EtOH Concentrations Research Articles

Effect of the allelic variant of alcohol dehydrogenase ADH1B*2 on ethanol metabolism.

It has been known that ADH1B*2 allele has a protective effect against the development of alcohol dependence. However, the protection mechanism is still unknown. We investigated whether ADH1B gene polymorphism affects ethanol (EtOH) metabolism. In a parent study, we conducted a randomized crossover trials on 24 healthy male subjects who were selected by genotyping: 12 with ALDH2*1/*1 (active form) and 12 with ALDH2*1/*2 (inactive form). In the present study, the 24 subjects were reclassified into 2 groups of 11 with ADH1B*1/*2 and 13 with ADH1B*2/*2 according to the ADH1B genotypes. Each subject was administered 1 of 3 doses of EtOH (0.25, 0.5, 0.75 g/kg) or a placebo in 4 trials. After the administration of alcohol, blood EtOH and acetaldehyde concentrations were measured 9 times over 4 hours. In the case of EtOH, the area under the concentration-time curve from 0 to 4 hours (AUC0-4 ) and the peak blood concentration of EtOH (Cmax ) in subjects with ADH1B*2/*2 were significantly higher than those in subjects with ADH1B*1/*2 at all 3 dosages before stratifying by ALDH2 genotype. However, after stratifying by ALDH2 genotype, a statistically significant difference between ADH1B*2/*2 and ADH1B*1/*2 was found only at the 0.5 g/kg dosage regardless of ALDH2 genotype. In the case of acetaldehyde, the AUC0-4 and Cmax of acetaldehyde of ADH1B*2/*2 after administration of 0.25 g/kg alcohol and the AUC0-4 of acetaldehyde of ADH1B*2/*2 at 0.5 g/kg were significantly higher than corresponding values of ADH1B*1/*2 only in the group of ALDH2*1/*2. Our findings indicate that the blood EtOH concentrations of ADH1B*2/*2 group are higher than those of ADH1B*1/*2 group regardless of ALDH2 genotype, and the blood acetaldehyde concentrations of ADH1B*2/*2 are also higher than those of ADH1B*1/*2 only in the ALDH2*1/*2 group. To our knowledge, this is the first report to demonstrate the association of ADH1B*2 allele with blood EtOH and acetaldehyde levels in humans, and these results suggest that higher blood EtOH and acetaldehyde concentrations in ADH1B*2/*2 may constitute the mechanism of protection against alcoholism by ADH1B*2/*2.

Chronic Alcohol Intake During Adolescence, but not Adulthood, Promotes Persistent Deficits in Risk-Based Decision Making

Adolescent alcohol use is a major public health concern and is strongly correlated with the development of alcohol abuse problems in adulthood. Adolescence is characterized by maturation and remodeling of brain regions implicated in decision making and therefore may be uniquely vulnerable to environmental insults such as alcohol exposure. We have previously demonstrated that voluntary alcohol consumption in adolescence results in maladaptive risk-based decision making in adulthood. However, it is unclear whether this effect on risk-based decision making can be attributed to chronic alcohol use in general or to a selective effect of alcohol use during the adolescent period. Ethanol (EtOH) was presented to adolescent (postnatal day [PND] 30 to 49) and adult rats (PND 80 to 99) for 20 days, either 24 hours or 1 h/d, in a gel matrix consisting of distilled water, gelatin, polycose (10%), and EtOH (10%). The 24-hour time course of EtOH intake was measured and compared between adolescent and adult animals. Following 20 days of withdrawal from EtOH, we assessed risk-based decision making with a concurrent instrumental probability-discounting task. Blood EtOH concentrations (BECs) were taken from trunk blood and assessed using the Analox micro-stat GM7 in separate groups of animals at different time points. Unlike animals exposed to EtOH during adolescence, animals exposed to alcohol during adulthood did not display differences in risk preference compared to controls. Adolescent and adult rats displayed similar EtOH intake levels and patterns when given either 24- or 1-hour access per day. In addition, while both groups reached significant BEC levels, we failed to find a difference between adult and adolescent animals. Here, we show that adolescent, but not adult, EtOH intake leads to a persistent increase in risk preference which cannot be attributed to differences in intake levels or BECs attained. Our findings support previous work implicating adolescence as a time period of heightened susceptibility to the long-term negative effects of alcohol exposure.

