EtOH Concentrations Research Articles

Ethanol Blocks Adenosine Uptake via Inhibiting the Nucleoside Transport System in Bronchial Epithelial Cells

Adenosine uptake into cells by nucleoside transporters plays a significant role in governing extracellular adenosine concentration. Extracellular adenosine is an important signaling molecule that modulates many cellular functions via 4 G-protein-coupled receptor subtypes (A(1), A(2A), A(2B), and A(3)). Previously, we demonstrated that adenosine is critical in maintaining airway homeostasis and airway repair and that airway host defenses are impaired by alcohol. Taken together, we hypothesized that ethanol impairs adenosine uptake via the nucleoside transport system. To examine ethanol-induced alteration on adenosine transport, we used a human bronchial epithelial cell line (BEAS-2B). Cells were preincubated for 10 minutes in the presence and absence of varying concentrations of ethanol (EtOH). In addition, some cells were pretreated with S-(4-Nitrobenzyl)-6-thioinosine (100 microM: NBT), a potent adenosine uptake inhibitor. Uptake was then determined by addition of [(3)H]-adenosine at various time intervals. Increasing EtOH concentrations resulted in increasing inhibition of adenosine uptake when measured at 1 minute. Cells pretreated with NBT effectively blocked adenosine uptake. In addition, short-term EtOH revealed increased extracellular adenosine concentration. Conversely, adenosine transport became desensitized in cells exposed to EtOH (100 mM) for 24 hours. To determine the mechanism of EtOH-induced desensitization of adenosine transport, cAMP activity was assessed in response to EtOH. Short-term EtOH exposure (10 minutes) had little or no effect on adenosine-mediated cAMP activation, whereas long-term EtOH exposure (24 hours) blocked adenosine-mediated cAMP activation. Western blot analysis of lysates from unstimulated BEAS-2B cells detected a single 55 kDa band indicating the presence of hENT1 and hENT2, respectively. Real-time RT-PCR of RNA from BEAS-2B revealed transcriptional expression of ENT1 and ENT2. Collectively, these data reveal that acute exposure of cells to EtOH inhibits adenosine uptake via a nucleoside transporter, and chronic exposure of cells to EtOH desensitizes the adenosine transporter to these inhibitory effects of ethanol. Furthermore, our data suggest that inhibition of adenosine uptake by EtOH leads to an increased extracellular adenosine accumulation, influencing the effect of adenosine at the epithelial cell surface, which may alter airway homeostasis.

Determining Degree of Saturation after Application of Transiently Supersaturated Metered Dose Aerosols for Topical Delivery of Corticosteroids

A transiently supersaturated drug delivery system has the potential to enhance topical drug delivery via heightened thermodynamic activity. The aim of this work was to quantify the degree of saturation (DS) for transiently supersaturated formulations using three traditional and one novel in vitro assessment methods. Metered dose aerosols (MDA) were formulated containing saturated levels of beclomethasone dipropionate monohydrate (BDP) or betamethasone 17‐valerate (BMV) within a pressurised canister, and included ethanol (EtOH), hydrofluoroalkane 134a propellant and poly(vinyl pyrrolidone). Attempts to determine the DS via the measurement of drug flux through synthetic membranes did not correlate and was shown to be dependent on the EtOH concentration. The inability of these methods to accurately assess the drug DS may be due to the transient nature of the formulation and the volatile solvents dehydrating the membrane. A mathematical equation that used the evaporation rate of the formulation was derived to determine the theoretical DS at various time points after MDA actuation. It was shown that the MDAs became supersaturated with a high DS, this enhanced drug release from the formulation and therefore these preparations have the potential to increase the amount of drug delivered into the skin. © 2008 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:543–554, 2009

Dose-Dependent Effects of Ethanol and E. Coli on Gut Permeability and Cytokine Production

