In vivo contribution of Class III alcohol dehydrogenase (ADH3) to alcohol metabolism through activation by cytoplasmic solution hydrophobicity

Alcohol metabolism in vivo cannot be explained solely by the action of the classical alcohol dehydrogenase, Class I ADH (ADH1). Over the past three decades, attempts to identify the metabolizing enzymes responsible for the ADH1-independent pathway have focused on the microsomal ethanol oxidizing system (MEOS) and catalase, but have failed to clarify their roles in systemic alcohol metabolism. In this study, we used Adh3-null mutant mice to demonstrate that Class III ADH (ADH3), a ubiquitous enzyme of ancient origin, contributes to alcohol metabolism in vivo dose-dependently resulting in a diminution of acute alcohol intoxication. Although the ethanol oxidation activity of ADH3 in vitro is low due to its very high Km, it was found to exhibit a markedly enhanced catalytic efficiency (kcat/Km) toward ethanol when the solution hydrophobicity of the reaction medium was increased with a hydrophobic substance. Confocal laser scanning microscopy with Nile red as a hydrophobic probe revealed a cytoplasmic solution of mouse liver cells to be much more hydrophobic than the buffer solution used for in vitro experiments. So, the in vivo contribution of high-Km ADH3 to alcohol metabolism is likely to involve activation in a hydrophobic solution. Thus, the present study demonstrated that ADH3 plays an important role in systemic ethanol metabolism at higher levels of blood ethanol through activation by cytoplasmic solution hydrophobicity.     

Mechanism of ethanol inhibition of fermentation in Zymomonas mobilis CP4.

Accumulation of alcohol during fermentation is accompanied by a progressive decrease in the rate of sugar conversion to ethanol. In this study, we provided evidence that inhibition of fermentation by ethanol can be attributed to an indirect effect of ethanol on the enzymes of glycolysis involving the plasma membrane. Ethanol decreased the effectiveness of the plasma membrane as a semipermeable barrier, allowing leakage of essential cofactors and coenzymes. This leakage of cofactors and coenzymes, coupled with possible additional leakage of intermediary metabolites en route to ethanol formation, is sufficient to explain the inhibitory effects of ethanol on fermentation in Zymomonas mobilis.     

Lack of first-pass metabolism of ethanol at blood concentrations in the social drinking range

Previously published data are displayed in a new manner and show that there is complete systemic availability of oral doses of 23-47 g of ethanol (0.35-0.75 g/kg or 30-60 ml of 95% ethanol) in man when administered in the fasting state relative to an intravenous infusion of the same doses administered over a 2-hr period. A previous report by other authors that oral ethanol (0.15 g/kg) in man had a mean systemic availability of only 29% is explained by the fact that the subjects were fed one hour prior to administration of the alcohol and that the intravenous dose was infused over only a 20 minute period.     

Evaluation of accelerated development of stable alcoholic motivation in rats for studying potential alcohol deterrents

The express technique reflecting an acquisition of a clear alcohol addiction during short-term voluntary alcoholization for further antialcoholic drugs testing was performed in male albino rats. By VARIMAX factor analysis of indexes related with preference of alcohol solutions with different tastes the conditions of short-term (2 months) voluntary alcoholization leading to persistent ethanol intake were studied. Isolation stress inducing a specific alcohol drive was excluded from rearing conditions. 0.1% saccharin solution in 15% ethanol was used for alcoholization. Statistical analysis revealed factor of "developed alcohol abuse" which may be detected in conditions of one-trail sweet ethanol intake after 3 days alcohol deprivation (similar to heavy drinking syndrome in humans). Using pharmacological drugs (pyrazidol, piracetam) validity of the method for specific drug design was confirmed.     

Source and avoidance of the "nothing dehydrogenase" effect, a spurious band produced during polyacrylamide gel electrophoresis of dehydrogenase enzymes from yeasts

During a study of the distribution of several NAD-linked dehydrogenase enzymes in various yeasts, in which polyacrylamide gel electrophoresis, followed by activity staining with phenazine methosulfate and a tetrazolium was used, a band was frequently detected, the production of which appeared to be independent of any added substrate (the "nothing dehydrogenase" effect). It has been shown that this effect is caused by alcohol dehydrogenase acting on traces of ethanol inadvertently introduced into the system. Two sources of ethanol were identified. They were (i) the enzyme extracts, which could be freed from ethanol by gel filtration, and (ii) the acrylamide used to prepare the gel, which could be freed from ethanol by recrystallization from ethanol-free chloroform. It is suggested that the use of commercial chloroform (stabilized with ethanol) as a recrystallizing solvent is the source of ethanol contamination in commercial preparations of acrylamide.     