Absorption and Peak Blood Alcohol Concentration After Drinking Beer, Wine, or Spirits

Both the amount and the rate of absorption of ethanol (EtOH) from alcoholic beverages are key determinants of the peak blood alcohol concentration (BAC) and exposure of organs other than gut and liver. Previous studies suggest EtOH is absorbed more rapidly in the fasting than in the postprandial state. The concentration of EtOH and the type of beverage may determine gastric emptying/absorption of EtOH. The pharmacokinetics of EtOH were measured in 15 healthy men after consumption of 0.5 g of EtOH/kg body weight. During this 3-session crossover study, subjects consumed in separate sessions, beer (5.1% v/v), white wine (12.5% v/v), or vodka/tonic (20% v/v) over 20 minutes following an overnight fast. BAC was measured by gas chromatography at multiple points after consumption. Peak BAC (Cmax ) was significantly higher (p < 0.001) after vodka/tonic (77.4 ± 17.0 mg/dl) than after wine (61.7 ± 10.8 mg/dl) or beer (50.3 ± 9.8 mg/dl) and was significantly higher (p < 0.001) after wine than beer. The time to Cmax occurred significantly earlier (p < 0.01) after vodka/tonic (36 ± 10 minutes) compared to wine (54 ± 14 minutes) or beer (62 ± 23 minutes). Six subjects exceeded a Cmax of 80 mg/dl after vodka/tonic, but none exceeded this limit after beer or wine. The area under the concentration-time curve (AUC) was significantly greater after drinking vodka/tonic (p < 0.001) than after wine or beer. Comparison of AUCs indicated the relative bioavailability of EtOH was lower after drinking beer. Findings indicate that BAC is higher after drinking vodka/tonic than beer or wine after fasting. A binge pattern is significantly more likely to result in BAC above 80 mg/dl after drinking vodka/tonic than beer or wine. Men drinking on an empty stomach should know BAC will vary depending on beverage type and the rate and amount of EtOH.

Effect of solvation on the thermodynamics of formation for 18-crown-6 ether complexes with glycine and triglycine in water-ethanol solutions at 298 K

Using data from calorimetric titration, standard thermodynamic parameters logK ○, Δr G ○, Δr H ○, and TΔr S ○ of the formation of 18-crown-6 ether (18C6) molecular complex with triglycine (3Gly), [3Gly18C6] in H2O-EtOH solvents with contents of ethanol x ranging between 0.0 and 0.5 mole fractions are calculated. Increasing the concentration of EtOH in the solvent is found to raise the reaction’s exothermicity from −5.9 to −21.0 kJ mol−1 and logK ○ [3Gly18C6] from 1.10 to 2.53. A comparative analysis of the effect the composition of H2O-EtOH solvent has on the reactions of [3Gly18C6] and [Gly18C6] formation is performed. As in case of [Gly18C6] formation, the changes in the complex’s enthalpy of solvation Δtr H ○([3Gly18C6]) are close to the Δtr H ○(18C6) parameter and differ considerably from the Δtr H ○(3Gly) value, testifying to the crucial role 18C6 plays in changing the [3Gly18C6] state of solvation. The ratio between solvation contributions from reagents to Δtr G ○ of [3Gly18C6] formation is found to differ from that the one between the corresponding contributions to Δtr H r o : in transferring from water to H2O-EtOH mixtures, the increase in the positive Δtr G ○(18C6) values is slight and therefore negligible when compared to Δtr G ○(3Gly).

Genetic Relationship Between Predisposition for Binge Alcohol Consumption and Blunted Sensitivity to Adverse Effects of Alcohol in Mice

Initial sensitivity to ethanol (EtOH) and the capacity to develop acute functional tolerance (AFT) to its adverse effects may influence the amount of alcohol consumed and may also predict future alcohol use patterns. The current study assessed sensitivity and AFT to the ataxic and hypnotic effects of EtOH in the first replicate of mice (HDID-1) selectively bred for high blood EtOH concentrations (BECs) following limited access to EtOH in the Drinking in the Dark (DID) paradigm. Naïve male and female HDID-1 and HS/Npt mice from the progenitor stock were evaluated in 3 separate experiments. In Experiments 1 and 2, EtOH-induced ataxia was assessed using the static dowel task. In Experiment 3, EtOH-induced hypnosis was assessed by using modified restraint tubes to measure the loss of righting reflex (LORR). HDID-1 mice exhibited reduced initial sensitivity to both EtOH-induced ataxia (p < 0.001) and hypnosis (p < 0.05) relative to HS/Npt mice. AFT was calculated by subtracting the BEC at loss of function from the BEC at recovery (Experiments 1 and 3) or by subtracting BEC at an initial recovery from the BEC at a second recovery following an additional alcohol dose (Experiment 2). The dowel test yielded no line differences in AFT, but HS/Npt mice developed slightly greater AFT to EtOH-induced LORR than HDID-1 (p < 0.05). These results suggest that HDID-1 mice exhibit aspects of blunted ataxic and hypnotic sensitivity to EtOH which may influence their high EtOH intake via DID, but do not display widely different development of AFT. These findings differ from previous findings with the high alcohol-preferring (HAP) selected mouse lines, suggesting that genetic predisposition for binge, versus other forms of excessive alcohol consumption, is associated with unique responses to EtOH-induced motor incoordination.

