High Levels Of Ethanol Research Articles

Effect on gluconeogenesis of mutants blocking two mitochondrial transport systems in the yeast Saccharomyces cerevisiae

Two mutants of Saccharomyces cerevisiae, ccr1 and tpy1, have been found to interfere with the transport of small molecules across the inner mitochondrial membrane. Both also have the effect of interfering with the synthesis of a number of cytoplasmically located enzymes involved in gluconeogenesis, even when the cells are released from glucose repression. The ccr1 mutant, defective in the transport of dicarboxylic acids across the inner membrane, represses the synthesis of gluconeogenic enzymes almost totally, but synthesis can be induced on complete medium without a carbon source. This mutant has low levels of intracellular malate under all growth conditions tested. The tpy1 mutant, defective in the transport of pyruvate across the inner membrane, shows repression of gluconeogenesis enzymes under some growth conditions, particularly high levels of ethanol in the medium. These conditions also lead to low levels of malate in the cells. Intracellular levels of malate in these mutants, and in the wild type, are correlated with the levels of gluconeogenic enzymes present. The ability of isolated mutant mitochondria to phosphorylate ADP is shown to be consistent with the interpretation that they are defective in inner membrane transport, although as yet no evidence is available that these defects are the primary lesions in the two mutants. The data are consistent with two general models. In one, the exhaustion of an extramitochondrial corepressor or introduction of a coinducer by mitochondrial activity triggers the induction of gluconeogenic enzyme synthesis. In the second, the mitochondria themselves trigger this induction, but only when the tricarboxylic acid cycle is able to operate at a high level.

High-Gravity Brewing: Production of High Levels of Ethanol without Excessive Concentrations of Esters and Fusel Alcohols

The influence of nutritional supplementation and the degree of anaerobiosis on the production of esters and fusel alcohols in very high gravity brewing was examined. Although fermentation in semian...

Effects of ethanol on reproduction and arterial hypertension in spontaneously hypertensive and normotensive rats: a preliminary communication.

Ethanol consumption and spontaneous (essential) hypertension are important fetal and maternal risk factors. Alone, they contribute to embryopathy (fetal alcohol syndrome) or maternal organ pathology and fetal loss in hypertensive pregnancies. Combined, the effects of ethanol consumption on the progress of a hypertensive pregnancy have not been adequately investigated. In the present study, groups of O-A strain genetic hypertensive (SHR: groups 1 and 2) and Wistar-Kyoto normotensive (WKY: groups 3 and 4) pregnant rats were given 20 ml/kg of distilled water by gavage to serve as controls [groups 1 (SHR) and 3 (WKY)] or 3.2 g/kg of ethanol [groups 2 (SHR) and 4 (WKY)] from days 6 to 15 of gestation. During acclimation, hypertension developed in SHR rats (WKY pressures were 105 to 114 mm Hg; SHR pressures were 137 to 148 mm Hg). From day 6 to 15 of gestation, ethanol-consuming rats (groups 2 and 4) had higher arterial pressures than controls (groups 1 and 3). Pregnant SHR rats given ethanol did not experience a prebirthing hypotension. On gestation day 20, most offspring (84%, group 2; 86%, group 4) of alcoholic dams were dead or malformed. Intrauterine growth retardation occurred in group 4. Hydrocephalus, microphthalmia, and mild hydronephrosis and hydroureter were common in live offspring of group 2 dams. Hydronephrosis and hydroureter were increased in group 4 pups. Variant cranial ossification was noted in group 2 and 4 pups. These preliminary data suggest an altered hypertensive response during pregnancy in alcohol-consuming rats and confirm the embryopathic effects of relatively high levels of ethanol consumed during the critical period of organogenesis in two additional strains of rats.

Alcohol dehydrogenase II and fructose-1,6-bisphosphatase appear to be co-regulated in wild-type yeast

An activity gel assay for fructose-1,6-bisphosphatase (FBP), the enzyme catalyzing the final step in gluconeogenesis in yeast, has been developed which can be used in conjunction with spectrophotometric assays to show that it is tightly co-regulated with the inducible alcohol dehydrogenase, ADHII. Both enzymes are repressed coordinately in aerobically grown yeast by the addition of high levels of glucose or ethanol, and induced on minimal medium by the addition of yeast extract. A mutant deficient in FBP segregates independently of the ADHII structural gene locus. This phenomenon is of interest because of the discovery of Ciriacy [(1979) Mol. Gen. Genet. 176, 427-431] of mutants (ccr, or carbon catabolite repression) which repress both FBP and ADHII simultaneously, along with several other enzymes.

