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ADH activity and ethanol tolerance in third chromosome substitution lines in Drosophila melanogaster.

In vitro ADH activity and ethanol tolerance were studied in males of a series of third chromosome substitution lines in Drosophila melanogaster. The lines were divided into those with a random third chromosome from a vineyard population (VO lines) and those with a selected third chromosome from males obtained after an egg-to-adult ethanol survival test on the F4 of the previous population (VE lines). Both ADH activity and ethanol tolerance varied significantly among the lines, but the characters showed no significant correlation. Ethanol tolerance (at the higher ethanol concentrations) was higher in the selected lines (VE lines) but ADH activity was not. In our lines, the in vitro ADH activity variability, linked to the regulatory genes (located on the third chromosome) and unrelated to the polymorphism of the Adh locus (located on the second chromosome), is not involved in the ethanol tolerance variability. The data suggest that in this population ethanol tolerance was acquired in nature, at least partially, by means other than increasing ADH activity.

Alcohol Dehydrogenase↗

Structural requirements for neurohypophyseal peptide maintenance of ethanol tolerance.

Tolerance to the hypnotic effect of ethanol in mice is prolonged by daily subcutaneous administration of arginine vasopressin and certain analogs of this hormone. The major structural requirement for maintenance of ethanol tolerance by these peptides appears to be the N-terminal "ring" structure of vasopressin containing two amino acid residues with aromatic side chains. Peptides structurally related to the C-terminal portion of the neurohypophyseal hormones are less active in maintaining tolerance than the intact hormones. The structure-activity pattern observed for the effects of peptides on ethanol tolerance is similar to that described for neurohypophyseal peptide inhibition of extinction of an active avoidance response, an action thought to reflect peptide effects on memory consolidation. The results are in line with our hypothesis that similar CNS recognition sites may mediate neurohypophyseal peptide effects on ethanol tolerance and certain memory processes. The neurohypophyseal hormones and analogs did not affect the hypnotic or hypothermic response to an acute injection of ethanol, indicating that the determination of tolerance was not influenced by a direct peptide-ethanol interaction. The hormones themselves, however, did cause a drop in body temperature in the mice, which could be a result of either central or peripheral hormonal actions.

Animals↗

Ethanol tolerance in the rat is learned.

Rats were trained to walk on a treadmill to avoid foot shock. The animals developed tolerance for ethanol if given subsequent practice while ethanol intoxicated. Rats given equivalent doses of ethanol after practice did not develop tolerance, nor did saline-treated controls. These results challenge the hypothesis that mere repeated doses of ethanol are sufficient to induce tolerance. It seems that tolerance does not develop unless the response used to measure tolerance is performed while the subject is intoxicated.

Animals↗

Anaerobiosis in Echinochloa crus-galli (Barnyard Grass) Seedlings : Intermediary Metabolism and Ethanol Tolerance.

Tolerance to ethanol and the ability to metabolize key intermediary substrates under anaerobiosis were studied in Echinochloa crus-galli (L.) Beauv. var oryzicola seeds to further characterize the mechanisms which enable it to germinate and grow without O(2).Our results indicate that E. crus-galli var oryzicola possesses an inherently high tolerance to ethanol and is able to metabolize low levels of ethanol in the absence of O(2). Concentrations of ethanol 45-fold greater than endogenous levels did not prove toxic to germinating seeds.Five-day anaerobically grown seedlings of E. crus-galli var oryzicola metabolized added [(14)C]sucrose primarily to CO(2) and ethanol. Of the soluble compounds labeled, the phosphorylated intermediates of glycolysis and the oxidative pentose phosphate pathway predominated more under anaerobiosis than in air. In addition, organic acids and lipids were labeled from [(14)C]sucrose, the latter indicating that metabolism of carbohydrate via acetyl-CoA occurred in the absence of O(2). Lipids were also labeled when seeds were supplied with [(14)C]ethanol or [(14)C]acetate. Labeling experiments using the above compounds plus [(14)C]NaHCO(3), showed further labeling of organic acids; succinate and citrate being labeled under nitrogen, while fumarate was formed in air.The above metabolic characteristics would allow for the maintenance of an active alcoholic fermentation system which, along with high alcohol dehydrogenase activity, would continue to recycle NAD and result in continued energy production without O(2). In addition, Echinochloa's ability to metabolize carbohydrate intermediates and to synthesize lipids indicates that mechanisms exist for providing the carbon intermediates for biosynthesis, particularly membrane synthesis for growth, even in the absence of O(2).