An Evolutionarily Conserved Mechanism of Calcium‐Dependent Neurotoxicity in a Zebrafish Model of Fetal Alcohol Spectrum Disorders

Fetal alcohol spectrum disorders (FASD) are a leading cause of neurodevelopmental disability. Nonhuman animal models offer novel insights into its underlying mechanisms. Although the developing zebrafish has great promise for FASD research, a significant challenge to its wider adoption is the paucity of clear, mechanistic parallels between its ethanol (EtOH) responses and those of nonpiscine, established models. Inconsistencies in the published pharmacodynamics for EtOH-exposed zebrafish, alongside the use of comparatively high EtOH doses, challenge the interpretation of this model's clinical relevance. To address these limitations, we developed a binge, single-exposure model of EtOH exposure in the early zebrafish embryo. Brief (3-hour) EtOH exposure is sufficient to cause significant neural crest losses and craniofacial alterations, with peak vulnerability during neurogenesis and early somitogenesis. These losses are apoptotic, documented using TUNEL assay and secA5-YFP-reporter fish. Apoptosis is dose dependent with an EC50 = 56.2 ± 14.3 mM EtOHint , a clinically relevant value within the range producing apoptosis in chick and mouse neural crest. This apoptosis requires the calcium-dependent activation of CaMKII and recapitulates the well-described EtOH signaling mechanism in avian neural crest. Importantly, we resolve the existing confusion regarding zebrafish EtOH kinetics. We show that steady-state EtOH concentrations within both chorion-intact and dechorionated embryos are maintained at 35.7 ± 2.8% of EtOHext levels across the range from 50 to 300 mM EtOHext , a value consistent with several published reports. Equilibrium is rapid and complete within 5 minutes of EtOH addition. The calcium/CaMKII mechanism of EtOH's neurotoxicity is shared between an amniote (chick) and teleost fish, indicating that this mechanism is evolutionarily conserved. Our data suggest that EtOHext concentrations >2% (v/v) for chorion-intact embryos and 1.5% (v/v) for dechorionated embryos have limited clinical relevance. The strong parallels with established models endorse the zebrafish's relevance for mechanistic studies of EtOH's developmental neurotoxicity.

Continuous Ethanol Fermentation in Immersed, Cross-Flow Microfiltration Membrane Bioreactor with Cell Retention

The key objective of this study was to devise a continuous ZeeWeed® membrane-based, immersed, microfiltration (MF) laboratory scale fermentation system for ethanol production with cell retention to achieve effective ethanol productivity, flux rates and sugar utilization. The new bioreactor was compared to the fermentation kinetics’ of the ultrafiltration unit.A synthetic glucose based medium was fermented by fresh, baker’s yeast to produce ethanol. The cells were not recycled; the medium was continuously withdrawn by filtration through an internal, immersed hollow-fiber cartridge. In this way, the inside of the membrane was exposed to the ethanolic solution, while broth with viable yeast cells remained outside the membrane. This design, with a cell retention system, provided much less membrane fouling (loss of about 76% of the original water flux after 96 hours of filtration) than while using the ultrafiltration (UF) external hollow-fiber membrane with cell recycling (loss of 97% of the original water flux after 2-3 hours of operation). Both modules converted at least 95% of glucose with biomass concentration of 30 g/L, and the final ethanol concentration of 62 g/L. However, the UF membrane became plugged after only 2 hrs of operation. The ZeeWeed® membrane operated successfully for 96 hrs with a final flux of 4 L/h m2 with ethanol concentration of 62.4 g/l, biomass yield 0.34 g/g and cell viability of 95.3%. This concept could be successfully used for biofuel production. A very strong positive correlation was observed between the biomass and EtOH concentration (R=0.98; at p