Preparation and characterization of immobilized growing cells of Zymomonas mobilis for ethanol production

Zymomonas mobilis cells were entrapped in K. carrageenan. Growth was observed with the immobilized cell preparation. The kinetic and yield parameters for the conversion of fructose to ethanol were nearly identical to free cells. The same preparation of immobilized cells was used in six repeated batch runs and at the end sixth-batch fructose was converted to ethanol more rapidly and efficiently with ethanol productivity of 14 g/L h and 96% conversion of fructose. The effect of high fructose and ethanol levels on specific fructose uptake rate and ethanol productivity was studied and quantitatively analyzed.

A correlation between voluntary ethanol consumption and brain catalase activity in the rat

The relationship between voluntary ethanol consumption and brain catalase activity was investigated in male Long Evans rats. In the first study, rats which were voluntarily consuming alcohol or water for 25 days were sacrificed by decapitation immediately (group A) or 15 days (group B) following withdrawal of alcohol and their brains analysed for catalase activity. Mean brain catalase activity did not differ among the two groups of rats exposed to ethanol and the ones exposed to water only. Furthermore, there were significant positive correlations between individual voluntary ethanol intake and catalase activity in both groups, (group A:r = .69, p less than or equal to 0.05; group B:r = .54, p less than or equal to 0.05). In the second study, rats were forced to drink high levels of ethanol presented as the only source of fluid for 25 days. Rats were sacrificed and brain, liver, muscle and heart tissue were extracted and analysed for catalase activity. There were no differences in mean brain catalase activity between water and forced ethanol drinking rats indicating that the enzyme was not inducible by high volume ethanol consumption. The results suggest that inherent differences in brain catalase activity may be one of the factors in determining an animal's propensity to voluntarily consume ethanol.

High-Gravity Brewing: Production of High Levels of Ethanol Without Excessive Concentrations of Esters and Fusel Alcohols

The influence of nutritional supplementation and the degree of anaerobiosis on the production of esters and fusel alcohols in very high gravity brewing was examined. Although fermentation in semianaerobic conditions led to a drastic reduction in ester production, acceptable ester levels were found under anaerobic conditions where worts were supplemented with lipids and assimilable nitrogen. Such fermentations were rapid, and resultant beers may be organoleptically acceptable to the consumer.

Genetic aspects of ethanol tolerance and production bySaccharomyces cerevisiae

We developed a method of hybrid selection between homothallic wild-type and heterothallic strains. The hybrids obtained were used to study the heredity of ethanol tolerance and production. Both characters segregated independently, but no ethanol-sensitive strains were able to produce high levels of ethanol. At least four genes are implicated in ethanol tolerance.

High-Gravity Brewing: Effects of Nutrition on Yeast Composition, Fermentative Ability, and Alcohol Production

A number of economic and product quality advantages exist in brewing when high-gravity worts of 16 to 18% dissolved solids are fermented. Above this level, production problems such as slow or stuck fermentations and poor yeast viability occur. Ethanol toxicity has been cited as the main cause, as brewers' yeasts are reported to tolerate only 7 to 9% (vol/vol) ethanol. The inhibitory effect of high osmotic pressure has also been implicated. In this report, it is demonstrated that the factor limiting the production of high levels of ethanol by brewing yeasts is actually a nutritional deficiency. When a nitrogen source, ergosterol, and oleic acid are added to worts up to 31% dissolved solids, it is possible to produce beers up to 16.2% (vol/vol) ethanol. Yeast viability remains high, and the yeasts can be repitched at least five times. Supplementation does not increase the fermentative tolerance of the yeasts to ethanol but increases the length and level of new yeast cell mass synthesis over that seen in unsupplemented wort (and therefore the period of more rapid wort attenuation). Glycogen, protein, and sterol levels in yeasts were examined, as was the importance of pitching rate, temperature, and degree of anaerobiosis. The ethanol tolerance of brewers' yeast is suggested to be no different than that of sake or distillers' yeast.