The gut may prime inflammatory responses following shock/trauma insults. Ethanol (EtOH) use is common in trauma patients and may impair intestinal barrier function. We compared varying concentrations of EtOH on proinflammatory cytokine production of Caco2 cell monolayers and the resultant changes in barrier function. We hypothesized that even low concentrations of EtOH would cause significant cytokine release and barrier dysfunction in vitro. Confluent Caco2 cell monolayers were grown in a two-chamber culture system and exposed to varying concentrations of EtOH (0.1%, 0.5%, 1.0%, 1.5%, and 2.0%) with/without Escherichia coli C-25 (EC). Supernatants were collected and TNF and IL6 quantified by ELISA (pg/mL). Monolayer integrity was assessed by apoptosis and permeability measurements. Caco2 production of TNF-alpha increased in a dose-dependent manner when incubated with increasing concentrations of EtoH. A synergistic effect was seen when E. coli was added to the apical chamber. A similar result was seen with the production of IL-6. A dose-dependent effect was also noted with EtOH with or without E. coli on apoptosis and permeability measurements. In addition to alterations in gut permeability, increasing concentrations of ethanol have a synergistic effect with E. coli on Caco2 production of proinflammatory cytokines TNF and IL-6. The creation of a proinflammatory cytokine milieu with an altered barrier integrity may be a mechanism by which ethanol may increase septic complications in the injured patient.

Ethanol increases the distribution of MDMA to the rat brain: possible implications in the ethanol-induced potentiation of the psychostimulant effects of MDMA

Ecstasy (3,4-methylenedioxymethylamphetamine; MDMA) is a popular club drug often taken with ethanol (EtOH). We recently found EtOH potentiated the psychomotor effects of MDMA in rats. This potentiation could reflect pharmacodynamic or/and pharmacokinetic processes. To test the latter hypothesis, rats were injected i.p. with 6.6 or 10 mg/kg MDMA with or without 1.5 g/kg EtOH, and were killed at 5, 15 or 60 min after injection. MDMA, its primary metabolite, 3,4-methylenedioxyamphetamine (MDA), and EtOH concentrations were determined in the plasma and the hippocampus, frontal cortex and striatum at each time-point. EtOH potentiated MDMA-induced hyperactivity mainly during the first 60 min post-administration. Fifteen and 60 min after treatment with MDMA and EtOH, MDMA concentrations were greater than after MDMA alone in the blood and the three brain regions examined. EtOH, however, did not increase the fraction of MDMA converted to MDA, as shown by unaltered MDA/MDMA ratios at either MDMA dose. Interestingly, when combined with EtOH, the distribution of MDMA and MDA in the brain was not homogeneous. Concentrations of both were much higher in the striatum and cortex, than in the hippocampus. Thus, at least part of the potentiation of the MDMA-induced hyperlocomotion by EtOH might be the result of a higher concentration of MDMA and metabolites in the blood and brain. Our results present clear evidence that EtOH increases brain and blood concentrations of MDMA and leads to the possibility of both enhanced MDMA-based neurotoxicity and increased liability for abuse.

Chronic candidosis and oral cancer in APECED‐patients: Production of carcinogenic acetaldehyde from glucose and ethanol by Candida albicans

According to our results all C. albicans isolates analysed were capable of producing carcinogenic levels of ACH from EtOH. The isolates from cancer and control subjects produced significantly higher amounts of ACH than those from APECED patients but the clinical relevance of this difference is questionable since all isolates produced very high amounts. The 11 mM EtOH concentration used can be found in saliva for hours after social alcohol consumption or naturally in many products produced by fermentation.17The main finding of this study is that C. albicans may play a role in metabolizing glucose to carcinogenic ACH in the mouth and could participate in the pathogenesis of oral cancer in non-alcohol drinkers. The observation supports the concept of a novel microbially mediated mechanism in the pathogenesis of oral cancer and could partly explain why chronic oral candidosis is carcinogenic in APECED patients and why they have a high risk for oral cancer at an early age.

Voluntary Ethanol Consumption Differs in Adolescent and Adult Male Rats Using a Modified Sucrose‐Fading Paradigm