The ameliorating effect of Rosa roxburghii against ethanol-induced psychomotor alterations in rats

Background: Ethanol (EtOH) is one of the oldest recreational substances known to man, primarily taken because it induces a sense of well-being (euphoric effects) and relaxation (anxiolytic effects). EtOH use entails various negative consequences. Of particular interest are EtOH-induced psychomotor alterations, because of its immediate manifestation and adverse consequences. Rosa roxburghii (RR), a wild plant of Southwest China, has gained attention on account of its numerous beneficial effects on the immune, nervous, and cardiovascular systems. Objective: In the present study we assessed the effects of Rosa roxburghii (RR) on EtOH-induced psychomotor alterations in rats. Methods: Sprague Dawley rats were orally administered distilled water (control group) or ethanol (4 g/kg BW) (EtOH-group) to induce psychomotor alterations. RR extract (25, 50, and 100 mg/kg, p.o.) was administered 30 min before EtOH treatment (RR-group). EtOH-induced psychomotor alterations were evaluated in the open-field, accelerating rotarod, hanging wire, and cold swimming tests. Behavioral evaluation and hematological analysis (EtOH and acetaldehyde concentration) were done at 1, 2, 4 and 8 hours after EtOH administration. Results: The EtOH group showed psychomotor alterations as compared with the control group. These EtOH-induced psychomotor alterations were directly related to the rise in blood ethanol and acetaldehyde concentrations. Pre-treatment of RR significantly improved EtOH-induced psychomotor alterations on open-field, accelerating rotarod, hanging wire, and cold swimming tests. These improvements in psychomotor performance coincided with the decreased blood ethanol and acetaldehyde levels observed in the RR-treated group. Conclusion: These results suggest that RR has ameliorating effects against EtOH-induced psychomotor alterations.

Controlling Carrier Densities in Photochemically Reduced Colloidal ZnO Nanocrystals: Size Dependence and Role of the Hole Quencher

Photodoped colloidal ZnO nanocrystals are model systems for understanding the generation and physical or chemical properties of excess delocalized charge carriers in semiconductor nanocrystals. Typically, ZnO photodoping is achieved photochemically using ethanol (EtOH) as a sacrificial reductant. Curiously, different studies have reported over an order of magnitude spread in the maximum number of conduction-band electrons that can be accumulated by photochemical oxidation of EtOH. Here, we demonstrate that this apparent discrepancy results from a strong size dependence of the average maximum number of excess electrons per nanocrystal, <nmax>. We demonstrate that <nmax> increases in proportion to nanocrystal volume, such that the maximum carrier density remains constant for all nanocrystal sizes. <nmax> is found to be largely insensitive to precise experimental conditions such as solvent, ligands, protons or other cations, photolysis conditions, and nanocrystal or EtOH concentrations. These results reconcile the broad range of literature results obtained with EtOH as the hole quencher. Furthermore, we demonstrate that <nmax> depends on the identity of the hole quencher, and is thus not an intrinsic property of the multiply reduced ZnO nanocrystals themselves. Using a series of substituted borohydride hole quenchers, we show that it is possible to increase the nanocrystal carrier densities over 4-fold relative to previous photodoping reports. When excess lithium and potassium triethylborohydrides are used in the photodoping, formation of Zn(0) is observed. The relationship between metallic Zn(0) formation and ZnO surface electron traps is discussed.

Molecular complexation of some amino acids and triglycine with 18-crown-6 ether in H2O-EtOH solvents at 298.15 K

The influence of composition of H2O-EtOH solvent on the reaction of formation of a molecular complex of 18-crown-6 ether (18C6) with triglycine (3Gly) has been studied at 298.15 K by a thermochemical method. The standard thermodynamic parameters (ΔrG°, ΔrH°, and TΔrS°) of the reaction of [3Gly18C6] complex formation in water-ethanol (H2O-EtOH) solvents having an EtOH mole fraction of 0.0, 0.1, 0.15, 0.2, 0.25, 0.30, and 0.50 have been calculated from the data of calorimetric measurements performed on a TAM III titration microcalorimeter. It has been found that an increase in EtOH concentration in the mixed solvent results in an increase in stability of [3Gly18C6] and in an enhancement in exothermicity of its formation reaction. The water-ethanol solvent has an analogous effect on the stability and energetics of the reactions of formation of molecular complexes of 18C6 with glycine, D,L-alanine, and L-phenylalanine.