Effects of different liquid diets and sustained ethanol release on alcohol metabolism

The effects of two liquid diets, Sustacal and Shorey-AIN, on liver alcohol dehydrogenase (ADH) activity and ethanol clearance were tested in rats under conditions of high ethanol exposure for nine days. High blood ethanol levels (BEL) were produced through a combination of an initial intubated dose of ethanol sustained ethanol release tube (SERT), and ethanol as 37% of total energy in the liquid diet. Under free-feeding conditions, rats consumed slightly more ethanol per unit body weight in the Shorey-AIN diet, a diet formulated for rodent nutrition, than in the Sustacal diet, a diet originally intended for human consumption. However, BEL were significantly higher in the Sustacal group than in the Shorey-AIN group. No differences in ethanol clearance rates were observed between the groups. On the other hand, total liver ADH activity was significantly reduced in both the Shorey AIN/ethanol and the Sustacal/ethanol groups, compared to lab chow controls. When the Sustacal diet was fortified with casein and methionine so that the protein content matched that of the Shorey AIN diet, the BEL were no longer significantly higher than those produced by the Shorey AIN/ethanol diet. The results demonstrate the effect of nutritional factors on BEL under conditions of high ethanol load. However, these factors do not appear to alter major characteristics of ethanol metabolism and clearance in our short-term experiments.

Interaction of ethanol with neural cells in culture: a model of intoxication, tolerance and withdrawal.

The interaction of ethanol with the nervous system produces acute intoxication, tolerance and withdrawal phenomena. Additionally, there are several alcohol-related neurological disorders which develop in alcoholic patients. Current evidence suggests that ethanol produces some of these changes by altering the structure and function of neural membranes. Therefore, neurotransmitter receptors and receptor-dependent molecular events in the nervous system may be highly sensitive to ethanol. The murine neuroblastoma X glioma hybrid cell line NG108-15 was used to study the acute and chronic interactions of ethanol with intact cells. Ethanol acutely inhibited opiate receptor binding, but after chronic exposure the cells exhibited an apparent adaptive increase in the number of opiate binding sites; this was reversible when ethanol was withdrawn. High levels of ethanol (200 mM) increased opiate binding after 18-24 hours; lower concentrations (25-50 mM) produced similar changes after two weeks. This model system has great potential for exploring the cellular and molecular mechanisms which underlie ethanol intoxication, tolerance and withdrawal.

The interaction of ethanol and exogenous arachidonic acid in the formation of leukotrienes and prostaglandin D 2 in mastocytoma cells

The present studies were undertaken to examine the hypothesis that ethanol could affect cellular biosynthesis in the murine mastocytoma cell of prostaglandins and leukotrienes, oxidative metabolites of arachidonic acid, at concentrations that could be encountered in vivo as well as during in vitro experiments. The effects of ethanol which encompass these concentration ranges (200-1000 mg%) can be summarized as follows: first in the absence of exogenous arachidonic acid, ethanol caused a dose dependent decrease in the production of leukotrienes which was statistically significant at 200 mg%. At 1000 mg%, ethanol caused a 20-50% decrease in leukotrienes and a 21% decrease in the amount of prostaglandin D2 (PGD2) formed in these cells. Secondly, when cells were incubated with exogenous arachidonic acid (14 micrograms/ml), large increases in both PGD2 and leukotrienes occurred. Under these conditions, ethanol caused a further increase in the amount of leukotrienes and a small increase in the amount of PGD2 formed. This stimulatory effect was specific for ethanol since neither t-butanol nor n-butanol caused the enhanced production of leukotrienes with exogenous arachidonic acid. Thus, these experiments suggest that ethanol affects metabolism of arachidonic acid at reasonably low doses (200-400 mg%) of ethanol in a manner dependent on the free arachidonic acid in the tissue. Also, in vitro experiments in which ethanol is used as a solvent for arachidonic acid could be greatly affected by high levels of ethanol (500-1000 mg%) which are frequently utilized.

Differential effects of long-chain alcohols in long- and short-sleep mice.

