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Role of acetaldehyde in the actions of ethanol on the brain — A review
Alcohol,Vol. 13, No. 2, pp. 147-151,1996 Copyright©1996ElsevierScienceInc. Printedin the USA.All rightsreserved 0741-8329/96$15.00 + .00 ELSEVIER
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Role of Acetaldehyde in the Actions of Ethanol on the B r a i n - A Review W A L T E R A. H U N T 1
Neurosciences and Behavioral Research Branch, National Institute on A l c o h o l A b u s e and Alcoholism, Bethesda, M D 20892-7003 Received l 0 M a r c h 1995; Accepted 18 A u g u s t 1995 HUNT, W. A. Role of acetaldehyde in the actions of ethanol on the brain-a review. ALCOHOL 13(2) 147-151, 1996.Over the last 30 years, acetaldehyde has been postulated to mediate various actions of ethanol on the brain. Experiments have studied ethanol consumption after acetaldehyde infusions into the brain, in rodents with high or low activities of hepatic and brain ethanol-metabolizing enzymes, and after treatment with drugs that alter the metabolism of acetaldehyde after ethanol ingestion. Evidence that acetaldehyde is involved in the actions of ethanol has been inconsistent because of the lack of knowledge of the brain acetaldehyde concentrations required to exert their effects, the lack of correlation between the activities of ethanol-metabolizing enzymes across strains of rodents and ethanol consumption, and the lack of specificity of drugs altering acetaldehyde metabolism. The formation of significant amounts of acetaldehyde the brain in vivo after ethanol ingestion and by what mechanism has not been clearly established, although catalase is a promising candidate. Future research needs to directly demonstrate in brain the formation of acetaldehyde in vivo, determine the concentrations in brain areas involved in ethanol consumption, and evaluate the possible actions of drugs other than an ability to block acetaldehyde metabolism. Alcohol dehydrogenase
Aldehyde dehydrogenase
Catalase
ETHANOL induces a multitude of effects on the brain that lead to changes in behavior, including euphoria, motor incoordination, aggression, tolerance and dependence, and brain damage. It is generally assumed that these actions are caused by ethanol itself. However, for 30 years a role of acetaldehyde (the first metabolite in the metabolism of ethanol) in some of the actions of ethanol on the brain has been postulated, but not established. Elevated blood acetaldehyde concentrations are generally considered aversive and are the basis for treating alcoholics with disulfiram (Antabuse), an inhibitor of acetaldehyde metabolism. However, some evidence suggests that acetaldehyde can have positively reinforcing effects as well as mediate actions of ethanol. This review will examine the available data supporting a role of acetaldehyde in the actions of ethanol on the brain. Also, possible sources of acetaldehyde will be assessed to determine if sufficient concentrations could exist in the brain. Finally, gaps in our knowledge will be identified in hopes of stimulating further research.
3-Amino-1,2,4-triazole
Thyroid function
BEHAVIORAL AND NEUROCHEMICALEFFECTS OF ACETALDEHYDE Numerous studies have suggested a role of acetaldehyde in the actions of ethanol on the brain [see review by Hunt (28)]. Results of these studies have been derived by several strategies, including intraventricular injections of acetaldehyde and measuring subsequent behavioral changes, comparing the activities of ethanol-metabolizing enzymes in strains of rodents with differing preferences for ethanol, and altering the metabolism of acetaldehyde after ethanol ingestion, thereby modifying its concentration in the blood and tissues. The most direct studies showing that acetaldehyde can be reinforcing infused it directly into the ventricular system of the brain. When acetaldehyde was infused over a period of days, ethanol preference increased, although the amount of ethanol consumed was modest, and no signs of intoxication were reported (13,41). Rats also responded on a lever to receive infusions of acetaldehyde intraventricularly (12,13), intraperitone-
Requests for reprints should be addressed to Dr. Walter A. Hunt, Division of Basic Research, National Institute on Alcohol Abuse and Alcoholism, Willco Bldg., Suite 402, 6000 Executive Blvd. MSC 7003, Bethesda, MD 20892-7003. 147
148 ally (42), and intravenously (59), and developed a conditioned taste preference (a measure of reinforcement) after acetaldehyde infusions (53). However, none of these studies determined the concentration of acetaldehyde in the brain. (In fairness, measuring acetaldehyde has been difficult, as discussed later, and has discouraged investigators from measuring it.) The blood acetaldehyde concentration is regulated in part by the activities of alcohol dehydrogenase, cytochrome P450, catalase, and aldehyde dehydrogenase. Alcohol dehydrogenase, cytochrome P450, and catalase convert ethanol to acetaldehyde, whereas aldehyde dehydrogenase converts acetaldehyde to acetate. The activities of these enzymes vary among rodent strains and can be altered by drugs, providing an opportunity to relate the actions of ethanol to these activities. Comparing enzymatic activities and altering blood acetaldehyde concentrations have been used primarily to examine the role of acetaldehyde in ethanol intake. Over the years, researchers have studied the relationship between enzymatic activity and ethanol consumption among rodent strains. High activities of alcohol dehydrogenase could lead to elevated acetaldehyde concentrations. However, no clear relationship with alcohol dehydrogenase activity has been found. Although ethanol-preferring C57BL mice have higher hepatic alcohol dehydrogenase activity than ethanolavoiding DBA mice (48), the opposite is true for ethanolpreferring AA and ethanol-avoiding ANA rats. AA rats have lower alcohol dehydrogenase activity than the ANA rats (17, 34,35). Results with aldehyde dehydrogenase have been more consistent than those from studies of alcohol dehydrogenase. Ethanol-preferring rodents have higher hepatic aldehyde dehydrogenase activity and lower blood acetaldehyde concentrations after ethanol ingestion than the ethanol-avoiding rodents (34, 35,48,49). Furthermore, brain aldehyde dehydrogenase correlated with ethanol consumption in Long-Evans rats (54). However, no differences in brain aldehyde dehydrogenase activity were detected between AA and ANA rats (29). High aldehyde dehydrogenase activity might accelerate the removal of synthesized acetaldehyde and increase ethanol consumption by reducing the aversive response. Several studies reported altered ethanol intake after drug treatment that inhibited acetaldehyde dehydrogenase and elevated blood acetaldehyde concentrations. Pretreatment with calcium cyanamide increased blood acetaldehyde concentrations and decreased ethanol consumption from 3-5 g/kg/day to < 1 g/kg/day (19,50,51) and ethanol-induced locomotor activity (56). These effects were either partially reversed by pretreatment with the alcohol dehydrogenase inhibitor 4methylpyrazole (50) or not reversed at all (56). On the other hand, 4-methylpyrazole alone reduced ethanol intake in the alcohol-preferring P rats, possibly because less ethanol consumption was needed to maintain a particular blood ethanol concentration (63). In all, the data from these studies suggested that elevated acetaldehyde concentrations were aversive under these experimental conditions. Brain catalase activity also correlated with ethanol consumption in Long-Evans rats (9). In rats consuming ethanol for 25 days, those drinking the most ethanol had the highest brain catalase activities. However, the opposite finding was observed in mice. Acatalasemic mice with catalase activity 4050°70 of normal exhibited more ethanol consumption than normal mice, especially high ethanol concentrations (> 12070), and less activation of motor activity (2,4). In fact, in a recent study with recombinant inbreds of C57BL/6J and DBA/2J mice,
