtOH and 76% vol. nPA. The addition of alcohol brought about 41% and 54% reductions in DNAPL viscosity at maximal concentrations of EtOH and nPA. Density of the PCB-DNAPL was relatively unaffected by the presence of alcohol. A series of seven experiments were conducted where successive slugs of nPA and xanthan gum polymer solutions were injected into a fractured shale sample. A 30% vol. nPA solution injected under a hydraulic gradient of 0.36 allowed enhanced PCB removal primarily through reduction of IFT and resulted in 72% DNAPL recovery. Several pore volumes of alcohol solution were necessary to displace all the potentially mobile non-wetting phase since the high-viscosity DNAPL was mobilized at a lower flow rate than the overall fluid velocity, illustrating non-piston displacement. The injection of a 95% vol. nPA alcohol solution, theoretically at a sufficient concentration to produce fully miscible displacement of the residual DNAPL at equilibrium, resulted in non-equilibrium partitioning of the PCB into the flushing solution, likely due to the high fluid velocities in the fracture. The injection of 200 pore volumes of 95% vol. nPA solution resulted in 94% DNAPL recovery. Alcohol floods operated below the miscibility envelope appear to be a valuable source zone remedial alternative where the objective is to reduce DNAPL mobility to zero, but it should be noted that DNAPL mobility is increased during the application of the technology and steps may need to be taken to prevent unwanted vertical mobilization.

Different Molecular Mechanisms Underlie Ethanol-Induced Bone Loss in Cycling and Pregnant Rats

Chronic ethanol (EtOH) consumption can result in osteopenia. In the current study, we examined the modulation of EtOH-induced bone loss during pregnancy. Nonpregnant and pregnant dams were intragastrically infused either control or EtOH-containing diets throughout gestation (gestation d 5 through 20 or an equivalent period of 15 d) by total enteral nutrition. The effects of EtOH (8.5 to 14 g/kg/d) on tibial bone mineral density (BMD), mineral content (BMC), and bone mineral area were assessed at gestation d 20 via peripheral quantitative computerized tomography. EtOH caused a dose-dependent decrease in BMD and BMC without affecting bone mineral area. Trabecular BMD and BMC were significantly lower in EtOH-treated, nonpregnant dams, compared with pregnant cohorts at the same infused dose of EtOH and urinary ethanol concentrations. Static histomorphometric analysis of tibiae from pregnant rats after EtOH treatment showed decreased osteoblast and osteoid surface, indicating inhibited bone formation, whereas EtOH-treated cycling rats showed higher osteoclast and eroded surface, indicative of increased bone resorption. Circulating osteocalcin and 1,25-dihydroxyvitamin D3 were lower in both EtOH-fed nonpregnant and pregnant rats. Gene expression of osteoclast markers, 70 kDa v-ATPase, and tartrate-resistant acid phosphatase were increased selectively in nonpregnant EtOH-treated rats but not pregnant rats. Moreover, only nonpregnant EtOH-fed rats showed induction in bone marrow receptor activator of nuclear factor-kappaB ligand mRNA and decreased circulating 17beta-estradiol levels. Our data suggest that EtOH-induced bone loss in pregnant rats is mainly due to inhibited bone formation, whereas in nonpregnant rats, the data are consistent with increased osteoclast activation and bone resorption concomitant with decreased estradiol levels.

Homer2 Is Necessary for EtOH-Induced Neuroplasticity

Homer proteins are integral to the assembly of proteins regulating glutamate signaling and synaptic plasticity. Constitutive Homer2 gene deletion [knock-out (KO)] and rescue with adeno-associated viral (AAV) transfection of Homer2b was used to demonstrate the importance of Homer proteins in neuroplasticity produced by repeated ethanol (EtOH) administration. Homer2 KO mice avoided drinking high concentrations of EtOH and did not develop place preference or locomotor sensitization after repeated EtOH administration. The deficient behavioral plasticity to EtOH after Homer2 deletion was paralleled by a lack of augmentation in the rise in extracellular dopamine and glutamate elicited by repeated EtOH injections. The genotypic differences in EtOH-induced change in behavior and neurochemistry were essentially reversed by AAV-mediated transfection of Homer2b into accumbens cells including, differences in EtOH preference, locomotor sensitization, and EtOH-induced elevations in extracellular glutamate and dopamine. These data demonstrate a necessary and active role for accumbens Homer2 expression in regulating EtOH-induced behavioral and cellular neuroplasticity.

Mitochondrially targeted vitamin E and vitamin E mitigate ethanol-mediated effects on cerebellar granule cell antioxidant defense systems

Ethanol (EtOH) disrupts the structure and function of the developing nervous system, sometimes leading to birth defects associated with fetal alcohol syndrome (FAS). Animal FAS models indicate that cellular membrane peroxidation, intracellular oxidant accumulation, and suppression of endogenous antioxidant enzymes contribute to the toxic effects of EtOH. Mitochondrially targeted vitamin E (MitoVit E), a chemically engineered form of vitamin E (VE) designed to accumulate in the mitochondria, has been shown to inhibit intracellular oxidant accumulation and cell death more effectively than VE. In previous investigations, we have shown that, in vivo, VE reduces neuronal death in the developing cerebellum of EtOH-exposed animals, and, in vitro, VE prevents apoptotic and necrotic death of EtOH-exposed cerebellar granule cells (CGCs). The present investigation shows that, in a FAS CGC model, 1 nM MitoVit E renders significant neuroprotection against EtOH concentrations as high as 1600 mg/dL. The present study also demonstrates that, in this same model, MitoVit E mitigates EtOH-induced accumulation of intracellular oxidants and counteracts suppression of glutathione peroxidase/glutathione reductase (GSH-Px/GSSG-R) functions, protein expression of gamma-glutamylcysteine synthetase (γ-GCS), and total cellular glutathione (GSH) levels. In the presence and absence of EtOH, VE amplifies the protein expression levels of γ-GCS, an enzyme that performs the rate-limiting step for GSH synthesis, and total GSH levels. These results suggest that MitoVit E and VE ameliorate EtOH toxicity through non-oxidant mechanisms–modulations of endogenous cellular proteins–and antioxidant means.