Observations that the long-sleep (LS) and short-sleep (SS( mouse lines differ in their depressant response to barbiturates, and that the difference between lines becomes greater as lipid solubility increases, prompted this investigation of the effects of alcohols that differ in lipid solubility. Results indicate that LS and SS mice differ significantly in their sleep time responses to propanol, butanol, and 3-methyl butanol, as well as ethanol: their hypothermic responses showed a similar pattern, but only the response to ethanol differed significantly between lines. For both sleep time and hypothermia, the difference between lines decreased with increasing lipid solubility. In all cases, the LS mice were more sensitive than the SS to the depressant effects of the alcohol. Similar ratios of SS:LS waking brain ethanol and butanol levels indicated that CNS sensitivity to long-chain alcohols is similar to that for ethanol. A pharmacokinetic study revealed higher ethanol levels for LS than for SS mice at all time points in blood, fat, and brain body compartments. Blood ethanol elimination curves showed that the SS mice eliminate ethanol at a faster rate than do the LS.

Teratogenesis after acute alcohol exposure in cultured rat embryos.

In order to investigate whether alcohol has teratogenic properties, rat embryos were cultured in vitro during their organogenetic period and exposed to ethanol at 200-800 mg% culture medium for the 48 hours of culture (0-24 somite stage) or to 600-800 mg% for 24 hours, or 6-hour periods. Exposure to alcohol throughout the entire 48-hour culture period or the first 24-hour period (0-12 somites) produced marked growth retardation, particularly of the head region in a dose-dependent manner, but did not prevent neural tube closure. Exposure to high levels of ethanol during specific 6-hour periods of early organogenesis (three to nine somites) prevented closure of the neural tube in 30% of cultured rat embryos, indicating a direct teratogenic action of ethanol. These results implied an effect of ethanol on embryonic development, independent of maternal metabolism. The 6-hour exposure experiments demonstrated that high doses of ethanol during specific periods of organogenesis can be teratogenic.

The influence of flooding pretreatment on metabolic changes in winter cereal seedlings during ice encasement

The survival of winter wheats during ice encasement is increased by pre-exposure to low temperature flooding. The response is duplicated by nitrogen exposure, indicating an anaerobic hardening process. During ice encasement, higher levels of ethanol accumulate in previously flooded plants than in nonflooded plants. Alcohol dehydrogenase activity is threefold higher at the end of a 2 week low temperature flooding period than in aerobically grown plants. The activity is lower after 3 days in ice. The decrease is similar in previously flooded and nonflooded plants, but is larger in Dover barley which is more susceptible to ice encasement stress than the wheats. Malate declines during flooding and is higher during ice encasement in nonflooded than in flooded plants. Previously flooded plants enter the more severe anaerobic stress of ice encasement with an accelerated glycolytic rate, and thus a higher survival potential.

A change in susceptibility of rat cerebellar Purkinje cells to damage by acetaldehyde during fetal, neonatal and adult life.

Phillips S.C. & Cragg B.G. 1982 Neuropathology a id Applied Neurobiology 8, 455–463A change in susceptibility of rat cerebellar Purkinje cells to damage by acetaldehyde during fetal neonatal and adult lifeThe sensitivity of rat cerebellar Purkinje cells to acetaldehyde during fetal, neonatal or adult life has been assessed by histological techniques. A pregnant female rat was exposed to ethanol vapour during the last 2 weeks of gestation while metabolism of acetaldehyde was blocked by injections of disulfiram (200mg/kg) on days E14, E16, E18 and E20. Purkinje cells were counted in the offspring 5 days after birth. The number of Purkinje cells in lobes I and VIII were 52% and 42% less than age matched controls. Smaller reductions were found in lobes V and X. The weight of the cerebellum was reduced by 42%. Neonatal rats received disulfiram (40mg) on the second day after birth and were exposed to ethanol vapour during daylight hours on the third and fourth days. Purkinje cells were counted at 5 days after birth, and losses similar to those described above were found. The weight of the cerebellum was reduced by 47%. Adult male rats were exposed to ethanol vapour for 2 weeks and given disulfiram (200mg/kg) on every second day. No reduction in cerebellar weight or loss of Purkinje cells was detected. When the dura overlying the cerebellum in adult rats was superfused with acetaldehyde solutions for 1 h, a 1–6 M solution caused degeneration whereas a 0–68 M solution did not. Thus the combination of high blood ethanol and acetaldehyde levels is damaging to perinatal Purkinje cells, whereas adult cells are resistant. We found the same ranking of susceptibility with ethanol alone (Philips & Cragg, 1982a and it was surprising that the cell losses with acetaldehyde were only marginally greater than those with ethanol alone, in view of the greater neurotoxicity of acetaldehyde during acute exposure (Phillips, 1981.