HUNT those mice drinking high amounts of ethanol had low brain catalase activity and those drinking low amounts had high activities (23). Thus, how catalase-mediated synthesis of acetaldehyde might contribute to ethanol consumption is presently unclear. Recent experiments have further assessed the role of catalase in the actions of ethanol by pretreating Long-Evans rats with the catalase inhibitor 3-amino-l,2,4-triazole. Pretreatment with 3-amino-l,2,4-triazole, at a dose that drastically lowered brain catalase activity (5) without affecting the blood ethanol concentration (60), significantly reduced ethanol consumption (3,33). In addition, it reduced ethanol-induced depression in locomotor activity (8), narcosis (7), and conditioned taste aversion (6) but had no effect on ethanol-induced hypothermia or the ethanol elimination rate (61). Moreover, tolerance to the depressant effects of ethanol but not to hypothermia or metabolic tolerance was also attenuated (61). Although these results are quite promising, one problem of using 3-amino-l,2,4-triazole as a catalase inhibitor in ethanol studies is its lack of specificity, an issue that needs careful consideration. 3-Amino-l,2,4-triazole has other effects that might be important to its ability to block actions of ethanol. Originally, 3-amino-l,2,4-triazole was developed as a herbicide. However, it is carcinogenic (57) and goitrogenic (22,37). Related to the latter effect, 3-amino-l,2,4-triazole exposure leads to a state of hypothyroidism, with serum thyroxine values significantly reduced (58). As a compensatory response, thyroid-stimulating hormone is elevated. Thyrotropin-releasing hormone (TRH) presumably would also increase, but effects of 3-amino-l,2,4-triazole on this hormone have not been reported. An effect of 3-amino-l,2,4-triazole on TRH could be significant to the interpretation of the results suggesting antagonism of a number of actions of ethanol. Pretreatment with TRH itself or the TRH analogue 1-methyl-(S)-4,5,dihydroorotyl-L-histidyl-L-prolineamide (TA-0910) has been reported to reduce ethanol preference in P rats (46), antagonize the impairment of the aerial righting reflex induced by ethanol (1 l), and diminish ethanol-induced hypothermia without altering brain ethanol concentrations (16). The effect of TA0910 on ethanol preference might be related to increases in dopamine release in the nucleus accumbens induced by TRH (27), similar to that found after ethanol ingestion (66). TA0910-induced changes in dopamine release might substitute for the effects of ethanol ingestion and reduce the need for ethanol to obtain a given level of reinforcement. Injections of TRH into the medial septum but not other areas of the brain produce the same results on ethanol-induced impairment (38). Other studies indicate that TRH levels increase in the medial septum after rats ingesting ethanol regained their righting reflex (40). These findings suggest that TRH increases in the medial septum in response to ethanol exposure, thereby contributing to the recovery from ethanol intoxication. If 3-amino-l,2,4-triazole administration was to increase TRH levels in the brain, especially in the medial septum, in response to a hypothyroidogenic effect, the antagonism of ethanol-induced actions by this drug could be due to actions of TRH rather than to catalase inhibition. However, most studies of 3-amino-l,2,4-triazole on thyroid function were performed after chronic exposure. But one study, using an acute dose 15070 of that used in ethanol studies (0.5-1.0 mg/kg, IP, 1-5 h before ethanol administration), reduced radioactive iodine uptake into the thyroid by almost 60070 10 h after treatment (21). A possible effect on thyroid function, especially on TRH levels, of a single dose of 3-amino-l,2,4-
ETHANOL AND ACETALDEHYDE IN THE BRAIN triazole, administered in the manner typically used in ethanol experiments, has not been reported and should be evaluated. SOURCES OF ACETALDEHYDEAND EXPECTED CONCENTRATIONS Whether acetaldehyde plays a significant role in the actions of ethanol on the brain ultimately depends on the availability of acetaldehyde after ethanol consumption in sufficient concentrations in the brain to be biologically effective. The primary site of the conversion of ethanol to acetaldehyde is in the liver. The liver being a relevant source of acetaldehyde for actions on the brain necessitates the presence of acetaldehyde in the blood, which would transport it to the brain. The measurement of blood acetaldehyde has been controversial for almost 20 years. Reported values of blood acetaldehyde have been confounded by artifacts, including nonenzymatic conversion of ethanol to acetaldehyde, binding to erythrocytes, and sample decomposition (18). Over the years, the methodology for measuring acetaldehyde has improved (20). As a result, using the most modern techniques, no measurable blood acetaldehyde concentrations (<0.5 /~M) were found after ethanol consumption by normal humans not taking aldehyde dehydrogenase inhibitors (20). Adding to the difficulty of finding a peripheral source of acetaldehyde for actions on the brain, a blood-brain barrier to acetaldehyde probably would reduce its penetration into the brain. Sippel (52) reported that blood acetaldehyde concentrations had to exceed 250 ~tM before measurable brain acetaldehyde concentrations could be detected. Furthermore, evidence suggests the presence of aldehyde dehydrogenase in the microvasculature in the brain (44). This enzyme would effectively destroy most acetaldehyde in the blood as it penetrated the brain. With no significant likelihood that peripherally derived acetaldehyde would reach the brain, a remaining source of acetaldehyde would be in the brain itself. Three general enzymatic systems catalyze the conversion of ethanol to acetaldehyde: alcohol dehydrogenase, cytochrome P450, and catalase. The existence of significant alcohol dehydrogenase activity in the brain has been disputed for many years. Early studies detected little alcohol dehydrogenase activity in the brain (45) or acetaldehyde in cerebrospinal fluid of intoxicated alcoholics (36). More recently, immunocytochemical methods found brain alcohol dehydrogenase only in neuronal cytoplasm of some neurons mostly in the mammillary bodies, periaqueductal gray, and the cerebral and cerebellar cortices (32). Such results suggest that high alcohol dehydrogenase activity could be of local importance to individual neurons, even though activity in general areas is low. The significance of localized alcohol dehydrogenase depends on the isozymes of the enzyme present. Alcohol dehydrogenase exists as several isozymes, distinguished by their affinity for ethanol and the ease with which the isozyme can be inhibited by pyrazole (62). Reports of the relative proportion of these isozymes in the brain have not been consistent. Although one study found the isozyme with high affinity (Km = 0.15 raM) for ethanol (14), all other studies found only low-affinity isozymes (Km > 500 mM) in several animal species, including humans (10,25,30,47). Thus, ethanol concentrations in the brain must be very high before alcohol dehydrogenase would play much of a role in the conversion of ethanol to acetaldehyde. Cytochrome P450 is an enzyme complex involved in the detoxification of drugs and toxins in the liver and can exist in many forms. The form most involved in ethanol metabolism
149 is cytochrome P450 2El. Cytochrome P450 2El as well as other cytochrome P450s have been identified in the brain and can be found in neurons and glia in several brain regions (26,59,64). The relative basal concentrations of these cytochrome P450s suggest that 2El represents less than I070of the activity of all cytochrome P450s (64). However, cytochrome P450 2El is inducible by ethanol, and increases of three- to fivefold in activity have been reported after ethanol administration (1,39,55,65). The importance of cytochrome P450 2El in the formation of acetaldehyde after ethanol consumption would, therefore, depend on how quickly the enzyme can be induced. To date, no studies have been published to address this question. Catalase, along with glutathione peroxidase, functions as a regulator of hydrogen peroxide levels, a metabolite formed during the detoxification of reactive oxygen radicals. In the presence of hydrogen peroxide, ethanol can be oxidized by purified rat liver catalase to acetaldehyde in vitro (31,43). This is accomplished by the catalase-hydrogen peroxide intermediate (Compound I) through a peroxidatic reaction in which acetaldehyde and water are formed from the intermediate and ethanol (a hydrogen donor). Evidence has been reported suggesting that catalase can oxidize ethanol to acetaldehyde in the brain (15,60). However, all of the results reported have depended on using 3-amino1,2,4-triazole, an irreversible inhibitor of catalase, to infer a catalase-mediated production of acetaldehyde and assess the effect of catalase inhibition on ethanol-induced alterations in behavior. Using methods of analysis that avoid artifactual formation of acetaldehyde, the most convincing study of catalase-mediated production of acetaldehyde in brain tissue demonstrated synthesis of acetaldehyde in the micromolar range (24). The formation of acetaldehyde was reduced by 3-amino1,2,4-triazole but not by metyrapone, a cytochrome P450 inhibitor, or 4-methylpyrazole, an alcohol dehydrogenase inhibitor, suggesting a role of catalase. Because these studies were