Involvement of Serotonin in the Pathogenesis of Alcohol Addiction

We studied the contents of serotonin (5-HT) in a few brain structures (hypothalamus, midbrain, and neocortex) and in blood of rats with genetically determined preference of either ethanol solution or water as a liquid for drinking (groups preferring ethanol, PE, or preferring water, PW, respectively). Rats of the PE group differed from PW animals by significantly higher levels of 5-HT in the hypothalamus and blood. Peroral introduction of 4 g/kg ethanol into PE rats resulted in rapid (in not more than 15 min) sharp increases in the 5-HT content in the hypothalamus, neocortex, and blood, but 45 min after ethanol introduction the 5-HT contents in the hypothalamus, midbrain, neocortex, and blood noticeably dropped. It is suggested that within this time interval condensation of 5-HT with acetaldehyde (AcAdh, the first metabolite of ethanol oxidation) is intensified. This results in the production of -carbolines, analogs of morphine-like alkaloids, which are ligands of the opioid receptors. Under conditions of the development of alcohol addiction (free access of PE animals to the ethanol solution and water for several months), the content of 5-HT in the brain structures and blood increased in a parallel manner with an increase in the daily consumption of alcohol. Our findings are proof of the significant involvement of the serotoninergic system in the development of the euphoria state after single alcohol consumption and motivation for its consumption in the course of formation of alcohol addiction.     

Inhibition of long myosin light-chain kinase activation alleviates intestinal damage after binge ethanol exposure and burn injury

Laboratory evidence suggests that intestinal permeability is elevated following either binge ethanol exposure or burn injury alone, and this barrier dysfunction is further perturbed when these insults are combined. We and others have previously reported a rise in both systemic and local proinflammatory cytokine production in mice after the combined insult. Knowing that long myosin light-chain kinase (MLCK) is important for epithelial barrier maintenance and can be activated by proinflammatory cytokines, we examined whether inhibition of MLCK alleviated detrimental intestinal responses seen after ethanol exposure and burn injury. To accomplish this, mice were given vehicle or a single binge ethanol exposure followed by a sham or dorsal scald burn injury. Following injury, one group of mice received membrane permeant inhibitor of MLCK (PIK). At 6 and 24 h postinjury, bacterial translocation and intestinal levels of proinflammatory cytokines were measured, and changes in tight junction protein localization and total intestinal morphology were analyzed. Elevated morphological damage, ileal IL-1β and IL-6 levels, and bacterial translocation were seen in mice exposed to ethanol and burn injury relative to either insult alone. This increase was not seen in mice receiving PIK after injury. Ethanol-exposed and burn-injured mice had reduced zonula occludens protein-1 and occludin localization to the tight junction relative to sham-injured mice. However, the observed changes in junctional complexes were not seen in our PIK-treated mice following the combined insult. These data suggest that MLCK activity may promote morphological and inflammatory responses in the ileum following ethanol exposure and burn injury.     

Interaction of ethanol and microwaves on the blood-brain barrier of rats

The combined effects of ethanol and microwaves on the permeation of Evans blue dye through the mammalian blood-brain barrier was studied in male Wistar rats. Anesthetized rats were infused through a cannula in the left femoral vein with 0.1, 0.3, 0.5 or 0.7 grams of absolute ethanol per kilogram of body mass. A control group was given 0.7 g/kg of isotonic saline. The left hemisphere of the brain was irradiated by 3.15-GHz microwave energy at 3.0 W/cm2 rms for 15 min. The rat's rectal temperature was maintained at 37.0 degrees C. Immediately after irradiation, 2% Evans blue dye in saline (2.0 ml/kg body mass) was injected through the cannula. The results show that as the quantity of alcohol was increased, the degree of staining was decreased or eliminated. The temperature of the irradiated area of the brain increased for the first 4 to 5 minutes of irradiation and then stabilized for the remainder of the irradiation period. The steady-state temperature was highest in animals receiving saline or the smallest dose of alcohol. As the quantity of alcohol was increased, the steady-state temperature was reduced. These results indicate that ethanol inhibits microwave-induced permeation of the blood-brain barrier through reduced heating of the brain.     