At low doses ethanol maintains blood–brain barrier (BBB) integrity after hypoxia and reoxygenation: a brain slice study

Post-ischemia ethanol (EtOH) treatments have been shown to exhibit neuroprotective effects in stroke. However, the mechanisms underlying these effects and those on blood–brain barrier (BBB) integrity have yet to be elucidated. In the present study, we determined whether administering differing concentrations of EtOH alter the expressions of BBB integral proteins, including aquaporins-4 and -9 (AQP-4, AQP-9), matrix metallopeptidases-2 and -9 (MMP-2, MMP-9), zonula occludens-1 (ZO-1), and basal lamina (laminin).We employed an organotypic brain slice culture model that utilizes oxygen–glucose deprivation followed by reoxygenation (OGD/R). Brain slices were obtained from 10-day-old Sprague–Dawley rats and divided into the following five groups (n = 8 subjects per group): (1) control, (2) hypoxia (OGD/R), no EtOH, (3) OGD/R and 10 mM EtOH, (4) OGD/R and 30 mM EtOH, and (5) OGD/R and 90 mM EtOH. To assess BBB integrity, levels of AQPs, MMPs, ZO-1, and laminin were determined by Western blot.Compared to control, OGD/R without EtOH significantly increased AQP-4, AQP-9, MMP-2, and MMP-9 levels, while decreasing ZO-1 and laminin levels. All EtOH concentration treatments (groups 3 through 5) significantly reduced the expressions of AQP-4, AQP-9, MMP-2, and MMP-9, compared to the OGD/R, non-alcohol treated slices. Furthermore, compared to the OGD/R without EtOH group, the 30 mM EtOH treatment significantly increased ZO-1 and laminin levels. In contrast, the 90 mM EtOH level neither enhanced the reduction in AQP and MMP levels nor increased ZO-1 or basal lamina expressions observed in the 30 mM treatment.In conclusion, at an optimal dose of 30 mM, EtOH improves the expressions of MMP-2, MMP-9, AQP-4, AQP-9, ZO-1, and basal laminin, previously altered by OGD/R. These effects may indicate a beneficial effect of EtOH on BBB integrity after stroke.

Impact of Combined Prenatal Ethanol and Prenatal Stress Exposure on Anxiety and Hippocampal-Sensitive Learning in Adult Offspring

Prenatal ethanol (EtOH) and prenatal stress have both been independently shown to induce learning deficits and anxiety behavior in adult offspring. However, the interactive effects of these 2 developmental teratogens on behavioral outcomes have not been systematically evaluated. We combined an established moderate prenatal EtOH consumption paradigm where Long-Evans rat dams voluntarily consume either a 0 or 5% EtOH solution in 0.066% saccharin water (resulting in a mean peak maternal serum EtOH concentration of 84mg/dl) with a novel prenatal stress paradigm. Pregnant rats were exposed to 3% 2,3,5-trimethyl-3-thiazoline (TMT) for 20minutes a day on gestational days 13, 15, 17, and 19. Adult female offspring were evaluated for anxiety-like behavior using an elevated plus-maze and hippocampal-sensitive learning using a 2-trial trace conditioning (TTTC) task. TMT exposure produced a threefold increase in maternal serum corticosterone compared to nonexposed, unhandled controls. Neither prenatal exposure paradigm, either alone or in combination, altered maternal weight gain, EtOH consumption, maternal care of litters, litter size, pup birth weight, or pup weight gain up to weaning. Offspring exposed to prenatal stress displayed significant increases in anxiety-like behavior in the elevated plus maze in terms of open arm entries and time spent on the open arms, with no significant effect of prenatal EtOH exposure and no interaction of the 2 prenatal exposures. Performance in a TTTC task revealed a significant effect of prenatal EtOH exposure on freezing behavior on the testing day, with no significant effect of prenatal stress exposure and no interaction of the 2 prenatal exposures. While each prenatal exposure independently produced different behavioral outcomes, the results indicate that there is no significant interaction of prenatal EtOH and prenatal stress exposures on learning or anxiety at the exposure levels employed in this dual exposure paradigm. Subsequent studies will examine whether similar outcomes occur in male offspring and whether other measures of anxiety or learning are differentially impacted by these prenatal exposure paradigms.

Circadian genes differentially affect tolerance to ethanol in Drosophila.