Effects of Finasteride on Chronic and Acute Ethanol Withdrawal Severity in the WSP and WSR Selected Lines

The neurosteroid allopregnanolone (ALLO) is a potent positive modulator of gamma-aminobutyric acidA (GABAA) receptors that can modulate ethanol (EtOH) withdrawal. The 5alpha-reductase inhibitor finasteride blocks the formation of ALLO from progesterone and was recently found to reduce certain effects of EtOH. Using the Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) selected lines, in the present studies we examined the effect of finasteride on acute and chronic EtOH withdrawal severity. In the first two studies, male WSP and WSR mice were exposed to 72-hr EtOH vapor or air and received four injections of finasteride (50 mg/kg intraperitoneal (IP) or vehicle 24 hr before and each day of the vapor exposure. After removal from the inhalation chamber, mice were scored for handling-induced convulsions (HICs) hourly for 12 hr and then again at 24 hr (study 1) or were tested on the elevated plus maze at 24 hr after removal from the inhalation chamber (study 2). In the third experiment, mice were pretreated with finasteride or vehicle 24 hr before an acute dose of EtOH (4 g/kg ip) or saline and then were tested for HICs as in the chronic study. In both chronic EtOH studies, finasteride pretreatment reduced EtOH withdrawal severity, measured by HICs, and anxiety-related behavior, but only in the WSP selected line. However, finasteride pretreatment also significantly decreased blood EtOH concentration on the initiation of withdrawal in both chronic EtOH studies in WSP and WSR mice. In contrast, pretreatment with finasteride slightly enhanced acute EtOH withdrawal severity in WSP mice, whereas there was no effect of finasteride or EtOH injection on HICs in WSR mice. Collectively, these findings indicate that the WSP line is more sensitive than the WSR line to the modulatory effects of finasteride in terms of both chronic and acute EtOH withdrawal severity. The differential effect of finasteride on acute versus chronic EtOH withdrawal severity may result from an indirect effect of finasteride on EtOH pharmacokinetics in the chronic paradigm.

Inhibition of acetaldehyde metabolism decreases acetylcholine release in medial frontal cortex of freely moving rats

The effect of high acetaldehyde (ACe) on acetylcholine (ACh) release was studied in vivo in the medial frontal cortex (mfc) of freely moving rats using brain microdialysis coupled with high performance liquid chromatography and an electrochemical detector. Ethanol (EtOH) and ACe concentrations were quantified simultaneously in the mfc of awake rats by in vivo microdialysis followed by head-space gas chromatography. Rats were treated intraperitoneally with saline, EtOH (1 and 2 g/kg) or cyanamide (CY, 50 mg/kg, a potent aldehyde dehydrogenase inhibitor) plus EtOH (1 and 2 g/kg). No significant effect on ACh levels was observed in saline groups, as compared to baseline value. The basal level of ACh in the dialysate was about 0.30 ± 0.04 pmol/20 μl, and this value was reduced significantly in the EtOH (1 and 2 g/kg) and CY + EtOH (1 and 2 g/kg) groups for 240 min after EtOH administration. The time courses of ACh release continued to decrease significantly after EtOH administration in the CY + EtOH (1 and 2 g/kg) groups compared to the values in the saline and EtOH (1 and 2 g/kg) groups. A significant decrease in ACh release was observed from 140 to 240 min after EtOH dosing in the EtOH (1 and 2 g/kg) groups, as compared to saline groups. EtOH and ACe concentrations in the mfc were first determined at 15 min after a dose of EtOH, reached a peak at 30 min and then gradually decreased in the CY + EtOH (1 and 2 g/kg) groups. The present study suggests that both EtOH and ACe concentration in the brain can decrease in vivo ACh release in the mfc of free-moving rats, and the ACe-induced decrease in ACh levels was significantly higher than EtOH.

Galanin Microinjection in the Third Ventricle Increases Voluntary Ethanol Intake

The neuropeptide galanin increases food intake. Chronic ethanol (EtOH) increases the expression of galanin in the hypothalamus. The research presented here examines the effects of microinjection of galanin in the third ventricle on voluntary alcohol intake. Male Sprague Dawley rats with a cannula in the third ventricle were given access to increasing concentrations of EtOH for 12 hr/day until all acquired a preference for 7% EtOH over water in a two-bottle choice. Rats then received a microinjection of galanin (0, 1, and 3 nmol) alone or in combination with the galanin antagonist M40 (1 nmol) and with M40 alone to determine the effects on EtOH and water intake. Tests were conducted during both the light and dark periods of a 12:12-hr light-dark cycle with food available ad libitum. As a control for galanin-induced calorie intake, both EtOH and food were measured in a subset of rats during the dark. Microinjections of galanin (1.0 and 3.0 nmol) increased EtOH consumption during both periods of the light-dark cycle. Galanin's effect on ethanol intake during the light was large relative to the very low intake of food and water during this period. Rats increased their intake of EtOH but not food. Receptor specificity for galanin (3 nmol) was shown by the galanin antagonist M40, which blocked the increase in EtOH intake. M40 alone decreased EtOH intake slightly. These data show that galanin injected in the third ventricle increases EtOH consumption and that the effect can occur during both the light and the dark periods of the diurnal cycle in the presence of food and water. This suggests that galanin may play a role in augmenting voluntary alcohol intake and perhaps the development of alcohol dependence.