Betaine prevents loss of sialic acid residues and peroxidative injury of erythrocyte membrane in ethanol-given rats

To evaluate chronic ethanol toxicity on erythrocyte membrane and preventive action of betaine as a methyl donor, 24 male Wistar albino rats were divided into three groups: control, ethanol and ethanol plus betaine group. Animals were fed 60 ml diet per day for two months. Rats in the ethanol group were fed ethanol 8 g/kg/day. The ethanol + betaine groups were fed ethanol plus betaine (0.5% w/v). After two months, all animals were killed. Malondialdehyde (MDA) and sialic acid (SA) levels were determined in plasma samples. Osmotic fragility tests were performed on whole blood samples and erythrocyte membrane thiol contents were determined using membrane suspensions. Plasma MDA levels in ethanol-given rats were increased significantly compared to the control group of rats (p < 0.05). MDA in the betaine group was significantly lower than that in the ethanol group (p < 0.05). Erythrocyte membrane thiol contents in ethanol group were decreased compared with those of the control group (p < 0.05). Thiol contents were increased slightly after betaine therapy, but this increase was not statistically significant (p > 0.05). Plasma sialic acid levels in the ethanol group were significantly higher than in the control group (p < 0.05). Sialic acid was decreased in the betaine group compared to the ethanol group (p < 0.05). In the osmotic fragility test, we observed that chronic ethanol consumption increased erythrocyte hemolysis. Betaine protected against ethanol-induced hemolysis. Our findings show that chronic ethanol administration affects erythrocyte membrane properties and this may be related to oxidative stress. Betaine protects erythrocyte membrane alterations against chronic ethanol toxicity. Therefore betaine as a nutritional agent, may protect ethanol induced clinical problems associated with membrane abnormalities.     

Ethanol-induced hepatotoxicity and protective effect of betaine.

The protective effects of betaine in ethanol hepatotoxicity were investigated in 24 female wistar albino rats. Animals were divided into three groups: control, ethanol and ethanol + betaine group. Animals were fed liquid diets and consumed approximately 60 diet per day. Rats were fed ethanol 8 kg(- 1) day(- 1). The ethanol + betaine group were fed ethanol plus betaine (0.5% w/v). All animal were fed for 2 months. Reduced glutathione, malondialdehyde and vitamin A were determined in the liver tissue. Alanine aminotransferase activities were also measured on intracardiac blood samples. GSH levels in the ethanol group were significantly lower than these in the control group (p < 0.001). GSH was elevated in the betaine group as compared to the ethanol group (p < 0.001). MDA in the ethanol group was significantly higher than that in the control group (p < 0.05). MDA was decreased in the betaine group as compared to the ethanol group (p < 0.05). Vitamin A in the ethanol group was significantly lower than that in the control group (p < 0.01), but, in the ethanol + betaine group it was high compared with the ethanol group (p < 0.01). ALT in the ethanol group was higher than that in the control group (p < 0.05). Oxidative stress may play a major role in the ethanol-mediated hepatotoxicity. Betaine may protect liver against injury and it may prevent vitamin A depletion. Therefore, it may be a useful nutritional agent in the prevention of clinical problems dependent on ethanol-induced vitamin A depletion and peroxidative injury in liver.     

Effects of Chronic Ethanol Administration on Serotonin Metabolism in the Various Regions of the Rat Brain

Changes in serotonin (5-HT) and 5-hydroxy indole acetic acid (5-HIAA), its major metabolite, in cerebral cortex, corpus striatum and hippocampus were investigated at 10^th and 21^st days of chronic ethanol ingestion in Wistar rats. Ethanol (7.2% v/v) was given to rats in a modified liquid diet. Biochemical analysis was performed in two groups of ethanol-treated and control rats (n = 6 for each group). Rats in each group were decapitated at the 10^th and 21^st days of ethanol consumption. Brains were removed and cerebral cortex, corpus striatum and hippocampus were dissected. 5-HT and 5-HIAA levels were measured in respective brain regions by using high performance liquid chromatography. In cerebral cortex and corpus striatum, 5-HT levels were significantly lower than control at the 10^th day of ethanol consumption. At the 21^st day, the levels tended to remain low, but not significantly different statistically. In hippocampus, 5-HIAA levels were significantly higher than control at 10^th day of ethanol consumption. Increased 5-HIAA level returned to control values at the 21^st day of ethanol consumption. Our results suggest that, 5-HT clearly seems to play a critical role in the brain at the 10^th day of chronic ethanol consumption.     

