Brain levels and turnover rates of presumptive neurotransmitters as influenced by administration and withdrawal of ethanol in mice

—Effects of acute or chronic administration of ethanol and its withdrawl on the steady-state levels and turnover rates of certain neurotransmitters have been investigated in mice. The influence of long-term administration of ethanol on the activities of enzymes involved in the metabolism of these transmitters has also been studied. Acute administration of ethanol or acetaldehyde or chronic administration of ethanol resulted in a decrease in the cerebral contents of acetylcholine, acetylCoA and CoA. Brain levels of 5-hydroxytryptamine, norepinephrine and choline remained unchanged after acute administration of ethanol. However, chronic administration of ethanol resulted in a decrease in the norepinephrine content without significantly affecting 5-hydroxytryptamine or choline contents. Cerebral levels of γ-aminobutyric acid increased with both acute or chronic administration of ethanol. The total incorporation of [³H]choline into acetylcholine in brain was depressed upon acute administration of ethanol. After withdrawal of ethanol for one day cerebral levels of norepinephrine returned to normal; however, γ-aminobutyric acid and acetylcholine returned to normal levels at 2 and 4 days after ethanol withdrawal, respectively. Pretreatment of mice with pyrazole, an inhibitor of alcohol dehydrogenase, prevented the ethanol-induced decrease in cerebral acetylcholine levels. The activities of cerebral choline acetyltransferase and glutamic decarboxylase were decreased after 2 weeks of chronic ethanol administration. However, the activities of acetyl cholinesterase and GABA-transaminase remained unaffected after 2 weeks of ethanol treatment     

Ethanol Administration in the Rat Decreases Prostacyclin Production by Isolated Brain Microvessels

The effects of short- and long-term ethanol administration to rats on basal levels and formation of prostacyclin (PGI2) measured as 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), and on lipid class content and fatty acid composition of isolated brain microvessels (BMV) were studied. After acute treatment (2 h, at the peak of plasma ethanol concentration) basal 6-keto-PGF1 alpha levels in BMV and release on incubation were reduced to 50% of control values. After chronic administration (15 days), PGI2 release was reduced to about 40% of control values, without changes in basal levels. Total lipid, phospholipid, and cholesterol levels in BMV, measured after prolonged administration of alcohol, were not modified. Also, only minor changes in the fatty acid composition of individual phospholipid classes were detected. The observed reduction of PGI2 synthesis in BMV thus could not be related to changes of the fatty acid precursor pool in the preparation. Precursor release and/or the biosynthetic pathways may be affected by ethanol administration.     

Activities of hepatic enzymes related to ethanol oxidation and effects of ethanol on hepatic metabolites in rats with chronic liver injury.

To study the effects of ethanol on liver chronically injured by CCl4, activities of hepatic enzymes related to ethanol oxidation, influences of ethanol on hepatic metabolites, and blood ethanol disappearance were observed. (1) Activities of alcohol dehydrogenase, low- and high-Km aldehyde dehydrogenase, microsomal ethanol-oxidizing system and drug-metabolizing enzyme were remarkably decreased in the injured liver. (2) Increases in lactate/pyruvate and beta-hydroxybutyrate/acetacetate ratios were shown in control liver 2 h after ethanol ingestion. Similar but less pronounced effects of ethanol on the 'redox state' were also seen in rats with chronic liver injury. (3) Delay in ethanol disappearance was not observed until 12 h after ethanol ingestion. The ethanol-induced changes in the redox state in the injured liver were similar to those in controls. Higher ethanol concentrations in blood from rats with chronic liver injury could be related to potentiate the injured liver.     