There is a strong relationship between circadian rhythms and ethanol (EtOH) responses. EtOH consumption has been shown to disrupt physiological and behavioral circadian rhythms in mammals (Alcohol Clin Exp Res 2005b, 29, 1550). The Drosophila central circadian pacemaker is composed of proteins encoded by the per, tim, cyc, and Clk genes. Using Drosophila mutant analysis, we asked whether these central components of the circadian clock make the equivalent contribution toward EtOH tolerance and whether rhythmicity itself is necessary for tolerance. We tested flies carrying mutations in core clock genes for the capacity to acquire EtOH tolerance. Tolerance was assayed by comparing the sedation curves of populations during their first and second sedation. Animals that had acquired tolerance sedated more slowly. Movement was also monitored as the flies breathe the EtOH vapor to determine if other facets of the EtOH response were affected by the mutations. Gas chromatography was used to measure internal EtOH concentration. Constant light was used to nongenetically destabilize the PER and TIM proteins. A group of circadian mutations, all of which eliminate circadian rhythms, do not disrupt tolerance identically. Mutations in per, tim, and cyc completely block tolerance. However, a mutation in Clk does not interfere with tolerance. Constant light also disrupts the capacity to acquire tolerance. These lines did not differ in EtOH absorption. Mutations affecting different parts of the intracellular circadian clock can block the capacity to acquire rapid EtOH tolerance. However, the role of circadian genes in EtOH tolerance is independent of their role in producing circadian rhythmicity. The interference in the capacity to acquire EtOH tolerance by some circadian mutations is not merely a downstream effect of a nonfunctional circadian clock; instead, these circadian genes play an independent role in EtOH tolerance.

Binge Ethanol‐Drinking Potentiates Corticotropin Releasing Factor R1 Receptor Activity in the Ventral Tegmental Area

Corticotropin releasing factor (CRF) and urocortin play an important role in many stress responses and also can regulate ethanol (EtOH) intake. Adaptations in CRF signaling in the central amygdala promote EtOH consumption after long-term EtOH intake in dependent animals and also after brief periods of binge EtOH intake. Thus, even brief episodes of EtOH consumption can alter the function of the CRF system, allowing CRF to regulate EtOH intake. Here, we examined whether brief binge EtOH consumption leads to CRF receptor adaptations within the ventral tegmental area (VTA), a structure involved in signaling rewarding and aversive events and important in the development and expression of drug and alcohol addiction. We utilized a mouse model of binge drinking known as drinking in the dark (DID), where C57BL/6J mice drink approximately 6 g/kg in 4 hours and achieve blood EtOH concentrations of approximately 100 mg/dl, which is equivalent to binge drinking in humans. We used ex vivo whole-cell recordings from putative VTA dopamine (DA) neurons to examine CRF regulation of NMDA receptor (NMDAR) currents. We also examined the impact of CRF receptor antagonist injection in the VTA on binge EtOH intake. Ex vivo whole-cell recordings from putative VTA DA neurons showed enhanced CRF-mediated potentiation of NMDAR currents in juvenile mice that consumed EtOH in the DID procedure. CRF-induced potentiation of NMDAR currents in EtOH-drinking mice was blocked by administration of CP-154,526 (3 μM), a selective CRF1 receptor antagonist. Furthermore, intra-VTA infusion of CP-154,526 (1 μg) significantly reduced binge EtOH consumption in adult mice. These results were not due to alterations of VTA NMDAR number or function, suggesting that binge drinking may enhance signaling through VTA CRF1 receptors onto NMDARs. Altered CRF1 receptor-mediated signaling in the VTA promotes binge-like EtOH consumption in mice, which supports the idea that CRF1 receptors may therefore be a promising pharmacological target for reducing binge drinking in humans.