Intermittent hypoxia conditioning prevents behavioral deficit and brain oxidative stress in ethanol-withdrawn rats.

Intermittent hypoxia (IH) has been found to protect brain from ischemic injury. We investigated whether IH mitigates brain oxidative stress and behavioral deficits in rats subjected to ethanol intoxication and abrupt ethanol withdrawal (EW). The effects of IH on overt EW behavioral signs, superoxide generation, protein oxidation, and mitochondrial permeability transition pore (PTP) opening were examined. Male rats consumed dextrin or 6.5% (wt/vol) ethanol for 35 days. During the last 20 days, rats were treated with repetitive (5-8 per day), brief (5-10 min) cycles of hypoxia (9.5-10% inspired O2) separated by 4-min normoxia exposures. Cerebellum, cortex, and hippocampus were biopsied on day 35 of the diet or at 24 h of EW. Superoxide and protein carbonyl contents in tissue homogenates and absorbance decline at 540 nm in mitochondrial suspensions served as indicators of oxidative stress, protein oxidation, and PTP opening, respectively. Although IH altered neither ethanol consumption nor blood ethanol concentration, it sharply lowered the severity of EW signs including tremor, tail rigidity, and startle response. Compared with dextrin and ethanol per se, in the three brain regions, EW increased superoxide and protein carbonyl contents and accelerated PTP opening in a manner ameliorated by IH. Administration of antioxidant N-acetylcysteine throughout the IH program abrogated the reductions in EW signs and superoxide content, implicating IH-induced ROS as mediators of the salutary adaptations. We conclude that IH conditioning during chronic ethanol consumption attenuates oxidative damage to the brain and mitigates behavioral abnormalities during subsequent EW. IH-induced ROS may evoke this powerful protection.     

Carnosine supplementation protects rat brain tissue against ethanol-induced oxidative stress

Ethanol causes oxidative stress and tissue damage. The aim of this study was to investigate the effect of antioxidant carnosine on the oxidative stress induced by ethanol in the rat brain tissue. Forty male rats were divided equally into four groups as control, carnosine (CAR), ethanol (EtOH), and ethanol plus carnosine (EtOH + CAR). Rats in the control group (n = 10) were injected intraperitoneally (i.p.) with 0.9% saline; EtOH group (n = 10) with 2 g/kg/day ethanol, CAR group (n = 10) received carnosine at a dose of 1 mg/kg/day and EtOH + CAR group (n = 10) received carnosine (orally) and ethanol (i.p.). All animals were sacrificed using ketamine and brain tissues were removed. Malondialdehyde (MDA), protein carbonyl (PCO) and tissue carnosine levels, and superoxide dismutase (SOD) activities were measured. Endogenous CAR levels in the rat brain tissue specimens were significantly increased in the CAR and EtOH groups when compared to the control animals. MDA and PCO levels in the EtOH group were significantly increased as compared to the other groups (P < 0.05). CAR treatment also decreased MDA levels in the CAR group as compared to the control group. Increased SOD activities were obtained in the EtOH + CAR group as compared to the control (P < 0.05). CAR levels in the rat brain were significantly increased in the CAR, EtOH and CAR + EtOH groups when compared to the control animals. These findings indicated that carnosine may appear as a protective agent against ethanol-induced brain damage.     

饲料蛋白水平对大规格草鱼生长、饲料利用和氮代谢相关酶活性的影响

为探讨饲料蛋白水平对草鱼（Ctenopharyngodon idellus）生长、饲料利用和氮代谢的影响,以鱼粉和酪蛋白为蛋白源制备蛋白水平分别为15%、20%、25%、30%和35%的5种等能（13.71 kJ/g）饲料饲养草鱼（20910） g 8周。结果表明:饲料蛋白水平对草鱼的增重率、特定生长率、饲料效率均影响显著（P0.05）,其中25%蛋白组最高。通过二次多项式的回归分析得出:当饲料蛋白水平为26.50%和27.20%的时候,特定生长率和饲料效率分别达到最高。15%蛋白组的摄食率显著高于其他蛋白组（P0.05）。蛋白质保留率和蛋白质效率随蛋白水平的升高而显著降低（P0.05）。各蛋白组的成活率、血清丙氨酸转氨酶活性和血清天冬氨酸转氨酶活性均无显著差异（P0.05）。血清总蛋白、血清尿素氮、肝脏天冬氨酸转氨酶活性、肝脏谷氨酸脱氢酶活性和肌肉腺苷酸脱氨酶活性随蛋白水平的升高显著增加（P0.05）,而肝脏丙氨酸转氨酶活性先增加后稳定,这表明高蛋白组（30%和35%蛋白组）相对于低蛋白组（15%和20%蛋白组）有较多的蛋白质用于分解代谢提供能量。       