Initiation of alcohol consumption during adolescence is high, which usually begins with consumption of highly concentrated sweetened alcoholic beverages in adolescent humans. Enhanced voluntary ethanol (EtOH) intake has been observed previously in adolescent relative to adult rats under continuous access conditions using sweetened EtOH solutions. The present set of experiments investigated patterns of voluntary EtOH intake in adolescent and adult rats using sweetened EtOH solutions in a limited access paradigm. Rats were trained with modified sucrose-substitution protocols that ended at either 5% sucrose-20% EtOH (5S/20E) (Exp. 1) or 5% sucrose-10% EtOH (5S/10E) (Exp. 2). Voluntary EtOH consumption differences between the 2 age groups were apparent at higher (i.e., 10 and 20%), but not lower (i.e., 2 and 5%) EtOH concentrations. Adolescent rats consumed more EtOH on a g/kg basis only at 20% EtOH (Exp. 1). Adolescent rats voluntarily consumed more EtOH than adults when maintained at 5S/10E (Exp. 2). To assess whether these age-related differences in voluntary EtOH intake were concentration dependent, rats were trained with 5S/20E and subsequently trained with decreasing EtOH concentrations (i.e., 5S/10E and 5S/5E). Adolescents consumed more EtOH when initially presented with the 5S/10E and 5S/20E EtOH concentrations, and subsequently at the lower 5S/5E EtOH concentration (Exp. 3). There were no differences in preference for the sucrose-only solution, however adolescents tended to consume more sucrose at the 5S sucrose concentration (Exp. 4). Given that adolescents consumed more EtOH at the 5S/10E and 5S/20E, but not at the 5S/5E EtOH concentrations, preference for sucrose does not solely explain the age differences in voluntary EtOH intake observed. Overall, results replicate previous work, demonstrating adolescent rats consume more EtOH relative to adults. However, the present results were observed using sweetened EtOH solutions in a limited access paradigm. The present modified sucrose-substitution paradigm may serve as a valid model of human adolescent drinking behavior.

Physiology and pharmacology of alcohol: the imidazobenzodiazepine alcohol antagonist site on subtypes of GABAA receptors as an opportunity for drug development?

Alcohol (ethanol, EtOH) has pleiotropic actions and induces a number of acute and long-term effects due to direct actions on alcohol targets, and effects of alcohol metabolites and metabolism. Many detrimental health consequences are due to EtOH metabolism and metabolites, in particular acetaldehyde, whose high reactivity leads to nonspecific chemical modifications of proteins and nucleic acids. Like acetaldehyde, alcohol has been widely considered a nonspecific drug, despite rather persuasive evidence implicating inhibitory GABA(A) receptors (GABA(A)Rs) in acute alcohol actions, for example, a GABA(A)R ligand, the imidazobenzodiazepine Ro15-4513 antagonizes many low-to-moderate dose alcohol actions in mammals. It was therefore rather surprising that abundant types of synaptic GABA(A)Rs are generally not responsive to relevant low concentrations of EtOH. In contrast, delta-subunit-containing GABA(A)Rs and extrasynaptic tonic GABA currents mediated by these receptors are sensitive to alcohol concentrations that are reached in blood and tissues during low-to-moderate alcohol consumption. We recently showed that low-dose alcohol enhancement on highly alcohol-sensitive GABA(A)R subtypes is antagonized by Ro15-4513 in an apparently competitive manner, providing a molecular explanation for behavioural Ro15-4513 alcohol antagonism. The identification of a Ro15-4513/EtOH binding site on unique GABA(A)R subtypes opens the possibility to characterize this alcohol site(s) and screen for compounds that modulate the function of EtOH/Ro15-4513-sensitive GABA(A)Rs. The utility of such drugs might range from novel alcohol antagonists that might be useful in the emergency room, to drugs for the treatment of alcoholism, as well as alcohol-mimetic drugs to harness acute positive effects of alcohol.

Effect of operational parameters on degradation of Malachite Green by ultrasonic irradiation

The aim of the present study was to apply ultrasonic technique to remove Malachite Oxalate Green (MG) from aqueous solution. An ultrasonic bath with frequency of 35 kHz was used to investigate the effect of different operational parameters such as MG concentration, power density, temperature, mechanical agitation and addition of EtOH, 2-PrOH and iso-BuOH. Decolorization of MG follows a first order kinetics and hydroxyl radicals have an important role in degradation of MG. The apparent reaction rate constant ( k ap) was influenced by variation of operational parameters. The activation energy was 30.95 kJ/mol in temperature range of 21–34 °C, suggesting a diffusion-controlled reaction. Alcohols act as hydroxyl radicals scavengers having undesirable contribution. UV–vis spectral change of MG showed hypsochromic shift occurred with increasing sonication time, proposing N-demethylation process of MG.