Deuterium isotope effects in the oxidation of alcohols in vitro and in vivo

The oxidation of ethanol and isopropanol by liver alcohol dehydrogenase was studied in vitro and in vivo. Oxidation of ethanol by horse liver alcohol dehydrogenase was carried out in the presence of lactaldehyde and other aldehydes which reoxidized enzyme-bound NADH. Under these conditions the oxidation of ethanol was accelerated 7 to 22-fold, depending on the nature of the aldehyde. (An acceleration of ethanol oxidation by lactaldehyde was previously reported by Gupta and Robinson [(1966) Biochim, Biophys. Acta118, 431]. In the presence of lactaldehyde ping-pong kinetics were observed and a deuterium isotope effect on V of 4.2 was seen. In the absence of acceptor aldehyde no, or small, isotope effects (Baker, R. H. (1962) Biochemistry1, 41) are observed. Therefore, when dissociation of NADH is no longer rate limiting the hydrogen transfer step becomes largely rate determining. Oxidation of isopropanol shows an isotope effect on V of 2.5 in the absence of acceptor aldehyde. With mouse liver alcohol dehydrogenase results similar to those obtained with the horse liver enzyme were obtained.When ethanol metabolism was examined in vivo, in mice by measuring blood alcohol levels, no isotope effect was observed with ethanol-1-d₂. On the other hand, an isotope effect of 2.0 was observed when the metabolism of isopropanol and isopropanol-2-d₁ were compared. This isotope effect is very close to that observed in vitro with the mouse liver enzyme. The relative rate of metabolism of ethanol and isopropanol in vivo was similar to that observed in vitro with the mouse liver enzyme (ethanol:isopropanol, 2.1 in vivo:2.2 in vitro). It was concluded that in the metabolism of ethanol and isopropanol, alcohol dehydrogenase is partially rate determining. Administration to mice of lactaldehyde, as well as other aldehydes, ketones, or fructose, simultaneously with ethanol produced no increase in the rate of ethanol metabolism.     

Electronic nose screening of ethanol release during sol-gel encapsulation. A novel non-invasive method to test silica polymerisation

Porous silica matrices prepared by sol-gel process yield biocompatible materials adequate for encapsulation of biomolecules or drugs. The procedure is simple and fast, but when alkoxyde precursors like tetraethoxysilane (TEOS) are used the polymerisation reaction leads to the formation of alcohol as a by-product, which can produce undesirable effects on the activity of entrapped enzymes or modify a drug release kinetic. Therefore, it is critical to determine that no remnant ethanol is left prior using or storing the obtained biomaterial. In this regard, the technique used in the alcohol determination should be non-invasive and non-destructive to preserve the encapsulation device intact and ready to use. In this work we have successfully used a portable electronic nose (e-nose) for the screening of silica polymerisation process during theophylline encapsulation. TEOS reaction was "smelt" since precursor pre-hydrolysis until the end of ethanol release, sensed directly at the headspace of matrices slabs. Measurements showed that ethanol was negligible since 10th day in polymeric slabs of 10 mm width and 2 cm diameter. This first use of e-nose following a polymerisation reaction opens a wide number of putative applications in pharmaceutical and biochemical fields.     

The effect of nitric oxide donors and L-arginine on the gastric electrolyte barrier.