Chronic ethanol feeding inhibits plasma levels of insulin-like growth factor-1

The purpose of this study was to determine whether the generalized catabolic effects of chronic ethanol may be associated with a decline in plasma levels of insulin-like growth factor-1 (IGF-1). Male Sprague-Dawley rats were fed a liquid diet containing 5% ethanol or pair-fed a diet made isocaloric with maltose-dextrin. Animals were maintained on this diet for either 12 days or 4.5 months. Another group of animals were fed control diet ad libitum for 2 weeks. After 12 days of feeding, plasma concentrations of IGF-1 in ad libitum fed rats were 771 +/- 41 ng/ml which was greater than concentrations in either pair-fed (595 +/- 23 ng/ml) or ethanol-fed (680 +/- 40 ng/ml) rats (P less than 0.05). After 4.5 months of feeding, plasma levels of IGF-1 in ad libitum and pair-fed rats were similar to the 12 day study (736 +/- 56 and 607 +/- 26 ng/ml, respectively). However, a significant decrease in plasma levels of IGF-1 was observed in ethanol-fed animals over the 4.5 month period (551 +/- 28 ng/ml, P less than 0.05). Results of a similar study in rats fed a high-fat diet for 4.5 months were similar to those found with the low-fat diet. These results indicate that 1) dietary restriction of the type routinely used in this pair-feeding regimen decreases plasma levels of IGF-1, 2) chronic ethanol feeding further decreases plasma IGF-1 levels compared to pair-fed rats, 3) the effects of ethanol on IGF-1 concentrations are not modified by dietary fat, and 4) the effects on IGF-1 are not directly dependent on elevated plasma ethanol concentrations. Our results suggest that IGF-1 secreting cells in the liver may be progressively damaged by chronic ethanol feeding.     

Effects of β‐carotene on antioxidant status in rats with chronic alcohol consumption

This study examined the effects of β‐carotene on antioxidant status in rats with chronic alcohol consumption. At the beginning of experiment (week 0), according to both the plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, rats (n = 24) were divided into 3 groups and fed with a standard diet (group C), a diet containing ethanol (group E), or a diet containing ethanol and β‐carotene (group E+B). After 10 weeks, plasma AST and ALT, fat accumulation in the liver, antioxidant enzyme activities in erythrocytes and the liver, malondialdehyde (MDA), and α‐tocopherol and retinol in plasma and hepatic samples were analyzed. The chronic alcohol diet significantly increased AST and ALT levels in plasma, and these changes were prevented by supplementing the diet with β‐carotene. Glutathione (GSH) in erythrocytes and in the liver was significantly elevated in rats fed with a diet containing β‐carotene. The results indicate that β‐carotene supplementation can prevent ethanol‐induced liver damage and increase GSH concentrations in erythrocytes and the liver. Copyright © 2009 John Wiley & Sons, Ltd.     

Enzymes of adenosine metabolism in the brain: diurnal rhythm and the effect of sleep deprivation