Direct measurements of the ozone formation potential from dairy cattle emissions using a transportable smog chamber

Tropospheric ozone continues to be an air pollution problem in the United States, particularly in California, Texas, and across the eastern seaboard. The obvious sources of ozone precursors have been largely controlled over the past several decades, leading to the critical examination of secondary sources. In particular, California has new air quality rules addressing agricultural sources of ozone precursors, including dairy farms. Some recent estimates predict that dairy cattle are second only to on-road vehicles as a leading source of ozone precursor emissions in California's San Joaquin Valley. The objective of this work was to directly measure the ozone formation potential from dairy housing. A transportable “smog” chamber was constructed and validated using organic gases known to be present in dairy emissions. The ozone formation potential of emissions from eight non-lactating dairy cows and their fresh waste was then directly evaluated in the field at a completely enclosed cow corral on the campus of the University of California, Davis. The results demonstrate that the majority of the ozone formation is explained by ethanol (EtOH) in the emissions from the dairy cows, not by acetone as previously thought. Ozone formation potential is generally small, with <20 ppb of ozone produced under typical conditions when EtOH concentrations were ∼200 ppb and NO x concentrations were ∼50 ppb. The results match our current understanding of atmospheric ozone formation potential, ruling out the possibility of unknown organic compounds in dairy emissions with significant ozone formation potential. Simulations carried out with a modified form of the Caltech Atmospheric Chemistry Mechanism verify that actual ozone formation from dairy emissions is much lower than what would be predicted using the current regulatory profiles. Based on these results, the ozone formation potential of emissions from dairy cattle in California seems to be lower than previously estimated.

Ethanol alters neutrophil membrane mechanics and adhesion molecule expression

Ethanol (EtOH) has been linked to both protective and damaging effects in inflammatory and immune responses of leukocytes. To characterize EtOH effect on neutrophil mechanics, we studied neutrophil rolling, tethering, and firm arrest behavior at physiologically relevant EtOH concentrations achieved in humans by alcohol consumption (&lt;0.5 v/v%). Neutrophils were perfused over parallel‐plate flow chambers with P‐selectin‐coated beads or confluent HAEC (human aortic endothelial cells) monolayer at a venous shear rate of 100 s−1. EtOH decreased the adhesion efficiency of neutrophils from 0.085 to 0.031, increased the average tether length and tether growth velocity fourfold, and decreased bond forces, suggesting a lower degree of membrane‐cytoskeleton association. When perfused over IL‐1‐activated HAEC, the fraction of neutrophils converting to firm arrest decreased from 36% to 24% with EtOH, and average rolling length and rolling velocity both increased with EtOH (24.8 to 33.7 μm and 2.5 to 3.4 m/sec, respectively). EtOH resulted in ~20% increase in L‐selectin and PSGL‐1 expression in quiescent neutrophils as measured by flow cytometry, and significantly inhibited the upregulation of CD11b and shedding of L‐selectin in fMLP‐activated neutrophils. Our results indicate that EtOH affects bond mechanics and surface molecules to change neutrophil response. This work was supported by NIH R01 HL56621.

In Vivo Time‐Course Changes in Ethanol Levels Sampled With Subcutaneous Microdialysis

The objective of this study was to determine time-course changes in in vivo ethanol (EtOH) concentrations using a novel subcutaneous (s.c.) microdialysis sampling technique. The hypothesis to be tested was that EtOH concentrations in the s.c. fluid would reflect blood EtOH concentrations. If this is the case, then s.c. microdialysis could allow a more detailed analysis of changes in in vivo levels of EtOH under different drinking paradigms. Adult male and female Wistar rats and male alcohol-preferring (P) rats were used in this study. A loop-style microdialysis probe was designed for s.c. applications. After initial in vitro characterization, probes were implanted under the skin between the shoulder blades. Animals were allowed to recover 4 to 24 hours prior to microdialysis collection (2.0 microl/min flow rate with isotonic saline). In vivo microdialysis experiments were then conducted to determine (i) the extraction fraction (or clearance) using EtOH no-net-flux (NNF) coupled with the alcohol clamp method, (ii) the dose-response and time-course effects after systemic EtOH administration and to compare with blood EtOH levels, and (iii) the time-course changes in EtOH levels during and after an EtOH drinking episode. In vivo probe recovery (extraction fraction) obtained using the alcohol clamp method was 69 +/- 3%, and was comparable to the in vitro recovery of 73 +/- 2%. For the EtOH dose-response experiment, rats injected i.p. with 0.5, 1.0, or 2.0 g/kg EtOH showed a clear dose-response effect in the s.c. dialysate samples. Peak concentrations (70, 123, and 203 mg%, respectively) were reached by 15 minutes after injection. In an experiment comparing levels of EtOH in s.c. dialysis and arterial blood samples in rats administered 1.0 g/kg EtOH, similar time-course changes in in vivo EtOH concentrations were observed with both i.g. and i.p. EtOH administration. In P rats drinking 15% EtOH during a 1-hour scheduled access period, EtOH levels in s.c. microdialysates rose rapidly over the session and peaked at approximately 50 mg% at 60 to 80 minutes. Overall, these experiments indicate that s.c. EtOH and blood EtOH concentrations follow a similar time course. Moreover, s.c. microdialysis can be useful as an experimental approach for determining detailed time-course changes in in vivo EtOH concentrations associated with alcohol drinking episodes.