performed in vitro, possible thyroidogenic effects of 3-amino1,2,4-triazole would not occur. CONCLUDINGREMARKS The role of acetaldehyde in the actions of ethanol on the brain has been investigated for almost 30 years. Although such a role is promising, its support is largely indirect and circumstantial, involving correlational studies and pharmacological evidence. Some of this evidence suggests that catalase mediates the formation of acetaldehyde from ethanol. The presence of catalase in the brain is not in itself proof that it catalyzes the conversion of ethanol to significant concentrations of acetaldehyde. This reaction occurs only in the presence of hydrogen peroxide. Whether sufficient concentrations of hydrogen peroxide are generated in the brain in vivo to support catalase-mediated production of acetaldehyde is unknown and needs further study. In addition, experiments with catalase inhibition are inconclusive because of the lack of specificity of 3-amino-l,2,4-triazole. The short-term effects of 3-amino-l,2,4-triazole on thyroid function require systematic evaluation. In addition, more specific catalase inhibitors are needed. Although catalase appears to be a likely source of any synthesized acetaldehyde after ethanol consumption, a role of cytochrome P450 2El cannot be completely discounted, especially during chronic ethanol exposure. As discussed earlier, cytochrome P450 2El can be induced in the brain by ethanol. Published reports did not study the time course of
150
HUNT
induction, which should be pursued. However, even with significant induction, it still needs to be established if this route of synthesis leads to significant concentrations of acetaldehyde in the brain. Ultimately, a significant role for acetaldehyde in the actions of ethanol on the brain requires the presence of acetaldehyde in the brain after ethanol consumption in quantities consistent with those used in experiments showing biological or behavioral changes. To date, the data are not compelling. What is required is a way in which acetaldehyde can be quantitated in the brain in vivo after ethanol administration. Finally, an unresolved issue is whether the results obtained represent a balance between rewarding and aversive properties of acetaldehyde. On the one hand, high alcohol dehydrogenase and/or catalase activities in the brains of some rodent strains might enhance the synthesis of acetaldehyde and thereby add to a possible rewarding effect of ethanol. On the other hand, high aldehyde dehydrogenase activity might decrease the acetaldehyde present and decrease the possible aversive effects of ethanol. The results suggest a narrow range
of acetaldehyde concentrations in modulating a rewarding action of ethanol ingestion. As brain acetaldehyde concentrations increase, the reinforcing properties would increase. However, once an upper limit of acetaldehyde concentrations is attained, the aversive effects of acetaldehyde would predominate, thereby reducing the reinforcing properties of ethanol. Although brain catalase and aldehyde dehydrogenase activities correlate with voluntary ingestion of alcohol, the relative contribution of these three variables to the degree of ethanol consumption in the same animals is not clear. Taken together, the results provide no clear relationship between activities of hepatic and brain ethanol-metabolizing enzymes per se and ethanol preference across rodent strains, let alone humans. Nevertheless, the role for these enzymes in causing differences in individual strains cannot be discounted. The role of acetaldehyde in the actions of ethanol on the brain has been studied for many years, but an exact role has not been conclusively demonstrated. There are still potentially fruitful areas of investigation to show once and for all whether such a role for acetaldehyde has any real importance.
REFERENCES 1. Anandatheerthavarada, H. K.; Shankar, S. K.; Bhamre, S.; Boyd, M. R.; Song, B.-J.; Ravindranath, V. Induction of brain cytochrome P450 IIEI by chronic ethanol treatment. Brain Res. 601:279-285; 1993. 2. Aragon, C. M. G.; Amit, Z. Differences in ethanol-induced behaviors in normal and acatalasemic mice: Systemic examination using a biobehavioral approach. Pharmacol. Biochem. Behav. 44:547-554; 1993. 3. Aragon, C. M. G.; Amit, Z. The effect of 3-amino-l,2,4-triazole on voluntary ethanol consumption: evidence for brain catalase involvement in the mechanism of action. Neuropharmacology 31 : 709-712; 1992. 4. Aragon, C. M. G.; Pesold, C. N.; Amit, Z. Ethanol-induced motor activity in normal and acatalasemic mice. Alcohol 9:207211; 1992. 5. Aragon, C. M. G.; Rogan, F.; Amit, Z. Dose- and timedependent effect of an acute 3-amino-1,2,4-triazole injection on rat brain catalase activity. Biochem. Pharmacol. 42:699-702; 1991. 6. Aragon, C. M. G.; Spivak, K.; Amit, Z. Blockade of ethanolinduced conditioned taste aversion by 3-amino-1,2,4-triazole: Evidence for catalase mediated synthesis of acetaldehyde in rat brain. Life Sci. 37:2077-2084; 1985. 7. Aragon, C. M. G.; Spivak, K.; Amit, Z. Effect of 3-amino-l,2,4triazole on ethanol-induced narcosis, lethality and hypothermia in rats. Pharmacol. Biochem. Behav. 39:55-59; 1991. 8. Aragon, C. M. G.; Spivak, K.; Amit, Z. Effects of 3-amino1,2,4-triazole on ethanol-induced open field activity: Evidence for brain cataiase mediation of ethanol's effects. Alcohol. Clin. Exp. Res. 13:104-108; 1989. 9. Aragon, C. M. G.; Sternklar, G.; Amit, Z. A correlation between voluntary ethanol consumption and brain catalase activity in the rat. Alcohol 2:353-356; 1985. 10. Beisswenger, T. B.; Holmquist, B.; Vallee, B. L. ADH is the sole alcohol dehydrogenase isozyme of mammalian brains: Implications and inferences. Proc. Natl. Acad. Sci. USA 82:8369-8373; 1985. 11. Breese, G. R.; Cott, J. M.; Cooper, B. R.; Prange, A. J.; Lipton, M. A. Antagonism of ethanol narcosis by thyrotropin releasing hormone. Life Sci. 14:1053-1063; 1974. 12. Brown, Z. W.; Amit, Z.; Rockman, G. E. Intraventricular selfadministration of acetaldehyde but not ethanol in naive laboratory rats. Psychopharmacology (Berlin) 64: 271-276; 1979. 13. Brown, Z. W.; Amit, Z.; Smith, B. Intraventricular self-ad-
14.
15. 16.
17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
ministration of acetaldehyde and voluntary consumption of ethanol in rats. Behav. Neural Biol. 28:150-155; 1980. Biihler, R.; Pestalozzi, D.; Hess, M.; von Wartburg, J. P. Immunohistochemical localization of alcohol dehydrogenase in human kidney, endocrine organs and brain. Pharmacol. Biochem. Behay. 18(Suppl. 1):55-59; 1983. Cohen, G.; Sinet, P. M.; Heikkila, R. Ethanol oxidation by rat brain in vivo. Alcohol. Clin. Exp. Res. 4:366-370; 1980. Cott, J. M.; Breese, G. R.; Cooper, B. R.; Barlow, S.; Prange, A. J. Investigations into the mechanism of reduction of ethanol sleep by thyrotropin-releasing hormone (TRH). J. Pharmacol. Exp. Thee 196:594-604; 1976. Eriksson, C. J. P. Ethanol and acetaldehyde metabolism in rat strains genetically selected for their ethanol preference. Biochem. Pharmacol. 22:2283-2292; 1973. Eriksson, C. J. P. Problems and pitfalls in acetaldehyde determinations. Alcohol. Clin. Exp. Res. 4:22-29; 1980. Eriksson, C. J. P.; Deitrich, R. A. Evidence against a biphasic effect of acetaldehyde on voluntary ethanol consumption in rats. Pharmacol. Biochem. Behav. 13(Suppl. 1):291-296; 1980. Eriksson, C. J. P.; Fukunaga, T. Human blood acetaldehyde (update 1992). Alcohol Alcohol. Suppl. 2:9-25; 1993. Fregly, M. J. Effect of aminotriazole on thyroid function in the rat. Toxicol. Appl. Pharmacol. 13:271-286; 1968. Gaines, T. B.; Kombrough, R. D.; Linder, R. E. The toxicity of amitrole in the rat. Toxicol. Appl. Pharmacol. 26:118-129; 1973. Gill, K.; Deitrich, R. A.; Erwin, V. G. Alcohol-related behaviors in the BXD recombinant inbred (RI) strains of mice. Alcohol. Clin. Exp. Res. 19:12A(Suppl.); 1995. Gill, K.; Menez, J. F.; Lucas, D.; Deitrich, R. A. Enzymatic production of acetaldehyde from ethanol in rat brain tissue. Alcohol. Clin. Exp. Res. 16:910-915; 1992. Giri, P. R.; Linnoila, M.; Neil, J. B. O.; Goldman, D. Distribution and possible metabolic role of Class III alcohol dehydrogenase in the human brain. Brain Res. 484:131-141; 1989. Hansson, T.; Tindberg, N.; Ingelman-Sundberg, M.; Kohler, C. Regional distribution of ethanol-inducible cytochrome P450 IIE1 in the rat central nervous system. Neuroscience 34:451-463; 1990. Heal, D. J.; Green, A. R. Administration of thyrotropin-releasing hormone (TRH) to rats releases dopamine in n. accumbens but not in n. caudatus. Neuropharmacology 18:23-31; 1979. Hunt, W. A. Biochemical bases for the reinforcing effects of ethanol. In: Cox, W. M., ed. Why people drink: Parameters of alcohol as a reinforcer. New York: Gardiner Press; 1990:51-70.