Real-Time Monitoring of Intracellular cAMP During Acute Ethanol Exposure

In previous studies, we have shown that ethanol (EtOH) enhances the activity of stimulatory G protein (Gs)-stimulated membrane-bound adenylyl cyclase (AC). The effect is AC isoform specific, and the type 7 AC (AC7) is most responsive to EtOH. In this study, we employed a fluorescence resonance energy transfer (FRET)-based cyclic AMP (cAMP) sensor, Epac1-camps, to examine real-time temporal dynamics of EtOH effects on cAMP concentrations. To our knowledge, this is the first report on real-time detection of the EtOH effect on intracellular cAMP. Hela cells were transfected with Epac1-camps, dopamine (DA) receptor D1a , and 1 isoform of AC (AC7 or AC3). Fluorescent images were captured using a specific filter set for cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and FRET, respectively, and FRET intensity was calculated on a pixel-by-pixel basis to examine changes in cAMP. During 2-minute stimulation with DA, the cytoplasmic cAMP level quickly increased and then decreased to a plateau, where the cAMP level was higher than the level prior to stimulation with DA. EtOH concentration dependently increased cytoplasmic cAMP in cells transfected with AC7, while EtOH did not have effect on cells transfected with AC3. Similar trends were observed for cAMP at the plasma membrane and in the nucleus during 2-minute stimulation with DA. Unexpectedly, when cells expressing AC7 were stimulated with DA or other Gs-coupled receptor's ligand plus EtOH for 5 seconds, EtOH reduced cAMP concentration. These results suggest that EtOH has 2 opposing effects on the cAMP-generating system in an AC isoform-specific manner, the enhancing effect on AC activity and the short-lived inhibitory effect. Thus, EtOH may have a different effect on cAMP depending on not only AC isoform but also the duration of exposure.

Embryonic Catalase Protects Against Ethanol-Initiated DNA Oxidation and Teratogenesis in Acatalasemic and Transgenic Human Catalase–Expressing Mice

Reactive oxygen species (ROS) are implicated in fetal alcohol spectrum disorders (FASD) caused by alcohol (ethanol, EtOH). Although catalase detoxifies hydrogen peroxide, embryonic catalase activity is only about 5% of maternal levels. To determine the roles of ROS and embryonic catalase in FASD, pregnant mice with enhanced (expressing human catalase, hCat) or deficient (acatalasemic, aCat) catalase activity, or their respective wild-type (WT) controls, were treated ip on gestational day 9 with 4 or 6g/kg EtOH or its saline vehicle, and embryos and fetuses were, respectively, evaluated for oxidatively damaged DNA and structural anomalies. Untreated hCat and aCat dams had, respectively, more and less offspring than their WT controls. hCat progenies were protected from all EtOH fetal anomalies at the low dose (p < .01) and from reduced head diameter and resorptions at the high dose (p < .001). Conversely, aCat progenies were more sensitive to dose-dependent EtOH fetal anomalies (p < .001) and exhibited a 50% increase in maternal lethality (p < .05) at the high dose. Maternal pretreatment of aCat mice with polyethylene glycol-conjugated catalase (PEG-Cat) reduced EtOH fetal anomalies (p < .001). EtOH-initiated embryonic DNA oxidation was reduced in hCat and WT mice pretreated with PEG-Cat and enhanced in aCat mice. Plasma concentrations of EtOH in catalase-altered mice were similar to controls, precluding a pharmacokinetic basis for altered EtOH teratogenesis. Endogenous embryonic catalase, despite its low level, is an important embryoprotective enzyme for EtOH teratogenesis and a likely determinant of individual risk.

Time-Course of Neuroendocrine Changes and Its Correlation with Hypertension Induced by Ethanol Consumption

Ethanol (ETOH) consumption has been associated with endocrine and autonomic changes, including the development of hypertension. However, the sequence of pathophysiological events underlying the emergence of this effect is poorly understood. This study aimed to establish a time-course correlation between neuroendocrine and cardiovascular changes contributing to the development of hypertension following ETOH consumption. Male adult Wistar rats were subjected to the intake of increasing ETOH concentrations in their drinking water (first week: 5%, second week: 10%, third and fourth weeks: 20% v/v). ETOH consumption decreased plasma and urinary volumes, as well as body weight and fluid intake. Furthermore, plasma osmolality, plasma sodium and urinary osmolality were elevated in the ETOH-treated rats. ETOH intake also induced a progressive increase in the mean arterial pressure (MAP), without affecting heart rate. Initially, this increase in MAP was correlated with increased plasma concentrations of adrenaline and noradrenaline. After the second week of ETOH treatment, plasma catecholamines returned to basal levels, and incremental increases were observed in plasma concentrations of vasopressin (AVP) and angiotensin II (ANG II). Conversely, plasma oxytocin, atrial natriuretic peptide, prolactin and the hypothalamus-pituitary-adrenal axis components were not significantly altered by ETOH. Taken together, these results suggest that increased sympathetic activity may contribute to the early increase in MAP observed in ETOH-treated rats. However, the maintenance of this effect may be predominantly regulated by the long-term increase in the secretion of other circulating factors, such as AVP and ANG II, the secretion of both hormones being stimulated by the ETOH-induced dehydration.