Putative Role of Brain Acetaldehyde in Ethanol Addiction

The putative contribution of brain acetaldehyde (AcH) to ethanol (EtOH) tolerance and dependence (addiction) is reviewed. Although the role of AcH in EtOH addiction has been controversial, there are data showing a relationship. AcH can be formed in the brain tissues through the peroxidatic activity of catalase and by oxidation via other oxidizing enzymes such as cytochrome P-4502E1. Significant formation of AcH occurs in vitro in brain tissue at concentrations of EtOH that can be achieved by voluntary consumption of EtOH by rodents. AcH itself possesses reinforcing properties, which suggests that some of the behavioral pharmacological effects attributed to EtOH may be a result of the formation of AcH, and supports the involvement of AcH in EtOH addiction. Modulation of aldehyde dehydrogenase (ALDH) and brain catalase activity can change EtOH-related addictive behaviors presumably by changing AcH levels. Moreover, some condensation reaction products of AcH may promote some actions of EtOH and its consumption. On the basis of the findings, it can be concluded that AcH may mediate some of the CNS actions of EtOH including tolerance and dependence, although further exploration the involvement of AcH in EtOH addiction is warranted.

Ethanol Increases Fetal Human Neurosphere Size and Alters Adhesion Molecule Gene Expression

Ethanol (ETOH) consumption by pregnant women can result in Fetal Alcohol Spectrum Disorder (FASD). To date, the cellular targets and mechanisms responsible for FASD are not fully characterized. Our aim was to determine if ETOH can affect fetal human brain-derived neural progenitor cells (NPC). Neural progenitor cells were isolated by positive selection from normal second trimester fetal human brains (n = 4) and cultured, for up to 72 hours, in mitogenic media containing 0, 1, 10, or 100 mM ETOH. From 48 to 72 hours in culture, neurospheres generated in these conditions were filmed using time-lapse video microscopy. At the end of 72 hours, neurosphere diameter and roundness were measured using videographic software. Mitotic phase analysis of cell-cycle activity and apoptotic cell count were also performed at this time, by flow cytometry using propidium iodide (PI) staining. Real-time PCR was used to estimate expression of genes associated with cell adhesion pathways. Neurosphere diameter correlated positively (r = 0.87) with increasing ETOH concentrations. There was no significant difference in cell-cycle activity and no significant increase in apoptosis with increasing ETOH concentrations. Time-lapse video microscopy showed that ETOH (100 mM) reduced the time for neurosphere coalescence. Real-time PCR analysis showed that ETOH significantly altered the expression of genes involved in cell adhesion. There was an increase in the expression of alpha and beta Laminins 1, beta Integrins 3 and 5, Secreted phosphoprotein1 and Sarcoglycan epsilon. No change in the expression of beta Actin was observed while the expression of beta Integrin 2 was significantly suppressed. ETOH had no effect on NPC apoptosis but, resulted in more rapid coalescence and increased volume of neurospheres. Additionally, the expression of genes associated with cell adhesion was significantly altered. ETOH induced changes in NPC surface adhesion interactions may underlie aspects of neurodevelopmental abnormalities in FASD.