Journal Article↗

Characteristics of ethanol tolerance in alcohol drinking (AA) and alcohol avoiding (ANA) rats.

The development of tolerance to ethanol was examined in two rat lines selected for high (AA) and low (ANA) ethanol consumption. In the first experiment, the acquisition of tolerance to the motor-impairment, hypothermic and hypnotic effects of ethanol produced by daily treatment with 5 g/kg ethanol for a period of 24 days was examined. Tolerance to these effects of ethanol was observed in the AA rats while marginal or no tolerance was demonstrated in the ANA rats. In the second experiment the development of rapid tolerance to the hypothermic and hypnotic effects of ethanol was examined. The hypothermic and hypnotic responses to IP injection of 3.5 g/kg ethanol were found to be attenuated in the AA but not the ANA rats by a single equivalent ethanol injection given 24 h earlier. These results suggest some relationship between the capacity to develop tolerance and voluntary ethanol intake.

Alcohol Drinking↗

[Study on ethanol tolerance of Saccharomyces cerevisiae X330 under very high gravity medium].

The impacts of osmoprotectants and nutrient components on both ethanol tolerance of Saccharomyces cerevisiae X330 and its ethanol fermentability were investigated when high gravity synthetic medium were used. The results indicate that nutrient limitation plays important role in the ethanol tolerance of Saccharomyces cerevisiae. When the nutritional requirements of Saccharomyces cerevisiae are satisfied, its ethanol tolerance increases, especially at high sugar concentrations. The effect of the individual nutrient component in the PYN medium on ethanol tolerance is different, which is yeast extract > peptone > magnesium sulfate > vitamin C = potassium phosphate > calcium chloride = ammonium sulfate. Osmoprotectants ( such as glycine and proline ) are effective in improving the ethanol tolerance of Saccharomyces cerevisiae X330, and the optimum concentrations of 20 mmol/L glycine and 10 mmol/L proline were obtained experimentally while glycine exerted a stronger enhancing effect than proline. After 3 h of exposure to 18% (V/V) ethanol at 30 degrees C, 57.1% and 50.0% remained viable for the cells grown in glycine-added and proline-added medium respectively.

Bioreactors↗

Calcium transport and fluorescence polarization of 1,6-diphenyl-1, 3,5-hexatriene in sarcoplasmic reticulum from normal and ethanol-tolerant rats.

Sarcoplasmic reticulum (SR) of high purity and functional integrity was isolated from skeletal muscle of normal and ethanol-tolerant rats. Ethanol at low concentrations (0.1 to 0.2 M), added in vitro to isolated SR, resulted in slight inhibition of both calcium loading and calcium-stimulated ATPase rates. Higher concentrations of ethanol resulted in further inhibition of calcium loading, but not of calcium-stimulated ATPase. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) in isolated SR membranes showed a small decrease in anisotropy with the addition of ethanol in vitro. Sarcoplasmic reticulum from normal and ethanol-tolerant rats did not differ in calcium pump function or in fluorescence polarization of DPH, in the presence or absence of ethanol.

Adenosine Triphosphatases↗

Parallel development of ethanol tolerance and operant compensatory behaviors in rats.

This experiment was designed to detect compensatory learning that has been suggested to occur during the course of tolerance development to ethanol's effects on operant performance. The effects of presession ethanol injections on the development of tolerance to ethanol's effects on operant performance in an afternoon Fixed-Ratio (FR) task was assessed in rats that were concurrently performing in a morning DRL task. Only presession saline injections were administered for the DRL task. A cumulative dosing procedure was used to establish initial and postethanol exposure dose-effect curves for both tasks. Daily presession ethanol administration produced a 3-fold shift-to-the-right in the dose-effect curve for FR-task performance. No changes were evident in the FR-task performance of controls that received daily saline injections. However, during the period of daily ethanol injections and during subsequent cumulative dose tests, the ethanol, but not the control, group displayed dose-related increases in total DRL-task responses relative to baseline. These DRL data were interpreted as reflecting the development of rate-increasing behaviors that compensated for and contributed to the tolerance of ethanol's rate-decreasing effects on FR-task performance.

Animals↗

Enhanced [3H]-noradrenaline release in synaptosomes from ethanol-tolerant animals: the role of nerve terminal calcium ion concentrations.