Flaccidity after Head Injury

Upon admission, 17 of 223 (8%) consecutive patients with severe head injury exhibited a flaccid, wholly unresponsive motor examination. In this study alcoholic intoxication neither caused depressed motor responsiveness in head-injured patients with high serum ethanol levels nor accounted for the motor examination in those exhibiting the flaccid state. Flaccidity was attributed principally to impaired ventilation in 4 patients, a major intracranial mass in 12, and a spinal cord injury in 1. Compared to the larger group of head-injured patients, the flaccid patients had a significantly greater incidence of hypercapnia (P less than 0.001), acidosis (P less than 0.01), and both elevated and uncontrollable intracranial pressure (ICP) (P less than 0.001). These findings and the high mortality rate (76%) in this study suggest that the magnitude of respiratory complications and the severity of mechanical brain injury are greater in flaccid patients. The flaccid patients undergoing surgical decompression for major intracranial mass lesions (11 cases) have all died and, although still small in number, this group may represent an important subset with a poor prognosis. Nonetheless, a protocol that encourages rapid radiological and electrophysiological assessment and vigorous surgical and ICP management until the probable cause of flaccidity is identified and treated has benefit. The flaccid state was reversed and a good recovery was attained after the restoration of blood pressure and/or ventilation in 2 patients who appeared to have sustained a very grave head injury. In another patient, absent somatosensory evoked potentials greatly facilitated the diagnosis of a spinal subdural hematoma. This program of prompt diagnosis and intense therapy did not result in a protracted course or undue numbers of severely brain-damaged survivors.

Absence of a role for salsolinol in the mechanism of ethanol teratogenicity.

Alcohol and its primary metabolite, acetaldehyde, are central nervous system teratogens. Acetaldehyde can further interact with endogenous biogenic amines under certain conditions to generate tetrahydroisoquinoline (TIQ) alkaloidal metabolites, some of which interfere with adrenergic neuronal function and structural integrity. The possible role of salsolinol (the TIQ derived from the condensation of acetaldehyde with dopamine) in the mechanism of alcohol teratogenicity was investigated in rats. Administration of 2 mg/kg of salsolinol to rats on the 15th day of gestation did not result in the recovery of this TIQ in the fetuses. Furthermore, acute oral administration of alcohol (3 g/kg x 3 doses) to rats on different days of gestation did not result in the synthesis of salsolinol by the fetuses despite the high maternal blood ethanol levels (124 +/- 22 mg%). These findings argue against a role for salsolinol in the mechanism of alcohol teratogenicity. Small amounts of endogenous salsolinol (3.6 ng/g) were detected in fetuses from control rats, in agreement with previous findings in neonatal rats. The physiological role (if any) of endogenous salsolinol in the fetal and neonatal rat is not known.

Subcutaneous silastic implants: maintenance of high blood ethanol levels in rats drinking a liquid diet.

A new subcutaneous form of ethanol exposure in rats is described. The Sustained Ethanol Release Tube (SERT) for rats is similar to an earlier device reported for mice, except that only one refill per day is required. This device, plus an intragastric loading dose for initially raising blood ethanol levels (BEL), is capable of maintaining high BEL for greater than 12 hours. Supplementation of SERT-released ethanol with a Sustacal chocolate-flavored diet with 37% of total energy as ethanol produces high, stable BEL for indefinite periods. Maintenance of such BEL for 9 days is sufficient to cause dramatic withdrawal signs when ethanol exposure is stopped. The method is useful as a model for conveniently and quickly producing physical dependence to ethanol in rats.

THE EFFECT OF OSMOTIC PRESSURE ON THE PRODUCTION AND EXCRETION OF ETHANOL AND GLYCEROL BY A BREWING YEAST STRAIN*

When the cells of a lager brewing yeast Saccharomyces uvarum (carlsbergensis) were grown in minimal media containing sucrose and a non-metabolized sugar sorbitol, significant levels of intracellular ethanol were obtained. Intracellular ethanol concentration decreased as the osmotic pressure of the medium was lowered and the proportion of extracellular ethanol increased. A reduction in cell viability occurred when there were high levels of intracellular ethanol. The total amount of glycerol produced increased with increased osmotic pressure, but glycerol diffused out of the cells faster than ethanol.