Microplasma discharge in ethanol solution: Characterization and its application to the synthesis of carbon microstructures

Radio-frequency (rf) microplasma discharge of argon was successfully generated within an ethanol (EtOH) solution and applied for the synthesis of carbon microstructures. The plasma state depends on the flow rate of argon as well as the EtOH concentration. The argon related species are consumed mostly for dissociating EtOH at concentration of over 10%. The carbon related microstructures such as particle and nanotubes (CNTs) were formed directly on c-Si utilizing a microplasma jet of argon generated within ethanol solution, the average size and number density of carbon related products could be controlled by adjusting the ethanol concentration. The determining factor of average size and number density of carbon microstructures are discussed in terms of the surface reaction within ethanol solution.

TiO2 nanowires array fabrication and gas sensing properties

A cheap nanofabrication process for titania (TiO2) polycrystalline nanowire array for gas sensing applications with lateral size ranging from 90 to 180 nm, and gas sensing characterizations are presented. Alternatively to typical pattern transfer techniques for submicron fabrication, authors focused on a standard 365 nm UV photolithographic process able to fabricate sol–gel nanostructured titania nanowires from a solid thin film. Main aim of present work is the experimental validation of enhanced gas sensing response of nanopatterned metal oxide thin film sensors. Two different kinds of gas sensor with nanopatterned sensitive area have been realized onto silicon substrates and tested towards different EtOH concentrations; experimental tests have been carried out with a contemporary output signals collection from a nanowires-based gas sensor and a second device with solid sensitive film without patterning, in order to validate effects of nanomachining on sensitive material response.

Matching Law Choice Analyses of Ethanol and Sucrose Consumption in Alcohol‐Preferring (P), Nonpreferring (NP), and Sprague–Dawley (SD) Rats

Quantitative models of choice, such as the matching law and behavioral economics, allow for the analysis of alcohol consumption within a choice context. Such models can provide further knowledge of phenotypic differences between selected lines of rats. The current study applied the generalized matching law to the ethanol (EtOH) and sucrose consumption of alcohol-preferring (P), nonpreferring (NP), and Sprague-Dawley (SD) rats. Male P, NP, and SD rats were trained to consume increasing concentrations of EtOH and water in a limited access, 2-bottle choice procedure. The animals were then given concurrent access to pairs of differing EtOH solutions (2, 4, 6, and 10%, v/v) for 1 h/d. A subset of animals from each rat type was subsequently given access to 8% sucrose for 1 h/d for 5 days, and then concurrent access to pairs of differing sucrose solutions (3, 8, and 15%, w/v) for 1 h/d. The generalized matching law was used to assess the relationship between the relative EtOH and sucrose concentrations and the relative volumes of solutions consumed. The P rats consumed more EtOH and sucrose than the NP and SD rats overall. However, only the P rats consistently consumed disproportionately greater quantities of the higher EtOH concentration than the lower one, indicating overmatching. Additionally, all 3 types of rats displayed overmatching in the sucrose choice experiment. These data provide further evidence of phenotypic differences between the P rats and other types of rats, and suggest that the generalized matching law can be a useful tool for describing rats' EtOH or sucrose consumption in a 2-bottle choice, limited access paradigm.

Catalase mediates acetaldehyde formation in the striatum of free-moving rats

Using brain microdialysis, we measured both ethanol (EtOH) and acetaldehyde (AcH) levels in the striatum of free-moving rats following the inhibition of EtOH oxidation pathways. Rats received intraperitoneal EtOH (1 g/kg) alone or in combination with 4-methylpyrazole (MP, 82 mg/kg, an alcohol dehydrogenase inhibitor), and/or catalase inhibitor sodium azide (AZ, 10 mg/kg) or 3-amino-1,2,4-triazole (AT, 1 g/kg), and/or cyanamide (CY, 50 mg/kg, an aldehyde dehydrogenase inhibitor). Results revealed that both EtOH and AcH concentrations reached a plateau at 30 min after a dose of EtOH, and then gradually decreased for 4 h. AcH was identified in the CY + EtOH, CY + AT/AZ + EtOH, and CY + 4-MP + EtOH groups. The CY + EtOH-induced peak AcH level was 195.2 ± 19.4 μM, and this level was significantly higher than the values in other groups studied. The catalase or ADH inhibitor in combination with CY lowered considerably the AcH concentration in the brain. The EtOH level reached a maximum of 25.9 ± 2.3 mM in the CY + 4-MP + EtOH group, and this level was markedly higher than in the EtOH group. No significant difference in brain EtOH levels was seen in any of the other groups examined. The findings strongly support the assumption that the enzyme catalase plays a significant role in AcH formation directly in the rat brain.