ETHANOL
AND ACETALDEHYDE
IN T H E B R A I N
29. Inoue, K.; Rusi, M.; Lindros, K. O. Brain aldehyde dehydrogenase activity in rat strains with high and low ethanol preferences. Pharmacol. Biochem. Behav. 14:107-111; 1975. 30. Julia, P.; Farres, J.; Pares, X. Characterization of three isozymes of rat alcohol dehydrogenase: Tissue distribution and physical and enzymatic properties. Eur. J. Biochem. 162:179-189; 1987. 31. Keilin, D.; Martree, E. F. Properties of catalase. Catalysis of coupled oxidation of alcohol. Biochem. J. 39:293-301; 1945. 32. Kerr, J. T.; Maxwell, D. S.; Crabb, D. W. Immunocytochemistry of alcohol dehydrogenase in the rat central nervous system. Alcohol. Clin. Exp. Res. 13:730-736; 1989. 33. Koechling, U. M.; Amit, Z. Effects of 3-amino-l,2,4-triazole on brain catalase in the mediation of ethanol consumption in mice. Alcohol 11:235-239; 1994. 34. Koivisto, T.; Eriksson, C. P. J. Hepatic aldehyde and alcohol dehydrogenases in alcohol-preferring and alcohol-avoiding rat lines. Biochem. Pharmacol. 48:1551-1558; 1994. 35. Koivula, T.; Koivusalo, M.; Lindros, K. O. Liver aldehyde and alcohol dehydrogenase activities in rat strains genetically selected for their ethanol preference. Biochem. Pharmacol. 24:1807-1811; 1975. 36. Lindros, K. O.; Hillbom, M. E. Acetaldehyde in cerebrospinal fluid: Its near-absence in ethanol-intoxicated alcoholics. Med. Biol. 57:246-247; 1979. 37. Mayberry, W. E. Antithyroid effects of 3-amino-l,2,4-triazole. Proc. Soc. Exp. Biol. Med. 129:551-556; 1968. 38. McCown, T. J.; Moray, L. J.; Kizer, J. S.; Breese, G. R. Interactions bet~veen TRH and ethanol in the medial septum. Pharmacol. Biochem. Behav. 24:1269-1274; 1986. 39. Montoliu, C.; Valles, S.; Renau-Piqueras, J.; Guerri, C. Ethanolinduced oxygen radical formation and lipid peroxidation in rat brain: Effect of chronic alcohol consumption. J. Neurochem. 63: 1855-1862; 1994. 40. Morzorati, S.; Kubek, M. J. Septal TRH in alcohol-naive P and NP rats and following alcohol challenge. Brain Res. Bull. 31:301304; 1993. 41. Myers, R. D.; Veale, W. L. Alterations in volitional alcohol intake produced in rats by chronic intraventricular infusions of acetaldehyde, paraldehyde or methanol. Arch. Int. Pharmacodyn. Ther. 180:100-113; 1969. 42. Myers, W. D.; Ng, K. T.; Singer, G. Ethanol preference in rats with a prior history of acetaldehyde self-administration. Experientia 40:1008-1010; 1984. 43. Oshino, N.; Oshino, R.; Chance, B. The characteristics of the peroxidatic reaction in ethanol oxidation. Biochem. J. 131:555567; 1973. 44. Petersen, D. R. Aldehyde dehydrogenase and aldehyde reductase in isolated bovine brain microvessels. Alcohol 2:79-83; 1985. 45. Raskin, N. H.; Sokoloff, L. Enzymes catalyzing ethanol metabolism in neural and somatic tissue of the rat. J. Neurochem. 19: 273-282; 1972. 46. Rezvani, A. H.; Garbutt, J. C.; Shimoda, K.; Garges, P. L.; Janowsky, D. S.; Mason, G. A. Attenuation of alcohol preference in alcohol-preferring rats by a novel TRH analogue, TA0910. Alcohol. Clin. Exp. Res. 16:326-330; 1992. 47. Rout, U. K. Alcohol dehydrogenases in the brain of mice. Alcohol. Clin. Exp. Res. 16:286-289; 1992. 48. Schlesinger, K. R.; Kakihana, R.; Bennett, E. L. Effects of tetraethylthiuramdisulfide (Antabuse) on the metabolism and consumption of ethanol in mice. Psychosom. Med. 28:514-520; 1966.
151 49. Sheppard, J. R.; Albersheim, P.; McClearn, G. Aldehyde dehydrogenase and ethanol preference in mice. J. Biol. Chem. 245: 2876-2882; 1970. 50. Sinclair, J. D.; Lindros, K. O. Suppression of alcohol drinking with brain aldehyde dehydrogenase inhibition. Pharmacol. Biochem. Behav. 14:377-383; 1981. 51. Sinclair, J. D.; Lindros, K. O.; Terho, K. Aldehyde dehydrogenase inhibitors and voluntary ethanol drinking in rats. In: Thurman, R. G., ed. Alcohol and aldehyde metabolizing systems- IV. New York: Plenum Press; 1980:481-487. 52. Sippel, H. W. The acetaldehyde content in rat brain during ethanol metabolism. J. Neurochem. 23:451-452; 1974. 53. Smith, B. R.; Amit, Z.; Splawinsky, J. Conditioned place preference induced by intraventricular infusions of acetaldehyde. Alcohol 1:193-195; 1984. 54. Socaransky, S. M.; Aragon, C. M.; Amit, Z. Brain ALDH as a possible modulator of voluntary ethanol intake. Alcohol 2:361365; 1985. 55. Sohda, T.; Shimizu, M.; Kamimura, S.; Okumura, M. Immunohistochemical demonstration of ethanol-inducible P450 2E 1 in rat brain. Alcohol Alcohol. Suppl. 1B:69-75; 1993. 56. Spivak, K.; Aragon, C. M.; Amit, Z. Alterations in brain aldehyde dehydrogenase activity modify the locomotor effects produced by ethanol in rats. Alcohol Drug Res. 7:481-491; 1987. 57. Steinhoff, D.; Weber, H.; Mohr, U.; Boehme, K. Evaluation of amitrole (aminotriazole) for potential carcinogenicity in orally dosed rats, mice, and golden hamsters. Toxicol. Appl. Pharmacol. 69:161-169; 1983. 58. Stringer, B.; Wynford-Thomas, D.; Jasani, B.; Williams, E. D. Effect of goitrogen administration on the circadian rhythm of serum thyroid stimulating hormone in the rat. Acta Endocrinol. (Copenh.) 98:396-401; 1981. 59. Takayama, S.; Uyeno, E. T. Intravenous self-administration of ethanol and acetaldehyde by rats. Yakubutsu Seishin Kodo 5: 329-334; 1985. 60. Tampier, L.; Mardones, J. Effect of 3-amino-l,2,4-triazole pretreatment on ethanol blood levels after intraperitoneal administration. Alcohol 3:181-183; 1986. 61. Tampier, L.; Quintanilla, M. E. Effect of 3-amino-l,2,4-triazole on the hypothermic effect of ethanol and on ethanol tolerance development. Alcohol 8:279-282; 1991. 62. Vallee, B. L.; Bazzone, T. J. Isozymes of human liver alcohol dehydrogenase. In: Rattazzi, M. C.; Scandalio, J. G.; Whirr, G. S., eds. lsozymes, current topics in biological and medical research. Vol. 8. New York: Alan R. Liss; 1983:219-244. 63. Waller, M. B.; McBride, W. J.; Lumeng, L.; Li, T.-K. Effects of intravenous ethanol and of 4-methylpyrazole on alcohol drinking in alcohol-preferring rats. Pharmacol. Biochem. Behav. 17:763768; 1982. 64. Warner, M.; Gustafsson, J.-.~. Effect of ethanol on cytochrome P450 in rat brain. Proc. Natl. Acad. Sci. USA 91:1019-1023; 1994. 65. Warner, M.; Str6mstedt, M.; Wyss, A.; Gustafsson, J.-A. Regulation of cytochrome P450 in the central nervous system. J. Steroid Biochem. Mol. Biol. 47:191-194; 1993. 66. Weiss, F.; Lorang, M. T.; Bloom, F. E.; Koob, G. F. Oral alcohol self-administration stimulates dopamine release in the rat nucleus accumbens: Genetic and motivational determinants. J. Pharmacol. Exp. Ther. 267:250-258, 1993.