Adenosine plays a role in promoting sleep, an effect that is thought to be mediated in the basal forebrain. Adenosine levels vary in this region with prolonged wakefulness in a unique way. The basis for this is unknown. We examined, in rats, the activity of the major metabolic enzymes for adenosine - adenosine deaminase, adenosine kinase, ecto- and cytosolic 5'-nucleotidase - in sleep/wake regulatory regions as well as cerebral cortex, and how the activity varies across the day and with sleep deprivation. There were robust spatial differences for the activity of adenosine deaminase, adenosine kinase, and cytosolic and ecto-5'-nucleotidase. However, the basal forebrain was not different from other sleep/wake regulatory regions apart from the tuberomammillary nucleus. All adenosine metabolic enzymes exhibited diurnal variations in their activity, albeit not in all brain regions. Activity of adenosine deaminase increased during the active period in the ventrolateral pre-optic area but decreased significantly in the basal forebrain. Enzymatic activity of adenosine kinase and cytosolic-5'-nucleotidase was higher during the active period in all brain regions tested. However, the activity of ecto-5'-nucleotidase was augmented during the active period only in the cerebral cortex. This diurnal variation may play a role in the regulation of adenosine in relationship to sleep and wakefulness across the day. In contrast, we found no changes specifically with sleep deprivation in the activity of any enzyme in any brain region. Thus, changes in adenosine with sleep deprivation are not a consequence of alterations in adenosine enzyme activity.     

Phospholipid and Ganglioside Composition in Rat Brain After Chronic Intake of Ethanol

Abstract: A total of 18 60-day-old male Wistar rats were divided into three groups of six animals each. One group was fed a basal diet containing high levels of protein, fat, carbohydrate, vitamins, and minerals and separately a solution of 25% sucrose-32% ethyl alcohol (wt/vol). A second group was offered water as the only drinking fluid and a similar solid diet, except that carbohydrate replaced ethanol isocalorically. A third group was maintained on the basal diet ad libitum . All groups of animals were killed in a sober state after 6 months of chronic ethanol treatment and lipid analyses were performed on brain homog-enates. Chronic treatment of the animals with ethanol produces statistically significant modification of the phospholipid and ganglioside patterns in rat brain. A statistically significant decrease of the total phospholipid content and of some of the investigated fractions, i.e., phos-phatidylcholine and phosphatidylserine, as well as an increase of phosphatidylinositol were observed. Chronic alcohol consumption was associated with a statistically significant increase in the total amount of ganglioside in rat brain. An increase in most of the investigated ganglioside fractions was indicated but the difference was statistically significant only for trisialoganglioside GT_1b. The amount of disialoganglioside GD_1a in these brains was decreased after chronic intake of ethanol.     

Mast cell stabilizator and antioxidant effects of epidermal growth factor (EGF) on gastric mucosal injury induced by ethanol in rats

The role of epidermal growth factor (EGF), a polypeptide containing 53 amino acids, on protection and repair of ethanol-induced gastric mucosal injury was investigated in rats. In addition, the effects of EGF on the gastric damage were evaluated histopathologically. We used 48 Spraque-Dawley rats which were divided into [corrected] three groups as control rats, ethanol treated rats and ethanol+EGF treated rats. The ethanol group was given a gastric gavage containing 1 ml of 80% ethanol (v/v) prepared in distilled water. EGF (100 microg/kg) was given by intragastric gavage 30 min before the administration of ethanol. We studied histopathological evaluation and the histochemical heterogeneity of mast cells and its degree of degranulation. Besides, gastric tissue malondialdehyde (MDA), protein sulfhydryl groups (SH), and protein carbonyl levels were measured. EGF treatment stabilized mast cells degranulation and had lower polymorphonuclear leukocytes (PMNL) infiltration, ulcer index, histamine, and MDA; protein carbonyl levels were also lower, compared to the non-treated animals. EGF exerts a protective effect on gastric mucosa to ethanol-induced gastric injury probably through antioxidant and mast cell stabilizing mechanism.     

The effects of chronic alcohol and vitamin E consumption on aging pigments and learning performance in mice

Chronic ethanol consumption further accelerates age-related impairment of shuttle box avoidance learning in mice. The hypothesis was tested that the behavioral impairment is a result of brain lipofuscin pigment deposition, which may be accelerated by ethanol consumption and prevented by the antioxidant effects of pharmacological doses of vitamin E. Feeding an ethanol-containing liquid diet for 5 months did not increase the lipofuscin content when compared with mice pair-fed a liquid diet containing isocaloric amounts of sucrose or standard solid laboratory food containing nutritionally adequate amounts of vitamin E. Supplementation of diets with vitamin E decreased brain lipofuscin content in all groups but failed to prevent the age- or ethanol-induced learning deficit. There was no effect of chronic ethanol consumption on brain weights, DNA, RNA, or protein content.It is concluded that the age-related impairment of avoidance learning is accelerated by chronic alcohol consumption. At the molecular level this acceleration is not caused by an increased brain lipofuscin deposition nor is it prevented by the antioxidant effects of vitamin E.     