The potential difference across the stomach wall (PD) is determined by the gastric mucosal barrier. The decrease in the PD evoked by "the barrier breakers", e.g. aspirin, ethanol or bile acids is believed as a sensitive index of the mucosal damage. The effect of glyceryl trinitrate (GTN), isosorbide dinitrate (IDN) and molsidomine (MOL)--all exogenous donors of nitric oxide (NO), as well as L-arginine (L-ARG), which is a substrate for NO-synthase and Nomega-nitro-L-arginine (L-NNA), a non-selective NO synthase inhibitor on the gastric electrolyte barrier were studied against the gastric damage induced by ethanol. All NO donors given intragastrically alone caused only moderate, not significant changes in the PD and failed to affect the mucosal barrier, while L-NNA slightly decreased the PD. The NO donors and L-arginine applied as pretreatment prior to ethanol resulted in diminishing of its damaging action that was similar for all these drugs, while L-NNA intensified both the injury and the drop in the PD values caused by ethanol. In summary, our results showed the protective effect of endogenous nitric oxide from L-ARG and that originating from GTN, MOL and IDN on the gastric electrolyte barrier, supporting involvement of nitric oxide in the mechanism of gastric protection in the stomach.     

Granulocyte/macrophage colony-stimulating factor treatment improves alveolar epithelial barrier function in alcoholic rat lung

Chronic alcohol abuse increases the risk of developing acute lung injury approximately threefold in septic patients, and ethanol ingestion for 6 wk in rats impairs alveolar epithelial barrier function both in vitro and in vivo. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a trophic factor for the alveolar epithelium, and a recent phase II clinical study suggests that GM-CSF therapy decreases sepsis-mediated lung injury. Therefore, we hypothesized that GM-CSF treatment could improve ethanol-mediated defects in the alveolar epithelium during acute stresses such as endotoxemia. In this study, we determined that recombinant rat GM-CSF improved lung liquid clearance (as reflected by lung tissue wet:dry ratios) in ethanol-fed rats anesthetized and then challenged with 2 ml of saline via a tracheostomy tube. Furthermore, GM-CSF treatment improved lung liquid clearance and decreased epithelial protein leak in both control-fed and ethanol-fed rats after 6 h of endotoxemia induced by Salmonella typhimurium lipopolysaccharide given intraperitoneally, but with the greater net effect seen in the ethanol-fed rats. Our previous studies indicate that chronic ethanol ingestion decreases lung liquid clearance by increasing intercellular permeability. Consistent with this, GM-CSF treatment in vitro decreased permeability of alveolar epithelial monolayers derived from both control-fed and ethanol-fed rats. As in the endotoxemia model in vivo, the effect of GM-CSF was most dramatic in the ethanol group. Together, these results indicate that GM-CSF treatment has previously unrecognized effects in promoting alveolar epithelial barrier integrity and that these salutary effects may be particularly relevant in the setting of chronic alcohol abuse.     

Myth, marula, and elephant: an assessment of voluntary ethanol intoxication of the African elephant (Loxodonta africana) following feeding on the fruit of the marula tree (Sclerocarya birrea)

Africa can stir wild and fanciful notions in the casual visitor; one of these is the tale of inebriated wild elephants. The suggestion that the African elephant (Loxodonta africana) becomes intoxicated from eating the fruit of the marula tree (Sclerocarya birrea) is an attractive, established, and persistent tale. This idea now permeates the African tourist industry, historical travelogues, the popular press, and even scholastic works. Accounts of ethanol inebriation in animals under natural conditions appear mired in folklore. Elephants are attracted to alcohol, but there is no clear evidence of inebriation in the field. Extrapolating from human physiology, a 3,000-kg elephant would require the ingestion of between 10 and 27 L of 7% ethanol in a short period to overtly affect behavior, which is unlikely in the wild. Interpolating from ecological circumstances and assuming rather unrealistically that marula fruit contain 3% ethanol, an elephant feeding normally might attain an ethanol dose of 0.3 g kg(-1), about half that required. Physiological issues to resolve include alcohol dehydrogenase activity and ethanol clearance rates in elephants, as well as values for marula fruit alcohol content. These models were highly biased in favor of inebriation but even so failed to show that elephants can ordinarily become drunk. Such tales, it seems, may result from "humanizing" elephant behavior.     