Cortical slices from brains of ethanol-tolerant rats have previously been shown to release a greater fraction of uptake [3H]-noradrenaline ([3H]-NA) on K+-depolarisation than slices from control animals. In the present experiments, when stimulated with the Ca2+ ionophore A23187 (30 microM), untreated cortical synaptosomes release only a very small fraction of uptaken [3H]-NA. Under these conditions synaptosomal preparations from ethanol-tolerant animals released a greater fraction of uptaken [3H]-NA but the difference was not significant. However, after incubation with ouabain (100 microM, to increase intrasynaptosomal [Ca2+]) A23187 now produced a much greater enhancement of [3H]-NA release in preparations from ethanol-tolerant rats. Further, after superfusion of the synaptosomal preparation with 1mM EGTA and A23187 for 30 min (a procedure which should reduce intrasynaptosomal [Ca2+]) the reintroduction of Ca2+ to the superfusing fluid caused a marked release of [3H]-NA which was also significantly greater in preparations from ethanol-tolerant animals. Ca2+/Mg2+-ATPase activity was also higher in these synaptosomes but no difference could be detected in the release of [3H]-NA from a combined synaptic vesicle: synaptic plasma membrane preparation. The results suggest that the development of ethanol tolerance is associated with a fundamental change in the dynamic control of Ca2+ concentrations within the nerve terminal which potentiates the depolarisation-induced release of neurotransmitters.

Alcoholism↗

Effect of treatment dose and test system on the development of ethanol tolerance and physical dependence.

The development of ethanol tolerance as a function of daily treatment dosage was examined on two different test systems. Tolerance to the motor-impairment effect of ethanol, as measured by the tilting-plane test, failed to develop after daily administration of 1-3 g/kg, but developed to equal degrees after 4 or 5 g/kg daily. On the other hand, daily treatment with 2, 4 or 6 g/kg all produced significant tolerance to the hypothermic effect, and the extent of tolerance was proportional to the treatment dosage. Twenty-four hours after the termination of ethanol treatment, there was a significant hypothermia in the group treated with 6 g/kg, and at 32 hr hyperthermia occurred in both the 4 and 6 g/kg groups. These results indicate that treatment dosage is an important determinant of the extent of ethanol tolerance and physical dependence, but that the relationship is complex and varies with the test system used.

Alcoholism↗

Role of phospholipid head groups in ethanol tolerance of Saccharomyces cerevisiae.

Pre-incubation of cells of Saccharomyces cerevisiae with 2 M-ethanol led to decreased rates of L-alanine uptake, H+ efflux and fermentation rate. However, these responses were modified in yeast cells with altered phospholipid composition. Using L-alanine transport and H+ efflux as indices of ethanol tolerance, it was observed that cells enriched with phosphatidylserine had greater tolerance to ethanol. This resulted from altered charge of membrane phospholipids rather than changes in membrane fluidity. It is suggested that the anion:zwitterion ratio of phospholipids may be one of the important determinants of ethanol tolerance in S. cerevisiae.

Alanine↗

The NMDA receptor antagonist dizocilpine differentially affects environment-dependent and environment-independent ethanol tolerance.

Antagonists of the N-methyl-D-aspartate subtype of glutamate receptor have been reported to block the development of tolerance to various effects of ethanol and opiates, using paradigms in which tolerance is believed to be governed by learning. There is considerable evidence to implicate the N-methyl-D-aspartate receptor in learning processes, and therefore the ability of the antagonists to block tolerance has been attributed to their effects on learning. To evaluate this hypothesis, we compared, in C57BL/6 mice, the effect of the uncompetitive N-methyl-D-aspartate receptor antagonist, dizocilpine, on environment-dependent (associative) tolerance to ethanol, which is governed by learning, and on environment-independent (nonassociative) ethanol tolerance, in which learning plays a minimal role. Environment-dependent tolerance was induced by repeated ethanol injections, and dizocilpine blocked the development of this type of tolerance to the hypothermic and incoordinating effects of ethanol. In contrast, when environment-independent ethanol tolerance was induced by feeding the mice an ethanol-containing liquid diet, dizocilpine treatment had no effect on the development of tolerance to the hypothermic, incoordinating or hypnotic effects of ethanol. The results support the hypothesis that the effect of N-methyl-D-aspartate receptor antagonists on ethanol tolerance reflects the more general role of this receptor in processes involving learning and memory.

Animals↗

Temporal correlation of changes in rat brain sialic acid and in inhibition of Na+-K+ ATPase with ethanol tolerance.