Biological effects and metabolic interactions after chronic and acute administration of 4-methylpyrazole and ethanol to rats

4-Methylpyrazole in a dose producing an inhibition of alcohol dehydrogenase of about 60% was given alone or in combination with ethanol (10%) as sole drinking fluid to growing rats in periods up to 38 weeks. No effects were observed on the weight curves. Hematologic analyses showed normal values for blood and bone marrow. Studies of liver function with transaminase, bilirubin and albumin did not reveal any functional changes. Kidney function was normal as judged by creatinine and normal electrolytes. Electronmicroscopy of liver, kidney, and heart did not reveal any changes related to treatment. Combined treatment of ethanol and 4-methylpyrazole caused an increase of the microsomal drug-metabolizing activity. Chronic administration of ethanol and 4-methylpyrazole indicated that there is a mutual interaction in the metabolism of ethanol and 4-methylpyrazole, leading to a higher concentration of both ethanol and 4-methylpyrazole in the blood. Acute experiments, where alcohol dehydrogenase is saturated with ethanol, indicated a much slower elimination of 4-methylpyrazole. Administration of ethanol and 4-methylpyrazole in acute experiments showed a lower concentration of 4-hydroxymethylpyrazole in the blood indicating that ethanol interferes with the 4-methylpyrazole- and/or 4-hydroxymethyl-pyrazole-metabolizing enzymes. The present investigation has shown that the acute and chronic toxicity of 4-methylpyrazole alone or in combination with ethanol is minimal at doses that are effective in blocking ethanol metabolism in the rat. Because of its low toxicity and powerful inhibitory capacity, 4-methylpyrazole should be a potential tool for experimental clinical investigation of alcohol metabolism and its effects. 4-Methylpyrazole is also a potential therapeutic agent in methanol or ethylene glycol poisoning.     

Effect of alcohol intoxication and aldehyde dehydrogenase inhibitors on lipid peroxidation in rat liver

A single intraperitoneal administration of ethanol (3.5 g/kg) to rats induced a marked increase in lipid peroxidation and a decrease of antioxidative activity in the liver after 1 h when assessed by chemi-luminescence in liver homogenates. The pretreatment with aldehyde dehydrogenase inhibitor, disulfiram (200 mg/kg 24 hr before ethanol), caused a 10-fold elevation of the blood acetaldehyde levels, with no effect on the hepatic lipid peroxidation compared to control. Cyanamide (50 mg/kg, 2 h before the ethanol) increased approximately 100-fold the acetaldehyde levels, however, the changes in lipid peroxidation were not significantly different from that produced by ethanol alone. The present results suggest, that the metabolism of acetaldehyde and not acetaldehyde itself is responsible for the in vivo activation of lipid peroxidation during acute alcohol intoxication. Disulfiram prevents the ethanol-induced lipid peroxidation in the rat liver.     

No effect of chronic ethanol administration on the activity of alcohol dehydrogenase and aldehyde dehydrogenases in the near-term pregnant guinea pig

The objective of this study was to determine the effect of chronic maternal administration of moderate-dose ethanol on alcohol dehydrogenase, low Km aldehyde dehydrogenase, and high Km aldehyde dehydrogenase activities in the guinea pig at near-term pregnancy. The activity of each enzyme in the maternal liver, fetal liver, and placenta of the guinea pig at 59 days of gestation (term, 66 days) was determined spectrophotometrically following chronic daily oral administration of two doses of 1 g ethanol/kg maternal body weight or isocaloric sucrose solution. There was no experimental evidence of ethanol-induced malnutrition in the mother or growth retardation in the fetus. There was a statistically significant increase (65%) in the microsomal cytochrome P-450 content of the maternal liver for the ethanol treatment compared with the sucrose treatment. The alcohol dehydrogenase, low Km aldehyde dehydrogenase, and high Km aldehyde dehydrogenase activities in the maternal liver, fetal liver, and placenta were not statistically different for the ethanol-treated compared with the sucrose-treated animals. This also was the case for the maternal blood and fetal blood ethanol and acetaldehyde concentrations, determined at 2h after maternal administration of 1 g ethanol/kg maternal body weight. These data demonstrate that the ethanol- and acetaldehyde-oxidizing enzyme activities in the maternal-placental-fetal unit of the guinea pig at near-term pregnancy were not changed by chronic administration of moderate-dose ethanol.     

The effects of cyclic 3'5' adenosine monophosphate on the acute tolerance induced by ethanol in male rats.