0741-8329(95)02026-8
Role of Acetaldehyde in the Actions of Ethanol on the B r a i n - A Review W A L T E R A. H U N T 1
Neurosciences and Behavioral Research Branch, National Institute on A l c o h o l A b u s e and Alcoholism, Bethesda, M D 20892-7003 Received l 0 M a r c h 1995; Accepted 18 A u g u s t 1995 HUNT, W. A. Role of acetaldehyde in the actions of ethanol on the brain-a review. ALCOHOL 13(2) 147-151, 1996.Over the last 30 years, acetaldehyde has been postulated to mediate various actions of ethanol on the brain. Experiments have studied ethanol consumption after acetaldehyde infusions into the brain, in rodents with high or low activities of hepatic and brain ethanol-metabolizing enzymes, and after treatment with drugs that alter the metabolism of acetaldehyde after ethanol ingestion. Evidence that acetaldehyde is involved in the actions of ethanol has been inconsistent because of the lack of knowledge of the brain acetaldehyde concentrations required to exert their effects, the lack of correlation between the activities of ethanol-metabolizing enzymes across strains of rodents and ethanol consumption, and the lack of specificity of drugs altering acetaldehyde metabolism. The formation of significant amounts of acetaldehyde the brain in vivo after ethanol ingestion and by what mechanism has not been clearly established, although catalase is a promising candidate. Future research needs to directly demonstrate in brain the formation of acetaldehyde in vivo, determine the concentrations in brain areas involved in ethanol consumption, and evaluate the possible actions of drugs other than an ability to block acetaldehyde metabolism. Alcohol dehydrogenase
Aldehyde dehydrogenase
Catalase
ETHANOL induces a multitude of effects on the brain that lead to changes in behavior, including euphoria, motor incoordination, aggression, tolerance and dependence, and brain damage. It is generally assumed that these actions are caused by ethanol itself. However, for 30 years a role of acetaldehyde (the first metabolite in the metabolism of ethanol) in some of the actions of ethanol on the brain has been postulated, but not established. Elevated blood acetaldehyde concentrations are generally considered aversive and are the basis for treating alcoholics with disulfiram (Antabuse), an inhibitor of acetaldehyde metabolism. However, some evidence suggests that acetaldehyde can have positively reinforcing effects as well as mediate actions of ethanol. This review will examine the available data supporting a role of acetaldehyde in the actions of ethanol on the brain. Also, possible sources of acetaldehyde will be assessed to determine if sufficient concentrations could exist in the brain. Finally, gaps in our knowledge will be identified in hopes of stimulating further research.
3-Amino-1,2,4-triazole
Thyroid function
BEHAVIORAL AND NEUROCHEMICALEFFECTS OF ACETALDEHYDE Numerous studies have suggested a role of acetaldehyde in the actions of ethanol on the brain [see review by Hunt (28)]. Results of these studies have been derived by several strategies, including intraventricular injections of acetaldehyde and measuring subsequent behavioral changes, comparing the activities of ethanol-metabolizing enzymes in strains of rodents with differing preferences for ethanol, and altering the metabolism of acetaldehyde after ethanol ingestion, thereby modifying its concentration in the blood and tissues. The most direct studies showing that acetaldehyde can be reinforcing infused it directly into the ventricular system of the brain. When acetaldehyde was infused over a period of days, ethanol preference increased, although the amount of ethanol consumed was modest, and no signs of intoxication were reported (13,41). Rats also responded on a lever to receive infusions of acetaldehyde intraventricularly (12,13), intraperitone-
Requests for reprints should be addressed to Dr. Walter A. Hunt, Division of Basic Research, National Institute on Alcohol Abuse and Alcoholism, Willco Bldg., Suite 402, 6000 Executive Blvd. MSC 7003, Bethesda, MD 20892-7003. 147
148 ally (42), and intravenously (59), and developed a conditioned taste preference (a measure of reinforcement) after acetaldehyde infusions (53). However, none of these studies determined the concentration of acetaldehyde in the brain. (In fairness, measuring acetaldehyde has been difficult, as discussed later, and has discouraged investigators from measuring it.) The blood acetaldehyde concentration is regulated in part by the activities of alcohol dehydrogenase, cytochrome P450, catalase, and aldehyde dehydrogenase. Alcohol dehydrogenase, cytochrome P450, and catalase convert ethanol to acetaldehyde, whereas aldehyde dehydrogenase converts acetaldehyde to acetate. The activities of these enzymes vary among rodent strains and can be altered by drugs, providing an opportunity to relate the actions of ethanol to these activities. Comparing enzymatic activities and altering blood acetaldehyde concentrations have been used primarily to examine the role of acetaldehyde in ethanol intake. Over the years, researchers have studied the relationship between enzymatic activity and ethanol consumption among rodent strains. High activities of alcohol dehydrogenase could lead to elevated acetaldehyde concentrations. However, no clear relationship with alcohol dehydrogenase activity has been found. Although ethanol-preferring C57BL mice have higher hepatic alcohol dehydrogenase activity than ethanolavoiding DBA mice (48), the opposite is true for ethanolpreferring AA and ethanol-avoiding ANA rats. AA rats have lower alcohol dehydrogenase activity than the ANA rats (17, 34,35). Results with aldehyde dehydrogenase have been more consistent than those from studies of alcohol dehydrogenase. Ethanol-preferring rodents have higher hepatic aldehyde dehydrogenase activity and lower blood acetaldehyde concentrations after ethanol ingestion than the ethanol-avoiding rodents (34, 35,48,49). Furthermore, brain aldehyde dehydrogenase correlated with ethanol consumption in Long-Evans rats (54). However, no differences in brain aldehyde dehydrogenase activity were detected between AA and ANA rats (29). High aldehyde dehydrogenase activity might accelerate the removal of synthesized acetaldehyde and increase ethanol consumption by reducing the aversive response. Several studies reported altered ethanol intake after drug treatment that inhibited acetaldehyde dehydrogenase and elevated blood acetaldehyde concentrations. Pretreatment with calcium cyanamide increased blood acetaldehyde concentrations and decreased ethanol consumption from 3-5 g/kg/day to < 1 g/kg/day (19,50,51) and ethanol-induced locomotor activity (56). These effects were either partially reversed by pretreatment with the alcohol dehydrogenase inhibitor 4methylpyrazole (50) or not reversed at all (56). On the other hand, 4-methylpyrazole alone reduced ethanol intake in the alcohol-preferring P rats, possibly because less ethanol consumption was needed to maintain a particular blood ethanol concentration (63). In all, the data from these studies suggested that elevated acetaldehyde concentrations were aversive under these experimental conditions. Brain catalase activity also correlated with ethanol consumption in Long-Evans rats (9). In rats consuming ethanol for 25 days, those drinking the most ethanol had the highest brain catalase activities. However, the opposite finding was observed in mice. Acatalasemic mice with catalase activity 4050°70 of normal exhibited more ethanol consumption than normal mice, especially high ethanol concentrations (> 12070), and less activation of motor activity (2,4). In fact, in a recent study with recombinant inbreds of C57BL/6J and DBA/2J mice,