Altered Pattern of Na,K-ATPase Activity and mRNA During Chronic Alcohol Consumption by Juvenile and Adolescent Rats

The effect of chronic ethanol consumption on cerebral cortical activity of Na,K-ATPase was determined in Long-Evans (LE) rats fed an ethanol-containing diet beginning at different stages of development. Na,K-ATPase activity was operationally resolved into α1 and α2/3 isozyme activities. There was no significant difference in Na,K-ATPase activities before and after alcohol consumption in the preparations from adult rats. However, for rats beginning alcohol consumption as adolescents, the α2/3 activity was significantly elevated following chronic alcohol consumption. Both LE and Sprague–Dawley rats showed this same selective increase in cortical α2/3 activity when rats began alcohol consumption as juveniles. The shift in cortical α2/3 activity was not observed in cerebellum or subcortical forebrain and was reversible when rats were fed ethanol throughout the normal adolescent period and then withdrawn and tested 2 weeks later (during the adult period). Levels of isoform-specific mRNA were determined in preparations of cerebral cortices of rats showing elevated α2/3 isozyme activities. In these preparations, isoform specific α2 and α3 mRNA was significantly elevated. There was no effect of ethanol feeding on cortical α1 mRNA. These findings indicate that the longer term effects of ethanol on the developing brain include elevated Na,K-ATPase activity and a mechanism that is pre-translational and isoform specific.     

Maternal and fetal plasma adenosine deaminase, xanthine oxidase and malondialdehyde levels in pre-eclampsia

The aim of this study was to evaluate maternal-fetal plasma adenosine deaminase, xanthine oxidase (ADA, XO) activity and malondialdehyde (MDA) levels and the relationship between them in pre-eclampsia. Maternal and umbilical cord whole blood samples were taken from 29 pre-eclamptic and 33 normal pregnants. The plasma ADA, XO activities as well as MDA levels were assayed by spectrophotometric methods. MDA levels and ADA, XO activities were found to be higher in maternal and fetal plasma in pre-eclamptics than in normal pregnancy. The differences were statistically significant between groups (p < 0.05). Increased maternal-fetal plasma XO and ADA activities, as a marker of immunological disorder, may be related to the pathogenesis of pre-eclampsia. In addition, increased MDA levels may be a reflection of increased oxidative stress in pre-eclamptics and their fetuses.     

Serum selenium levels and oxidative balance as differential markers in hepatic damage caused by alcohol

Aims

Antioxidant system abnormalities have been associated with ethanol consumption. This study examines the effects of chronic ethanol consumption on oxidative balance, including selenium (Se) levels in alcoholic patients with or without liver disease, and if these measurements could be indicative of liver disease.

Main methods

Serum Se levels, antioxidant enzymes' activities, malondialdehyde (MDA) and protein carbonyl (PC) were determined in three groups of patients: alcoholics without liver disease, alcoholics with liver disease, and non-alcoholics with liver disease; and in healthy volunteers.

Key findings

Serum Se levels were lower in alcoholic patients and in patients affected by liver disease and especially lower in the alcoholic liver disease group. These values were correlated with the activity of glutathione peroxidase (GPx), the antioxidant selenoprotein. The antioxidant activities of the glutathione reductase (GR) and superoxide dismutase (SOD) were also lower in the three non-healthy groups. However, GR activity decreased and SOD activity increased in the non-alcoholic liver disease group versus alcoholic groups. Higher concentrations of PC in serum were found in non-healthy groups and were higher in alcoholic patients who also showed higher MDA levels. The highest MDA and PC levels were found in the alcoholic liver disease group.

Significance

We conclude that serum Se levels are drastically decreased in alcoholic liver disease patients, showing that this element has a direct correlation with GPx activity, and lipid oxidation, suggesting that the serum Se/MDA ratio could be an indicator of hepatic damage caused by alcohol consumption, and pointing to Se as a possible antioxidant therapy.     