Cytotoxicity of Insulin within its Self-assembly and Amyloidogenic Pathways

Solvational perturbations were employed to selectively tune the aggregational preferences of insulin at 60 °C in vitro in purely aqueous acidic solution and in the presence of the model co-solvent ethanol (EtOH) (at 40%(w/w)). Dynamic light scattering (DLS), thioflavin T (ThT)-fluorescence, Fourier transform infrared (FTIR) and atomic force microscopy (AFM) techniques were employed to characterize these pathways biophysically with respect to the pre-aggregational assembly of the protein, the aggregation kinetics, and finally the aggregate secondary structure and morphology. Using cell viability assays, the results were subsequently correlated with the cytotoxicity of the insulin species that form in the two distinct aggregation pathways. In the cosolvent-free solution, predominantly dimeric insulin self-assembles via the well-known amyloidogenic pathway, yielding exclusively fibrillar aggregates, whereas in the solution containing EtOH, the aggregation of predominantly monomeric insulin proceeds via a pathway that leads to exclusively non-fibrillar, amorphous aggregates. Initially present native insulin assemblies as well as partially unfolded monomeric species and low molecular mass oligomeric aggregates could be ruled out as direct and major cytotoxic species. Apart from the slower overall aggregation kinetics under amorphous aggregate promoting conditions, which is due to the chaotropic nature of high EtOH concentrations, however, both pathways were unexpectedly found to evoke insulin aggregates that were cytotoxic to cultured rat insulinoma cells. The observed kinetics of the decrease of cell viabilities correlated well with the results of the DLS, ThT, FTIR and AFM studies, revealing that the formation of cytotoxic species correlated well with the formation of large-sized, β-sheet-rich assemblies (>500 nm) of both fibrillar and amorphous nature. These results suggest that large-sized, β-sheet-rich insulin assemblies of both fibrillar and amorphous nature are toxic to pancreatic β-cells. In the light of the ongoing discussion about putative cytotoxic effects of prefibrillar and fibrillar amyloid aggregates, our results support the hypothesis that, in the case of insulin, factors other than the specific secondary or quarternary structural features of the various different aggregates may define their cytotoxic properties. Two such factors might be the aggregate size and the aggregate propensity to expose hydrophobic surfaces to a polar environment.

Sex differences in the effect of finasteride on acute ethanol withdrawal severity in C57BL/6J and DBA/2J mice

The neurosteroid allopregnanolone (ALLO) is a potent positive modulator of GABA A receptors that can modulate ethanol (EtOH) withdrawal. The 5α-reductase inhibitor finasteride can block the formation of ALLO and other GABAergic neurosteroids and also reduce certain effects of EtOH. Treatment with finasteride during chronic EtOH exposure decreased EtOH withdrawal severity and blood EtOH concentrations (BECs), suggesting an additional effect of finasteride on EtOH pharmacokinetics. Thus, the purpose of the present study was to determine the effect of finasteride on acute EtOH withdrawal severity, to minimize the effect of finasteride on EtOH metabolism. Male and female C57BL/6J and DBA/2J mice received a pretreatment of finasteride (50 mg/kg i.p.) or vehicle 24 h prior to an injection of EtOH (4 g/kg i.p.) or saline. Handling-induced convulsions (HICs) were scored at baseline, and then over a 24 h period after EtOH or saline injection. In another experiment, plasma estradiol and corticosterone levels were assessed at selected time points (0, 2, 8, and 24 h). In a final study, retro-orbital blood samples were collected at 30, 60, 120, and 240 min post-EtOH administration to access finasteride’s effects on EtOH clearance parameters. Pretreatment with finasteride increased acute EtOH withdrawal severity in female C57BL/6J and DBA/2J mice but decreased withdrawal severity in male mice of both strains. Finasteride did not alter BECs, EtOH clearance, estradiol, or corticosterone concentrations in a manner that appeared to contribute to the sex difference in finasteride’s effect on acute EtOH withdrawal severity. These findings suggest that male and female C57BL/6J and DBA/2J mice differ in their sensitivity to changes in ALLO or other GABAergic neurosteroid levels during acute EtOH withdrawal. Sex differences in the modulation of GABAergic 5α-reduced steroids may be an important consideration in understanding and developing therapeutic interventions in alcoholics.

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.