HUNT those mice drinking high amounts of ethanol had low brain catalase activity and those drinking low amounts had high activities (23). Thus, how catalase-mediated synthesis of acetaldehyde might contribute to ethanol consumption is presently unclear. Recent experiments have further assessed the role of catalase in the actions of ethanol by pretreating Long-Evans rats with the catalase inhibitor 3-amino-l,2,4-triazole. Pretreatment with 3-amino-l,2,4-triazole, at a dose that drastically lowered brain catalase activity (5) without affecting the blood ethanol concentration (60), significantly reduced ethanol consumption (3,33). In addition, it reduced ethanol-induced depression in locomotor activity (8), narcosis (7), and conditioned taste aversion (6) but had no effect on ethanol-induced hypothermia or the ethanol elimination rate (61). Moreover, tolerance to the depressant effects of ethanol but not to hypothermia or metabolic tolerance was also attenuated (61). Although these results are quite promising, one problem of using 3-amino-l,2,4-triazole as a catalase inhibitor in ethanol studies is its lack of specificity, an issue that needs careful consideration. 3-Amino-l,2,4-triazole has other effects that might be important to its ability to block actions of ethanol. Originally, 3-amino-l,2,4-triazole was developed as a herbicide. However, it is carcinogenic (57) and goitrogenic (22,37). Related to the latter effect, 3-amino-l,2,4-triazole exposure leads to a state of hypothyroidism, with serum thyroxine values significantly reduced (58). As a compensatory response, thyroid-stimulating hormone is elevated. Thyrotropin-releasing hormone (TRH) presumably would also increase, but effects of 3-amino-l,2,4-triazole on this hormone have not been reported. An effect of 3-amino-l,2,4-triazole on TRH could be significant to the interpretation of the results suggesting antagonism of a number of actions of ethanol. Pretreatment with TRH itself or the TRH analogue 1-methyl-(S)-4,5,dihydroorotyl-L-histidyl-L-prolineamide (TA-0910) has been reported to reduce ethanol preference in P rats (46), antagonize the impairment of the aerial righting reflex induced by ethanol (1 l), and diminish ethanol-induced hypothermia without altering brain ethanol concentrations (16). The effect of TA0910 on ethanol preference might be related to increases in dopamine release in the nucleus accumbens induced by TRH (27), similar to that found after ethanol ingestion (66). TA0910-induced changes in dopamine release might substitute for the effects of ethanol ingestion and reduce the need for ethanol to obtain a given level of reinforcement. Injections of TRH into the medial septum but not other areas of the brain produce the same results on ethanol-induced impairment (38). Other studies indicate that TRH levels increase in the medial septum after rats ingesting ethanol regained their righting reflex (40). These findings suggest that TRH increases in the medial septum in response to ethanol exposure, thereby contributing to the recovery from ethanol intoxication. If 3-amino-l,2,4-triazole administration was to increase TRH levels in the brain, especially in the medial septum, in response to a hypothyroidogenic effect, the antagonism of ethanol-induced actions by this drug could be due to actions of TRH rather than to catalase inhibition. However, most studies of 3-amino-l,2,4-triazole on thyroid function were performed after chronic exposure. But one study, using an acute dose 15070 of that used in ethanol studies (0.5-1.0 mg/kg, IP, 1-5 h before ethanol administration), reduced radioactive iodine uptake into the thyroid by almost 60070 10 h after treatment (21). A possible effect on thyroid function, especially on TRH levels, of a single dose of 3-amino-l,2,4-
ETHANOL AND ACETALDEHYDE IN THE BRAIN triazole, administered in the manner typically used in ethanol experiments, has not been reported and should be evaluated. SOURCES OF ACETALDEHYDEAND EXPECTED CONCENTRATIONS Whether acetaldehyde plays a significant role in the actions of ethanol on the brain ultimately depends on the availability of acetaldehyde after ethanol consumption in sufficient concentrations in the brain to be biologically effective. The primary site of the conversion of ethanol to acetaldehyde is in the liver. The liver being a relevant source of acetaldehyde for actions on the brain necessitates the presence of acetaldehyde in the blood, which would transport it to the brain. The measurement of blood acetaldehyde has been controversial for almost 20 years. Reported values of blood acetaldehyde have been confounded by artifacts, including nonenzymatic conversion of ethanol to acetaldehyde, binding to erythrocytes, and sample decomposition (18). Over the years, the methodology for measuring acetaldehyde has improved (20). As a result, using the most modern techniques, no measurable blood acetaldehyde concentrations (<0.5 /~M) were found after ethanol consumption by normal humans not taking aldehyde dehydrogenase inhibitors (20). Adding to the difficulty of finding a peripheral source of acetaldehyde for actions on the brain, a blood-brain barrier to acetaldehyde probably would reduce its penetration into the brain. Sippel (52) reported that blood acetaldehyde concentrations had to exceed 250 ~tM before measurable brain acetaldehyde concentrations could be detected. Furthermore, evidence suggests the presence of aldehyde dehydrogenase in the microvasculature in the brain (44). This enzyme would effectively destroy most acetaldehyde in the blood as it penetrated the brain. With no significant likelihood that peripherally derived acetaldehyde would reach the brain, a remaining source of acetaldehyde would be in the brain itself. Three general enzymatic systems catalyze the conversion of ethanol to acetaldehyde: alcohol dehydrogenase, cytochrome P450, and catalase. The existence of significant alcohol dehydrogenase activity in the brain has been disputed for many years. Early studies detected little alcohol dehydrogenase activity in the brain (45) or acetaldehyde in cerebrospinal fluid of intoxicated alcoholics (36). More recently, immunocytochemical methods found brain alcohol dehydrogenase only in neuronal cytoplasm of some neurons mostly in the mammillary bodies, periaqueductal gray, and the cerebral and cerebellar cortices (32). Such results suggest that high alcohol dehydrogenase activity could be of local importance to individual neurons, even though activity in general areas is low. The significance of localized alcohol dehydrogenase depends on the isozymes of the enzyme present. Alcohol dehydrogenase exists as several isozymes, distinguished by their affinity for ethanol and the ease with which the isozyme can be inhibited by pyrazole (62). Reports of the relative proportion of these isozymes in the brain have not been consistent. Although one study found the isozyme with high affinity (Km = 0.15 raM) for ethanol (14), all other studies found only low-affinity isozymes (Km > 500 mM) in several animal species, including humans (10,25,30,47). Thus, ethanol concentrations in the brain must be very high before alcohol dehydrogenase would play much of a role in the conversion of ethanol to acetaldehyde. Cytochrome P450 is an enzyme complex involved in the detoxification of drugs and toxins in the liver and can exist in many forms. The form most involved in ethanol metabolism
149 is cytochrome P450 2El. Cytochrome P450 2El as well as other cytochrome P450s have been identified in the brain and can be found in neurons and glia in several brain regions (26,59,64). The relative basal concentrations of these cytochrome P450s suggest that 2El represents less than I070of the activity of all cytochrome P450s (64). However, cytochrome P450 2El is inducible by ethanol, and increases of three- to fivefold in activity have been reported after ethanol administration (1,39,55,65). The importance of cytochrome P450 2El in the formation of acetaldehyde after ethanol consumption would, therefore, depend on how quickly the enzyme can be induced. To date, no studies have been published to address this question. Catalase, along with glutathione peroxidase, functions as a regulator of hydrogen peroxide levels, a metabolite formed during the detoxification of reactive oxygen radicals. In the presence of hydrogen peroxide, ethanol can be oxidized by purified rat liver catalase to acetaldehyde in vitro (31,43). This is accomplished by the catalase-hydrogen peroxide intermediate (Compound I) through a peroxidatic reaction in which acetaldehyde and water are formed from the intermediate and ethanol (a hydrogen donor). Evidence has been reported suggesting that catalase can oxidize ethanol to acetaldehyde in the brain (15,60). However, all of the results reported have depended on using 3-amino1,2,4-triazole, an irreversible inhibitor of catalase, to infer a catalase-mediated production of acetaldehyde and assess the effect of catalase inhibition on ethanol-induced alterations in behavior. Using methods of analysis that avoid artifactual formation of acetaldehyde, the most convincing study of catalase-mediated production of acetaldehyde in brain tissue demonstrated synthesis of acetaldehyde in the micromolar range (24). The formation of acetaldehyde was reduced by 3-amino1,2,4-triazole but not by metyrapone, a cytochrome P450 inhibitor, or 4-methylpyrazole, an alcohol dehydrogenase inhibitor, suggesting a role of catalase. Because these studies were performed in vitro, possible thyroidogenic effects of 3-amino1,2,4-triazole would not occur. CONCLUDINGREMARKS