Comparison between different combinations of chemical treatment on parthenogenetic activation of mouse oocytes and its subsequent embryonic development

Combination of different chemicals has been an effective method to activate oocytes. The objectives of this study were to evaluate the optimal: (1) concentration (Experiment 1) and duration (Experiment 2) of strontium chloride (SrCl2) in the presence of cytochalasin B (CB), (2) secondary activation agent [6-dimethylaminopurine (6-DMAP), cycloheximide (CHX) and CB] in combination with calcium ionophore (CaI) (Experiment 3) and (3) ethanol (EtOH) concentration in combination with 6-DMAP (Experiment 4). There were no significant (P>0.05) differences in cleavage (86.82–89.06%) and blastocyst (33.42–46.83%) rates when oocytes were treated with various concentrations of SrCl2 (2–10 mM). Three-hour duration generally showed the highest cleavage (93.83%) and blastocyst rates (60.42%) compared to other durations. Comparing with various combinations, CaI +6-DMAP showed the highest cleavage at all stages of development and significantly (P<0.05) higher from other combinations at 8-cell (50.58%), morula (30.29%) and blastocyst (23.28%) stages. When comparing the EtOH concentrations, 8% EtOH gave significantly (P<0.05) highest cleavage rates at 8-cell stage (39.70%), morula (21.50%) and blastocyst (13.86%) stages followed by 9% and 7% EtOH. In conclusion, 10 mM SrCl2+5 µg/ml CB in calcium-free Chatot Ziomek Bavister medium for 3-h incubation duration gave the highest cleavage rates of parthenotes mouse oocytes.

Calpain Inhibition Prevents Ethanol-Induced Alterations in Spinal Motoneurons

Long-term exposure of ethanol (EtOH) alters the structure and function in brain and spinal cord. The present study addresses the mechanisms of EtOH-induced damaging effects on spinal motoneurons in vitro. Altered morphology and biochemical changes of such damage were demonstrated by in situ Wright staining and DNA ladder assay. EtOH at low to moderate (25-50mM) concentrations induced damaging effects in the motoneuronal scaffold which involved activation of proteases like μ-calpain and caspase-3. Caspase-8 was seen only at higher (100mM) EtOH concentration. Further, pretreatment with calpeptin, a potent calpain inhibitor, confirmed the involvement of active proteases in EtOH-induced damage to motoneurons. The lysosomal enzyme cathepsin D was also elevated in the motoneurons by EtOH, and this effect was significantly attenuated by inhibitor treatment. Overall, EtOH exposure rendered spinal motoneurons vulnerable to damage, and calpeptin provided protection, suggesting a critical role of calpain activation in EtOH-induced alterations in spinal motoneurons.

Inactivation of the Tulane Virus, a Novel Surrogate for the Human Norovirus

Human noroviruses (HuNoVs) are the major cause of nonbacterial gastroenteritis epidemics. The culturable feline calicivirus and murine norovirus have been used extensively as surrogates to study HuNoV biology, as HuNoV does not grow in vitro. Additional efforts to identify new surrogates are needed, because neither of these common surrogates are truly intestinal pathogens. The newly described Tulane virus (TV) is a typical calicivirus, it is isolated from macaque stools, is cultivable in vitro, and recognizes human histo-blood group antigens. Therefore, TV is a promising surrogate for HuNoVs. In this study, we evaluated the resistance or stability of TV under various physical and environmental conditions by measuring a 50% reduction of tissue culture infective dose (TCID50) by using a TV cell culture system. Due to the nature of this virus, it is hard to produce a high-titer stock through tissue culture. In our study, the maximal reduction in virus titers was 5D (D = 1 log) in heat-denaturation and EtOH experiments, and 4D in UV, chlorine, and pH-stability experiments. Therefore in this study, we defined the inactivation of TV as reaching a TCID50/ml of 0 (a 4- to 5-D reduction in TCID50, depending on the detection limit). TV was inactivated after incubation at 63 °C for 5 min, incubation at 56 °C for 30 min (5D), exposure to 60 mJ/cm2 of UVC radiation (4D), or incubation at 300 ppm of free chlorine for 10 min (4D). TV was shown to be stable from pH 3.0 to 8.0, though an obvious reduction in virus titer was observed at pH 2.5 and 9.0, and was inactivated at pH 10.0 (4D). TV was resistant to a low concentration of EtOH (40% or lower) but was fully inactivated (5D) by 50 to 70% EtOH after a short exposure (20 s). In contrast, quantitative real-time PCR was unable to detect, or poorly detected, virus titer reductions between treated and untreated samples described in this study.