The effect of cyclic 3′5′ adenosine monophosphate (cAMP) on the acute tolerance induced by ethanol was studied in male rats. The acute tolerance was measured with a hexobarbital anesthesia method, where the dose of hexobarbital needed to obtain a burst suppression of 1 second or more in EEG is determined. Ethanol 2.0 g/kg was given ip 0.25 or 3 h prior to the threshold determination. cAMP 10 mg/kg or saline was given iv 6 h prior to the threshold determination.After saline pre-treatment less hexobarbital was needed 0.25 h after ethanol administration compared to 3 h after ethanol administration, although the blood levels were similar. An acute tolerance had developed. Pre-treatment with cAMP had no effect on the dose of hexobarbital needed without ethanol nor on the dose needed 3.0 h after ethanol administration. 0.25 h after ethanol more hexobarbital was needed in the animals pre-treated with cAMP compared with the corresponding saline treated animals. The dose of hexobarbital was as large as the one needed 3.0 h after ethanol. Thus cAMP seems to facilitate the induction of acute tolerance to ethanol while the hexobarbital threshold as such is uninfluenced.     

Biochemical changes in rat liver after 18.5 days of spaceflight

The effect of "weightlessness" on liver metabolism was examined using tissue from rats flown in earth orbit for 18.5 days aboard the Soviet Cosmos 936 biosatellite. Changes in the activities of certain carbohydrate and lipid enzymes were noted. Of the 28 hepatic enzyme activities assayed, two, palmitoyl-CoA desaturase and lactate dehydrogenase, increased, whereas five, glycogen phosphorylase, 6-phosphogluconate dehydrogenase, both acyltransferases which act on alpha-glycerolphosphate and diglycerides, and aconitate hydratase decreased. The remaining enzyme activities measured were unchanged. In addition, increased levels of liver glycogen and palmitoleate were noted which probably resulted from the lowered glycogen phosphorylase and increased palmitoyl-CoA desaturase activities, respectively, in those animals that experienced weightlessness. These changes caused by weightlessness were transient since all of the aforementioned alterations returned to normal values when measured in the livers of other rats which had flown in the biosatellite 25 days after recovery.     

The effects of 1,1-di(p-chlorophenyl)-2-chloroethylene on plasma enzymes and blood constituents in the Japanese quail.

Glutamate oxaloacetate transminase (GOT), glutamate dehydrogenase (GDH), sorbitol dehydrogenase (SDH), pseudo-cholinesterase (ChE) and various blood constituents were measured in the plasma of Japanese quail fed 1,1-di(p-chlorophenyl)-2-chloroethylene (DDMU) at low levels for periods ranging from 2 to 32 days. Previous work has shown that DDMU is a potent inducer of hepatic microsomal enzymes causing marked structural changes in the liver. A rapid increase in plasma GOT was observed within 4 days accompanied by an increase in relative liver weight. Plasma GDH and SDH increased to a maximum between 16 and 24 dyas which seems to be associated with hepatic cell proliferation. Plasma ChE showed a steady increase over the time course of DDMU administration. The level of plasma lipid was reduced after 4 days whereas the hepatic lipid content was substantially increased suggesting that the fatty liver condition may be caused by decreased release of triglyceride from the liver. Plasma glucose was reduced at 8 days but there was no evidence of a hyperglycaemic state. The changes noted after 2 days of DDMU diet were confirmed by measurements on birds 18 h after oral dosing the DDMU. The study demonstrates the value of plasma enzyme measurements for the early detection of toxic effects and indicates that DDMU administration leads to extrahepatic effects in addition to those previously described in the liver.     

ENZYMES CATALYSING ETHANOL METABOLISM IN NEURAL AND SOMATIC TISSUES OF THE RAT

Abstract— The enzymes catalysing ethanol metabolism, alcohol dehydrogenase (EC 1.l.1.1) and aldehyde dehydrogenase (EC 1.2.1.3), were assayed in a variety of neural and somatic tissues of the rat, the human counterparts of which are known to be vulnerable to excessive ethanol. The activity of alcohol dehydrogenase was assayed by the coupled oxidation of ethanol and reduction of lactaldehyde, a method which we have recently found to be sufficiently sensitive and specific to measure the relatively low levels of activity in whole brain. Detectable activities of these enzymes were found in peripheral nerve, skeletal muscle, retina, optic nerve and various regions of brain, as well as in a variety of non-neural tissues. The levels of the enzymic activities in all tissues were markedly lower than those of liver, but probably sufficient to perform a local function in the metabolism of ethanol or other endogenous substrates. The activity of alcohol dehydrogenase in the various tissues, like that of liver, was confined to the cytosol and exhibited kinetic properties and responses to inhibitors almost identical to those of the liver enzyme. We consider the results to be consistent with the hypothesis that the pathological effects of alcohol may be related, at least in part, to local mechanisms for the metabolism of alcohol.     