The role of acetaldehyde in the actions of ethanol on the brain has been investigated for almost 30 years. Although such a role is promising, its support is largely indirect and circumstantial, involving correlational studies and pharmacological evidence. Some of this evidence suggests that catalase mediates the formation of acetaldehyde from ethanol. The presence of catalase in the brain is not in itself proof that it catalyzes the conversion of ethanol to significant concentrations of acetaldehyde. This reaction occurs only in the presence of hydrogen peroxide. Whether sufficient concentrations of hydrogen peroxide are generated in the brain in vivo to support catalase-mediated production of acetaldehyde is unknown and needs further study. In addition, experiments with catalase inhibition are inconclusive because of the lack of specificity of 3-amino-l,2,4-triazole. The short-term effects of 3-amino-l,2,4-triazole on thyroid function require systematic evaluation. In addition, more specific catalase inhibitors are needed. Although catalase appears to be a likely source of any synthesized acetaldehyde after ethanol consumption, a role of cytochrome P450 2El cannot be completely discounted, especially during chronic ethanol exposure. As discussed earlier, cytochrome P450 2El can be induced in the brain by ethanol. Published reports did not study the time course of
150
HUNT
induction, which should be pursued. However, even with significant induction, it still needs to be established if this route of synthesis leads to significant concentrations of acetaldehyde in the brain. Ultimately, a significant role for acetaldehyde in the actions of ethanol on the brain requires the presence of acetaldehyde in the brain after ethanol consumption in quantities consistent with those used in experiments showing biological or behavioral changes. To date, the data are not compelling. What is required is a way in which acetaldehyde can be quantitated in the brain in vivo after ethanol administration. Finally, an unresolved issue is whether the results obtained represent a balance between rewarding and aversive properties of acetaldehyde. On the one hand, high alcohol dehydrogenase and/or catalase activities in the brains of some rodent strains might enhance the synthesis of acetaldehyde and thereby add to a possible rewarding effect of ethanol. On the other hand, high aldehyde dehydrogenase activity might decrease the acetaldehyde present and decrease the possible aversive effects of ethanol. The results suggest a narrow range
of acetaldehyde concentrations in modulating a rewarding action of ethanol ingestion. As brain acetaldehyde concentrations increase, the reinforcing properties would increase. However, once an upper limit of acetaldehyde concentrations is attained, the aversive effects of acetaldehyde would predominate, thereby reducing the reinforcing properties of ethanol. Although brain catalase and aldehyde dehydrogenase activities correlate with voluntary ingestion of alcohol, the relative contribution of these three variables to the degree of ethanol consumption in the same animals is not clear. Taken together, the results provide no clear relationship between activities of hepatic and brain ethanol-metabolizing enzymes per se and ethanol preference across rodent strains, let alone humans. Nevertheless, the role for these enzymes in causing differences in individual strains cannot be discounted. The role of acetaldehyde in the actions of ethanol on the brain has been studied for many years, but an exact role has not been conclusively demonstrated. There are still potentially fruitful areas of investigation to show once and for all whether such a role for acetaldehyde has any real importance.
REFERENCES 1. Anandatheerthavarada, H. K.; Shankar, S. K.; Bhamre, S.; Boyd, M. R.; Song, B.-J.; Ravindranath, V. Induction of brain cytochrome P450 IIEI by chronic ethanol treatment. Brain Res. 601:279-285; 1993. 2. Aragon, C. M. G.; Amit, Z. Differences in ethanol-induced behaviors in normal and acatalasemic mice: Systemic examination using a biobehavioral approach. Pharmacol. Biochem. Behav. 44:547-554; 1993. 3. Aragon, C. M. G.; Amit, Z. The effect of 3-amino-l,2,4-triazole on voluntary ethanol consumption: evidence for brain catalase involvement in the mechanism of action. Neuropharmacology 31 : 709-712; 1992. 4. Aragon, C. M. G.; Pesold, C. N.; Amit, Z. Ethanol-induced motor activity in normal and acatalasemic mice. Alcohol 9:207211; 1992. 5. Aragon, C. M. G.; Rogan, F.; Amit, Z. Dose- and timedependent effect of an acute 3-amino-1,2,4-triazole injection on rat brain catalase activity. Biochem. Pharmacol. 42:699-702; 1991. 6. Aragon, C. M. G.; Spivak, K.; Amit, Z. Blockade of ethanolinduced conditioned taste aversion by 3-amino-1,2,4-triazole: Evidence for catalase mediated synthesis of acetaldehyde in rat brain. Life Sci. 37:2077-2084; 1985. 7. Aragon, C. M. G.; Spivak, K.; Amit, Z. Effect of 3-amino-l,2,4triazole on ethanol-induced narcosis, lethality and hypothermia in rats. Pharmacol. Biochem. Behav. 39:55-59; 1991. 8. Aragon, C. M. G.; Spivak, K.; Amit, Z. Effects of 3-amino1,2,4-triazole on ethanol-induced open field activity: Evidence for brain cataiase mediation of ethanol's effects. Alcohol. Clin. Exp. Res. 13:104-108; 1989. 9. Aragon, C. M. G.; Sternklar, G.; Amit, Z. A correlation between voluntary ethanol consumption and brain catalase activity in the rat. Alcohol 2:353-356; 1985. 10. Beisswenger, T. B.; Holmquist, B.; Vallee, B. L. ADH is the sole alcohol dehydrogenase isozyme of mammalian brains: Implications and inferences. Proc. Natl. Acad. Sci. USA 82:8369-8373; 1985. 11. Breese, G. R.; Cott, J. M.; Cooper, B. R.; Prange, A. J.; Lipton, M. A. Antagonism of ethanol narcosis by thyrotropin releasing hormone. Life Sci. 14:1053-1063; 1974. 12. Brown, Z. W.; Amit, Z.; Rockman, G. E. Intraventricular selfadministration of acetaldehyde but not ethanol in naive laboratory rats. Psychopharmacology (Berlin) 64: 271-276; 1979. 13. Brown, Z. W.; Amit, Z.; Smith, B. Intraventricular self-ad-
14.
15. 16.
17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
ministration of acetaldehyde and voluntary consumption of ethanol in rats. Behav. Neural Biol. 28:150-155; 1980. Biihler, R.; Pestalozzi, D.; Hess, M.; von Wartburg, J. P. Immunohistochemical localization of alcohol dehydrogenase in human kidney, endocrine organs and brain. Pharmacol. Biochem. Behay. 18(Suppl. 1):55-59; 1983. Cohen, G.; Sinet, P. M.; Heikkila, R. Ethanol oxidation by rat brain in vivo. Alcohol. Clin. Exp. Res. 4:366-370; 1980. Cott, J. M.; Breese, G. R.; Cooper, B. R.; Barlow, S.; Prange, A. J. Investigations into the mechanism of reduction of ethanol sleep by thyrotropin-releasing hormone (TRH). J. Pharmacol. Exp. Thee 196:594-604; 1976. Eriksson, C. J. P. Ethanol and acetaldehyde metabolism in rat strains genetically selected for their ethanol preference. Biochem. Pharmacol. 22:2283-2292; 1973. Eriksson, C. J. P. Problems and pitfalls in acetaldehyde determinations. Alcohol. Clin. Exp. Res. 4:22-29; 1980. Eriksson, C. J. P.; Deitrich, R. A. Evidence against a biphasic effect of acetaldehyde on voluntary ethanol consumption in rats. Pharmacol. Biochem. Behav. 13(Suppl. 1):291-296; 1980. Eriksson, C. J. P.; Fukunaga, T. Human blood acetaldehyde (update 1992). Alcohol Alcohol. Suppl. 2:9-25; 1993. Fregly, M. J. Effect of aminotriazole on thyroid function in the rat. Toxicol. Appl. Pharmacol. 13:271-286; 1968. Gaines, T. B.; Kombrough, R. D.; Linder, R. E. The toxicity of amitrole in the rat. Toxicol. Appl. Pharmacol. 26:118-129; 1973. Gill, K.; Deitrich, R. A.; Erwin, V. G. Alcohol-related behaviors in the BXD recombinant inbred (RI) strains of mice. Alcohol. Clin. Exp. Res. 19:12A(Suppl.); 1995. Gill, K.; Menez, J. F.; Lucas, D.; Deitrich, R. A. Enzymatic production of acetaldehyde from ethanol in rat brain tissue. Alcohol. Clin. Exp. Res. 16:910-915; 1992. Giri, P. R.; Linnoila, M.; Neil, J. B. O.; Goldman, D. Distribution and possible metabolic role of Class III alcohol dehydrogenase in the human brain. Brain Res. 484:131-141; 1989. Hansson, T.; Tindberg, N.; Ingelman-Sundberg, M.; Kohler, C. Regional distribution of ethanol-inducible cytochrome P450 IIE1 in the rat central nervous system. Neuroscience 34:451-463; 1990. Heal, D. J.; Green, A. R. Administration of thyrotropin-releasing hormone (TRH) to rats releases dopamine in n. accumbens but not in n. caudatus. Neuropharmacology 18:23-31; 1979. Hunt, W. A. Biochemical bases for the reinforcing effects of ethanol. In: Cox, W. M., ed. Why people drink: Parameters of alcohol as a reinforcer. New York: Gardiner Press; 1990:51-70.