Administration of ethanol to rats by gavage, in daily doses increasing gradually from 3 to 6 g/kg, led to progressive development of tolerance to the hypothermic effect of test doses of ethanol (3 g/kg, i.p.), between days 4 and 21 of treatment. Tolerance was decreased by day 3 of withdrawal, and had disappeared by day 7. Basal levels of brain microsomal Na+-K+ ATPase activity increased progressively during tolerance development, and returned to normal by 4 days after ethanol withdrawal. Conversely, inhibition of the ATPase by ethanol alone (0.22-0.66 M), or by 1 microM norepinephrine (NE) combined with much lower concentrations of ethanol (0.0125-0.1 M), decreased progressively during tolerance development, and returned to normal after withdrawal. The best temporal correlation with the degree of tolerance was shown by the magnitude of NE sensitization of the enzyme to ethanol inhibition. The chronic sucrose control treatment resulted in an unexplained increase in the NE sensitization. Since synaptosomal membrane concentration of sialic acid showed a similar time course during ethanol treatment, it is possible that changes in membrane sialic acid contribute to the alteration in NE sensitization by altering NE receptor properties.

Animals↗

Ethanol tolerance and enhanced calcium/calmodulin-dependent phosphorylation of synaptic membrane proteins.

The chronic effects of ethanol on synaptic membrane proteins was studied in ethanol-tolerant rats. Synaptic plasma membranes and postsynaptic densities were prepared from homogenates of forebrain and incubated in vitro with [gamma-32P]adenosine triphosphate in the presence and absence of calcium and calmodulin. In ethanol-tolerant animals, enhanced phosphorylation of synaptic plasma membrane but not postsynaptic density proteins was demonstrated in the presence of calcium and calmodulin. These results suggest that the development of tolerance to ethanol may involve alteration in neuronal sensitivity to calcium.

Animals↗

Genetic dissection of ethanol tolerance in the budding yeast Saccharomyces cerevisiae.

Uncovering genetic control of variation in ethanol tolerance in natural populations of yeast Saccharomyces cerevisiae is essential for understanding the evolution of fermentation, the dominant lifestyle of the species, and for improving efficiency of selection for strains with high ethanol tolerance, a character of great economic value for the brewing and biofuel industries. To date, as many as 251 genes have been predicted to be involved in influencing this character. Candidacy of these genes was determined from a tested phenotypic effect following gene knockout, from an induced change in gene function under an ethanol stress condition, or by mutagenesis. This article represents the first genomics approach for dissecting genetic variation in ethanol tolerance between two yeast strains with a highly divergent trait phenotype. We developed a simple but reliable experimental protocol for scoring the phenotype and a set of STR/SNP markers evenly covering the whole genome. We created a mapping population comprising 319 segregants from crossing the parental strains. On the basis of the data sets, we find that the tolerance trait has a high heritability and that additive genetic variance dominates genetic variation of the trait. Segregation at five QTL detected has explained approximately 50% of phenotypic variation; in particular, the major QTL mapped on yeast chromosome 9 has accounted for a quarter of the phenotypic variation. We integrated the QTL analysis with the predicted candidacy of ethanol resistance genes and found that only a few of these candidates fall in the QTL regions.

Chromosome Mapping↗

Ethanol tolerance of Saccharomyces cerevisiae and its relationship to lipid content and composition.

Saccharomyces cerevisiae strain 14-12 is a highly ethanol-tolerant organism. It can grow in the presence of 13% ethanol but growth is completely prevented at 14% ethanol. A relationship was detected between yeast lipids and ethanol tolerance. A gradual decrease of lipid content was recorded as the concentration of supplemented ethanol increased. Moreover, free fatty acids were comparatively decreased in these lipid extracts. When separately added to media with 14% ethanol different lipids produced varied stimulatory effects on yeast growth. Maximum yield of yeast growth was obtained at 14% ethanol in the presence of lecithin, palmitic acid and cholesterol. Yeast lipids produced in the presence of these fractions are characterized by a relatively high percentage of free fatty acids. The change in the percentage of free fatty acids was shown to be the controlling factor in ethanol tolerance.

Drug Tolerance↗

Relationship between ethanol tolerance, lipid composition and plasma membrane fluidity in Saccharomyces cerevisiae and Kloeckera apiculata.

The lipid composition of a strain of each of two yeasts, Saccharomyces cerevisiae and Kloeckera apiculata, with different ethanol tolerances, was determined for cells grown with or without added ethanol. An increase in the proportion of ergosterol, unsaturated fatty acid levels and the maintenance of phospholipid biosynthesis seemed to be responsible for ethanol tolerance. The association of ethanol tolerance of yeast cells with plasma membrane fluidity, measured by fluorescence anisotropy, is discussed. We propose that an increase in plasma membrane fluidity may be correlated with a decrease in the sterol:phospholipid and sterol:protein ratios and an increase in unsaturation index.

Bacterial Proteins↗