Serotonin content in different sections of the brain, liver, intestines and blood of rats predisposed and not predisposed to alcohol consumption

Experiments were made with white random-bred rats (males) exposed to ethanol. The content of serotonin measured by spectrofluorometry was higher in the hypothalamus, brain stem and intestine, and was lower in the thalamus, striatum liver and blood in the animals predisposed to voluntary alcohol consumption and with lateral position duration 62 +/- 18 min as compared with the animals not predisposed to alcohol consumption and with lateral position duration 196 +/- 23 min, the dose of ethanol being 4.5 g/kg i. p. Thirty minutes after ethanol administration in a dose of 2.5 g/kg i. p. to the alcohol-predisposed rats there was a lowering of the serotonin content in the hypothalamus and an increase in the thalamus, brain stem, liver and blood. Meanwhile in the rats not predisposed to alcohol consumption, the serotonin content rose in the hypothalamus, brain stem, liver, intestine and blood and fell in the thalamus and striatum. It is assumed that the serotoninergic system of the brain may play a role in the formation of "positive" or "negative" attitudes to ethanol in the population of white random-bred rats.     

Experimental alcohol blastopathy

Experimental data are presented with respect to "experimental alcohol blastopathy" performed in our laboratory. As in our interpretation the notion of blastopathy involves both pathological changes during preimplantation development due to previous, preconceptional or preimplantation influences and later, pre- or postnatal effects induced by factors active during the preimplantation period, up to now the following experimental models were applied (on rats and mice): chronic and acute maternal, biparental or paternal ethanol alcoholization; preimplantation treatment with acetaldehyde or disulfiram followed by ethanol administration; acute ethanol intoxication before implantation on the background of chronic maternal ethanol intake; chronic maternal intake of various beverages. The main components of experimental alcohol blastopathy detected (by using a complex control methodology) were: pathological changes during the preimplantation developmental stages (lower mean number of embryos/animal, retardation of development, lowered migration rate of the embryos from the oviduct to the uterus, higher number of pathological morphological features), delayed implantation, disturbances of the early postimplantation development, retarded late foetal and placental growth. The effect of ethanol may be direct (ethanol being detectable in the oviductal and uterine fluid after both acute and chronic alcoholization) or indirect, via changes of the maternal macro- or microenvironment. The increase of the maternal blood acetaldehyde level may contribute to the appearance of alcohol blastopathy. Chronic beer and wine intake and acute intoxication with cognac suggest - up to now - the enhancing effect of beverage congeners. The noxious effect of acute ethanol intoxication superposed to chronic alcoholization is more marked that the separate effect of the two kinds of treatment. The chronic ethanol intake of fertilizing males (in mice) leads, both in the case of treated or untreated females, to lowered fertilization efficiency, to retardation of development (not occurring in the experimental model with chronic alcoholization of females) and to an enhanced increase of the number of pathological features. The cytogenetic control of preimplantation embryos (after chronic, acute or combined treatment with ethanol) does not reveal significant chromosomal changes. A possible alcohol blastopathy in humans must be taken into account (i.e. a noxious effect during the very early period of pregnancy when it is ignored).     

Effect of chronic alcohol consumption on rat brain microsome lipid composition, membrane fluidity and Na+-K+-ATPase activity

The present study reports differences in phospholipid classes, fatty acids of individual phospholipids, and changes in membrane fluidity and Na+-K+-ATPase activity in brain microsomes of rats maintained on an alcohol diet for 35 days compared to sex, age and weight-matched control rats maintained on a calorically-equivalent, non-alcohol diet. Although no difference in Na+-K+-ATPase activity was found in microsomes from alcohol vs control rats when measured in the absence of added alcohol, the presence of low concentrations of ethanol (less than 100 mM) stimulated, while high concentrations (greater than 100 mM) inhibited enzyme activity. The stimulation was differentially expressed in that the microsomal enzyme from alcohol rats was stimulated to a lesser extent than the enzyme from control rats. However, the inhibiting effect of high concentrations of alcohol was similar in microsomes from both alcohol and control rats. Also in membranes from alcohol rats, there was a lower quantity of phosphatidylethanolamine (PE) and higher quantities of phosphatidylserine (PS) and phosphatidylinositol (PI) compared to membranes from control rats. The major change in fatty acid composition was a reduction in the level of polyunsaturated fatty acids, which was particularly evident in PI and PS. The linoleic acid: arachidonic acid ratio (18:2/20:4) and the saturation:unsaturation ratio were also increased in PI and PS in membranes from alcohol animals. However, the ratio of n-6/n-3 fatty acids remained the same or was reduced in membranes from alcoholic animals. Although no difference in the inherent "fluidity" of membranes from alcohol vs control rats could be demonstrated by electron paramagnetic resonance, molecular tolerance to ethanol was demonstrated in the membranes from alcohol rats by the resistance to the disordering effects of added ethanol.     