Fetal alcohol exposure alters neurosteroid levels in the developing rat brain

Neurosteroids are modulators of neuronal function that may play important roles in brain maturation. We determined whether chronic prenatal ethanol exposure altered neurosteroid levels in the developing brain. Rat dams were exposed to: (i) a 5% ethanol-containing liquid diet that produces peak maternal blood alcohol levels near the legal intoxication limit (approximately 0.08 g/dL); (ii) an isocaloric liquid diet containing maltose-dextrin instead of ethanol with pair-feeding; (iii) rat chow ad libitum. Neurosteroid levels were assessed in offspring brains using radioimmunoassay or gas chromatography-mass spectrometry techniques. A prenatal ethanol exposure-induced increase in pregnenolone sulfate levels, but not dehydroepiandrosterone sulfate levels, was evident at the earliest time point studied (embryonic day 14). This effect lasted until post-natal day 5. Levels of other neurosteroids were assessed at embryonic day 20; pregnenolone levels, but not allopregnanolone levels, were elevated. Pregnenolone sulfate levels were not altered in the maternal brain. Neither pregnenolone nor pregnenolone sulfate levels were significantly altered in the fetal liver, placenta and maternal blood, indicating that the effect of ethanol is not secondary to accumulation of peripherally-produced steroids. Fetal ethanol exposure has been shown to decrease both cellular and behavioral responsiveness to neurosteroids, and our findings provide a plausible explanation for this effect.     

Oxidative Stress and Paraoxonase Activity in Experimental Selenosis: Effects of Betaine Administration

The present study was undertaken on male rats to elucidate the selenosis induced by sodium selenite and the role played by betaine in alleviating selenium toxicity. Rats were treated with sodium selenite (6 mg/kg body weight/day) with or without betaine (240 mg/kg body weight/day). Selenotoxicosis was evident from the elevated plasma levels of total bilirubin, transaminases, and alkaline phosphatase activities. Moreover, the total protein levels decreased, and this decrease associated with a decreased albumin level, whereas the globulin level increased in selenium-intoxicated rats. The development of selenosis corresponded well with the induction of oxidative stress evident from decrease of total thiol level and glutathione content. Furthermore, activities of glutathione reductase, glucose-6-phosphate dehydrogenase, catalase, and paraoxonase-1 were decreased in selenium-treated rats. In contrast, superoxide dismutase and glutathione peroxidase activities were increased by excess selenium administration compared with control animals. As well, malondialdehyde and protein carbonyl were elevated in rats treated with selenium. Supplementation of betaine simultaneously with selenium caused less marked alteration in the investigated parameters. Betaine attenuated the selenotoxicosis by restoring thiol levels that preserve enzymatic antioxidants activity and attenuate the oxidation of lipids and proteins.     

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     

Role of Extracellular Adenosine in Ethanol-Induced Desensitization of Cyclic AMP Production

Abstract: The decrease in receptor-stimulated cyclic AMP production after chronic ethanol exposure was suggested previously to be secondary to an ethanol-induced increase in extracellular adenosine. The present study was undertaken to ascertain whether a similar mechanism was responsible for the ethanol-induced desensitization of cyclic AMP production in PC12 pheochromocytoma cells. The acute addition of ethanol in vitro significantly increased both basal cyclic AMP content and extracellular levels of adenosine. A 4-day exposure to ethanol decreased basal as well as 2-chloroadenosine- and forskolin-stimulated cyclic AMP contents. No change in cyclic AMP content was observed after a 2-day exposure of PC12 cells to ethanol. Inclusion of adenosine deaminase during the chronic ethanol treatment significantly decreased extracellular levels of adenosine, yet the percentage decrease in 2-chloroadenosine- and forskolin-stimulated cyclic AMP levels after chronic ethanol exposure was not changed by the inclusion of the adenosine deaminase. Similar results were obtained when the chronic treatment was carried out with serum-free defined media. The ethanol-induced desensitization could not be mimicked by chronic exposure of PC12 cells to adenosine analogues. A 24-h exposure of PC12 cells to 2-chloroadenosine resulted in a decrease in the subsequent ability of this adenosine analogue to stimulate cyclic AMP content, but basal and forskolin-stimulated cyclic AMP levels were increased. Similar results were obtained after a 4-day exposure of PC12 cells to 2-chloroadenosine or 5'- N -ethylcarboxamido-adenosine. The present results indicate that the ethanol-induced decrease in receptor-stimulated cyclic AMP content in PC12 cells is not due to an increase in extracellular adenosine.