EFFECTS OF ETHANOL ON SEROTONIN METABOLISM IN BRAIN

The effect of ethanol on serotonin metabolism in brains of mice was determined both after a single injection and ‘chronic’ administration of ethanol. Behavioral effects were also monitored.‘Chronic’ administration of ethanol by inhalation to mice resulted in an increased susceptibility to Metrazole induced seizures. This susceptibility was evident for 48 h after ‘withdrawal’ of mice from ethanol chambers. No differences in brain 5-HT levels between control and ethanol treated mice were evident during withdrawal. However, a significant elevation in brain 5-HIAA levels was noted during this period. Short lived increases in brain 5-HIAA levels were also noted after a single injection of ethanol. Ethanol treatment produced no significant changes in the activity of brain MAO, aldehyde dehydrogenase, or aldehyde reductase. Other mechanisms for ethanol induced increases in brain 5-HIAA are discussed.     

Ethanol feeding to rats reversibly decreases hepatic carnitine palmitoyltransferase activity and increases enzyme sensitivity to malonyl-CoA

The effects of prolonged ethanol feeding on both carnitine palmitoyltransferase I activity and enzyme sensitivity to inhibition by malonyl-CoA were studied in rat liver, heart, skeletal muscle and kidney cortex mitochondria. Heart and skeletal muscle enzymes showed the highest specific activity and sensitivity to malonyl-CoA. Carnitine palmitoyltransferase I in extrahepatic tissues showed no changes on ethanol feeding. Only the liver enzyme activity was altered after long term ethanol administration, by suffering a progressive decrease in activity and a parallel increase in sensitivity to malonyl-CoA. These alterations reversed after 10 days of ethanol withdrawal. These results are discussed in relation to the control of carnitine palmitoyltransferase I and the effects of ethanol on fatty acid metabolism.     

Effect of ethanol on adenosine triphosphatase and enolase activities in rat brain and in cultured nerve cells

The effect of alcohol on enzymes involved in energy metabolism of nervous tissue were analyzed, in vivo after acute and chronic ethanol administration to rats and in vitro by addition of 50 mM and 100 mM ethanol to the medium of cultured nerve cells: chick neurons, chick glial cells, a neuronal cell line (MT17) and a glial tumoral cell line (C6). The parameters we measured were (Na⁺,K⁺), Mg²⁺ and ecto Ca²⁺,Mg²⁺ ATPase activities involved in transport phenomena and enolase activities (non neuronal NNE and neuron specific enolase NSE) as markers of nerve cell maturation. In vivo, after chronic ethanol administration (Na⁺,K⁺) ATPase activity was increased while Mg²⁺ dependent activity was not affected. Enolase activity was decreased. Acute ethanol administration decreased (Na⁺,K⁺) ATPase activity, while Mg²⁺ dependent activity was not affected. In cultured nerve cells ethanol effect was dose, time and cell type dependent; alterations of the cell membrane by trypsinization of the tissue before seeding modifies the effect of ethanol on the enzymes we analyzed. Our results suggest that alcohol effect on nerve cells depends mainly on the lipoprotein structure of the cell membranes which may have different properties from one cell type to another.     