Caffeine does not modulate nutritive blood flow to rat gastric submucosa--a microdialysis study

Background and Aims: Coffee irritates the gastric mucosa disrupting its barrier and increasing the risk of peptic ulcers. However, caffeine's contribution to these effects has not yet been elucidated. In this study we looked at the local effect of caffeine on the microcirculation and nitric oxide production in rats together with systemic marker of oxidative stress malondialdehyde as possible mechanisms whereby caffeine might participate in mucosal barrier impairment. Materials and Methods: Four groups of rats were anesthetized and administered as a bolus four different intraperitoneal doses of caffeine (0, 1, 10 and 50 mg kg(-1) b.wt.). The gastric submucosal microcirculation and nitric oxide production were then recorded for 2.5 hours by in situ microdialysis using the flow marker ethanol. At the completion of the experiments, plasma caffeine and malondialdehyde levels as well as morphological mucosal injury were determined. Results: There were no major differences in the macro- or microscopic pictures of the mucosa among the groups. Local microcirculatory (ethanol out/in ratio) and nitric oxide monitoring failed to demonstrate statistically significant changes as did measurement of plasma malondialdehyde in response to caffeine injections. Conclusions: Caffeine per se seems unlikely to contribute to the gastric mucosal barrier injury associated with coffee consumption by alterations in nutritive blood flow, nitric oxide production or aggravation of systemic oxidative stress. This information is relevant for better understanding of the mechanisms involved in caffeine-mediated influences on gastric physiology in relation to the irritant effects of coffee.     

Aldehyde dismutase activity of yeast alcohol dehydrogenase

Yeast alcohol dehydrogenase (EC 1.1.1.1) is able to catalyze the oxidation of acetaldehyde by NAD+ with a concomitant formation of ethanol, at pH 8.8 and pH 7.1; the stoichiometry of aldehyde oxidation vs. ethanol formation is 2:1. This enzymatic reaction obeys the Michaelis-Menten kinetics and was characterized by a high KM for acetaldehyde (68 mM) and a low kcat (2.3 s–1), at pH 8.8, 22°C. There is no visible burst of NADH during the reaction, from pH 7.1–10.1. Therefore, we have concluded that the enzyme catalyzes an apparent dismutation of two molecules of acetaldehyde into a molecule of acetic acid and a molecule of ethanol.     

Breakdown of the blood–brain barrier to proteins in white matter of the developing brain following systemic inflammation

Compromised blood–brain barrier permeability resulting from systemic inflammation has been implicated as a possible cause of brain damage in fetuses and newborns and may underlie white matter damage later in life. Rats at postnatal day (P) 0, P8 and P20 and opossums (Monodelphis domestica) at P15, P20, P35, P50 and P60 and adults of both species were injected intraperitoneally with 0.2–10 mg/kg body weight of 055:B5 lipopolysaccharide. An acute-phase response occurred in all animals. A change in the permeability of the blood–brain barrier to plasma proteins during a restricted period of postnatal development in both species was determined immunocytochemically by the presence of proteins surrounding cerebral blood vessels and in brain parenchyma. Blood vessels in white matter, but not grey matter, became transiently permeable to proteins between 10 and 24 h after lipopolysaccharide injection in P0 and P8 rats and P35–P60 opossums. Brains of Monodelphis younger than P35, rats older than P20 and adults of both species were not affected. Permeability of the blood–cerebrospinal fluid (CSF) barrier to proteins was not affected by systemic inflammation for at least 48 h after intraperitoneal injection of lipopolysaccharide. These results show that there is a restricted period in brain development when the blood–brain barrier, but not the blood–CSF barrier, to proteins is susceptible to systemic inflammation; this does not appear to be attributable to barrier immaturity but to its stage of development and only occurs in white matter.This work was supported by NIH grant number R01 NS043949-01A1.