Changes induced by Fe deficiency and Fe resupply in the organic acid metabolism of sugar beet (Beta vulgaris) leaves

The effects of iron deficiency and iron resupply on the metabolism of leaf organic acids have been investigated in hydroponically grown sugar beet. Organic acid concentrations and activities in leaf extracts of several enzymes related to organic acid metabolism were measured. Enzymes assayed included phosphoenol pyruvate carboxylase (PEPC; EC 4.1.1.31), different Krebs cycle enzymes: malate dehydrogenase (MDH; EC 1.1.1.37), aconitase (EC 4.2.1.3), fumarase (EC 4.2.1.2), citrate synthase (CS; EC 4.1.3.7) and isocitrate dehydrogenase (ICDH; EC 1.1.1.42), glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and two enzymes related to anaerobic metabolism (lactate dehydrogenase [LDH]; EC 1.1.1.27, and pyruvate decarboxylase [PDC]; EC 4.1.1.1). Iron concentration in leaves was severely decreased by iron deficiency. Iron resupply caused an increase in iron concentrations, reaching levels similar to the controls in 96 h. Iron deficiency induced a 2.3-fold (from 16 to 37 mmol m⁻²) increase in leaf total organic acid concentration. Organic anion concentrations were still 4-fold higher than the controls 24 h after resupply and decreased to values similar to those found in the controls after 96 h. All measured enzymes had increased activities in extracts of iron-deficient leaves when compared to the controls and generally decreased to control values 24 h after iron addition. These data provide evidence that organic acid accumulation in iron-deficient leaves is likely not due to an enhancement in leaf carbon fixation. Instead, this accumulation could be associated with organic acid export from the roots to the leaves via xylem.     

S-adenosylmethionine (SAMe) protects against acute alcohol induced hepatotoxicity in mice small star, filled

Although S-Adenosylmethionine (SAMe) has beneficial effects in many hepatic disorders, the effects of SAMe on acute alcohol-induced liver injury are unknown. In the present study, we investigated effects of SAMe on liver injury in mice induced by acute alcohol administration. Male C57BL/6 mice received ethanol (5 g/kg BW) by gavage every 12 hrs for a total of 3 doses. SAMe (5 mg/kg BW) was administrated i.p. once a day for three days before ethanol administration. Subsequent serum ALT level, hepatic lipid peroxidation, enzymatic activity of CYP2E1 and hepatic mitochondrial glutathione levels were measured colorimetrically. Intracellular SAMe concentration was measured by high-performance liquid chromatography (HPLC). Histopathological changes were assessed by H&E staining. Our results showed that acute ethanol administration caused prominent microvesicular steatosis with mild necrosis and an elevation of serum ALT activity. SAMe treatment significantly attenuated the liver injury. In association with the hepatocyte injury, acute alcohol administration induced significant decreases in both hepatic SAMe and mitochondrial GSH levels along with enhanced lipid peroxidation. SAMe treatment attenuated hepatic SAMe and mitochondrial GSH depletion and lipid peroxidation following acute alcohol exposure. These results demonstrate that SAMe protects against the liver injury and attenuates the mitochondrial GSH depletion caused by acute alcohol administration. SAMe may prove to be an effective therapeutic agent in many toxin-induced liver injuries including those induced by alcohol.     

Acute ethanol exposure in pregnancy alters the insulin-like growth factor axis of fetal and maternal sheep

Maternal ethanol intake during pregnancy impairs fetal growth, but mechanisms are not clearly defined. Reduced IGF abundance or bioavailability in the fetus and/or mother may contribute to this growth restriction. We hypothesized that an episode of acute ethanol exposure, mimicking binge drinking would restrict fetal growth and perturb the maternal and fetal IGF axes. Pregnant sheep were infused intravenously with saline or ethanol (1 g/kg maternal wt) over 1 h, on days 116, 117, and 118 of gestation (start of 1st infusion = time 0, term is 147 days). Maternal and fetal plasma IGF and IGF-binding protein (IGFBP) concentrations were measured before and after each infusion. Compared with controls, ethanol exposure reduced fetal weight at day 120 by 19%, transiently reduced maternal plasma IGF-I (-35%) at 30 h, and decreased fetal plasma IGF-II (-28%) from 24 to 54 h after the first infusion. Ethanol exposure did not alter maternal or fetal plasma concentrations of IGFBP-2 and IGFBP-3, measured by Western ligand blotting. We conclude that suppression of maternal and fetal IGF abundance may contribute to fetal growth restriction induced by acute or binge ethanol exposure.     

Effects of Ethanol and Polychlorinated Biphenyls in Combination on Ascorbic Acid Metabolism,Liver Drug-metabolizing Enzymes,and Lipid Metabolism in Rats

We investigated the combined effects of ethanol and polychlorinated biphenyls (PCB) on ascorbic acid metabolism, liver drug-metabolizing enzymes, and lipid metabolism in rats fed on a diet containing by 36% by energy of ethanol and 0.005% of PCB, either singly or in combination, for 49 days. Ethanol and PCB given together synergistically affected the amount of ascorbic acid excreted in the urine and the serum concentration of ascorbic acid. This synergistic effect was also observed in the activity of aniline hydroxylase in the liver. Ethanol and PCB given together seem to have had different effects on lipid metabolism. These results suggest that the effect of ethanol on the metabolism of ascorbic acid and of drugs may be enhanced by combined administration with PCB, and that the ascrobic acid deficiency and/or modification of the drug metabolism may become more serious.     

Vasopressin pressor receptor-mediated activation of HPA axis by acute ethanol stress in rats

The plasma arginine vasopressin (AVP), ACTH, and corticosterone levels and the hypothalamic corticotropin-releasing hormone (CRH) content were measured after oral administration of 1 ml of 75% ethanol to rats, a model known to induce acute gastric erosions and stress. Elevated plasma AVP, ACTH, and corticosterone levels were detected 1 h after ethanol administration. Treatment with the vasopressin pressor (V(1)) receptor antagonist [d(CH(2))(5)Tyr(Me)-AVP] before ethanol administration significantly reduced the ACTH and corticosterone level increases. A higher hypothalamic CRH content was measured at 30 or 60 min after ethanol administration. V(1) receptor antagonist injection, 5 min before ethanol administration, inhibited the rise in hypothalamic CRH content. The protein synthesis blocker cycloheximide prevented the hypothalamic CRH content elevation after stress. The AVP-, CRH-, and AVP + CRH-induced in vitro ACTH release in normal anterior pituitary tissue cultures was also prevented by pretreatment with the V(1) receptor antagonist. The results support the hypothesis that stress-induced AVP may not only act directly on the ACTH producing anterior pituitary cells but also indirectly at the hypothalamic level via the synthesis and release of CRH.     

Effects of ethanol feeding on hepatic lipid synthesis

Rats were fed a high-fat, liquid diet containing either 36% of total calories as ethanol or an isocaloric amount of sucrose, for a period up to 35 days. At different time intervals we measured the effects of ethanol administration on the activities of a number of key enzymes involved in hepatic lipid synthesis. At the start of the experimental period the activities of acetyl-CoA carboxylase and fatty acid synthase, measured in liver homogenates, increased in the control as well as in the ethanol-fed group. After 35 days these enzyme activities were still elevated but there were no significant differences between the two groups. In hepatocytes isolated from controls as well as from ethanol-fed rats, short-term incubations with ethanol induced an increase in the rate of fatty acid synthesis and in the activities of acetyl-CoA carboxylase and fatty acid synthase. However, no alterations in the regulation of these enzymes by short-term modulators of lipogenesis were apparent in hepatocytes isolated from alcohol-treated animals. The results do not indicate a major role for the enzymes of de novo fatty acid synthesis in the development of the alcoholic fatty liver. The amount of liver triacylglycerols increased in ethanol-fed rats during the entire treatment period, whereas the hepatic levels of phosphatidylcholine and phosphatidylethanolamine were not affected by ethanol ingestion. Ethanol administration for less than 2 weeks increased the activities of phosphatidate phosphohydrolase, diacylglycerol acyltransferase, and microsomal phosphocholine cytidylyltransferase, whereas the cytosolic activity of phosphocholine cytidylyltransferase was slightly decreased. Upon prolonged ethanol administration the activities of these enzymes were slowly restored to control values after 35 days, suggesting development of some kind of adaptation. It is interesting that, although the activities of phosphatidate phosphohydrolase and diacylglycerol acyltransferase were restored to the levels found in the control rats, this effect was not accompanied by a stabilization or decrease of the concentration of hepatic triacylglycerols.