ETHANOL
AND ACETALDEHYDE
IN T H E B R A I N
29. Inoue, K.; Rusi, M.; Lindros, K. O. Brain aldehyde dehydrogenase activity in rat strains with high and low ethanol preferences. Pharmacol. Biochem. Behav. 14:107-111; 1975. 30. Julia, P.; Farres, J.; Pares, X. Characterization of three isozymes of rat alcohol dehydrogenase: Tissue distribution and physical and enzymatic properties. Eur. J. Biochem. 162:179-189; 1987. 31. Keilin, D.; Martree, E. F. Properties of catalase. Catalysis of coupled oxidation of alcohol. Biochem. J. 39:293-301; 1945. 32. Kerr, J. T.; Maxwell, D. S.; Crabb, D. W. Immunocytochemistry of alcohol dehydrogenase in the rat central nervous system. Alcohol. Clin. Exp. Res. 13:730-736; 1989. 33. Koechling, U. M.; Amit, Z. Effects of 3-amino-l,2,4-triazole on brain catalase in the mediation of ethanol consumption in mice. Alcohol 11:235-239; 1994. 34. Koivisto, T.; Eriksson, C. P. J. Hepatic aldehyde and alcohol dehydrogenases in alcohol-preferring and alcohol-avoiding rat lines. Biochem. Pharmacol. 48:1551-1558; 1994. 35. Koivula, T.; Koivusalo, M.; Lindros, K. O. Liver aldehyde and alcohol dehydrogenase activities in rat strains genetically selected for their ethanol preference. Biochem. Pharmacol. 24:1807-1811; 1975. 36. Lindros, K. O.; Hillbom, M. E. Acetaldehyde in cerebrospinal fluid: Its near-absence in ethanol-intoxicated alcoholics. Med. Biol. 57:246-247; 1979. 37. Mayberry, W. E. Antithyroid effects of 3-amino-l,2,4-triazole. Proc. Soc. Exp. Biol. Med. 129:551-556; 1968. 38. McCown, T. J.; Moray, L. J.; Kizer, J. S.; Breese, G. R. Interactions bet~veen TRH and ethanol in the medial septum. Pharmacol. Biochem. Behav. 24:1269-1274; 1986. 39. Montoliu, C.; Valles, S.; Renau-Piqueras, J.; Guerri, C. Ethanolinduced oxygen radical formation and lipid peroxidation in rat brain: Effect of chronic alcohol consumption. J. Neurochem. 63: 1855-1862; 1994. 40. Morzorati, S.; Kubek, M. J. Septal TRH in alcohol-naive P and NP rats and following alcohol challenge. Brain Res. Bull. 31:301304; 1993. 41. Myers, R. D.; Veale, W. L. Alterations in volitional alcohol intake produced in rats by chronic intraventricular infusions of acetaldehyde, paraldehyde or methanol. Arch. Int. Pharmacodyn. Ther. 180:100-113; 1969. 42. Myers, W. D.; Ng, K. T.; Singer, G. Ethanol preference in rats with a prior history of acetaldehyde self-administration. Experientia 40:1008-1010; 1984. 43. Oshino, N.; Oshino, R.; Chance, B. The characteristics of the peroxidatic reaction in ethanol oxidation. Biochem. J. 131:555567; 1973. 44. Petersen, D. R. Aldehyde dehydrogenase and aldehyde reductase in isolated bovine brain microvessels. Alcohol 2:79-83; 1985. 45. Raskin, N. H.; Sokoloff, L. Enzymes catalyzing ethanol metabolism in neural and somatic tissue of the rat. J. Neurochem. 19: 273-282; 1972. 46. Rezvani, A. H.; Garbutt, J. C.; Shimoda, K.; Garges, P. L.; Janowsky, D. S.; Mason, G. A. Attenuation of alcohol preference in alcohol-preferring rats by a novel TRH analogue, TA0910. Alcohol. Clin. Exp. Res. 16:326-330; 1992. 47. Rout, U. K. Alcohol dehydrogenases in the brain of mice. Alcohol. Clin. Exp. Res. 16:286-289; 1992. 48. Schlesinger, K. R.; Kakihana, R.; Bennett, E. L. Effects of tetraethylthiuramdisulfide (Antabuse) on the metabolism and consumption of ethanol in mice. Psychosom. Med. 28:514-520; 1966.
151 49. Sheppard, J. R.; Albersheim, P.; McClearn, G. Aldehyde dehydrogenase and ethanol preference in mice. J. Biol. Chem. 245: 2876-2882; 1970. 50. Sinclair, J. D.; Lindros, K. O. Suppression of alcohol drinking with brain aldehyde dehydrogenase inhibition. Pharmacol. Biochem. Behav. 14:377-383; 1981. 51. Sinclair, J. D.; Lindros, K. O.; Terho, K. Aldehyde dehydrogenase inhibitors and voluntary ethanol drinking in rats. In: Thurman, R. G., ed. Alcohol and aldehyde metabolizing systems- IV. New York: Plenum Press; 1980:481-487. 52. Sippel, H. W. The acetaldehyde content in rat brain during ethanol metabolism. J. Neurochem. 23:451-452; 1974. 53. Smith, B. R.; Amit, Z.; Splawinsky, J. Conditioned place preference induced by intraventricular infusions of acetaldehyde. Alcohol 1:193-195; 1984. 54. Socaransky, S. M.; Aragon, C. M.; Amit, Z. Brain ALDH as a possible modulator of voluntary ethanol intake. Alcohol 2:361365; 1985. 55. Sohda, T.; Shimizu, M.; Kamimura, S.; Okumura, M. Immunohistochemical demonstration of ethanol-inducible P450 2E 1 in rat brain. Alcohol Alcohol. Suppl. 1B:69-75; 1993. 56. Spivak, K.; Aragon, C. M.; Amit, Z. Alterations in brain aldehyde dehydrogenase activity modify the locomotor effects produced by ethanol in rats. Alcohol Drug Res. 7:481-491; 1987. 57. Steinhoff, D.; Weber, H.; Mohr, U.; Boehme, K. Evaluation of amitrole (aminotriazole) for potential carcinogenicity in orally dosed rats, mice, and golden hamsters. Toxicol. Appl. Pharmacol. 69:161-169; 1983. 58. Stringer, B.; Wynford-Thomas, D.; Jasani, B.; Williams, E. D. Effect of goitrogen administration on the circadian rhythm of serum thyroid stimulating hormone in the rat. Acta Endocrinol. (Copenh.) 98:396-401; 1981. 59. Takayama, S.; Uyeno, E. T. Intravenous self-administration of ethanol and acetaldehyde by rats. Yakubutsu Seishin Kodo 5: 329-334; 1985. 60. Tampier, L.; Mardones, J. Effect of 3-amino-l,2,4-triazole pretreatment on ethanol blood levels after intraperitoneal administration. Alcohol 3:181-183; 1986. 61. Tampier, L.; Quintanilla, M. E. Effect of 3-amino-l,2,4-triazole on the hypothermic effect of ethanol and on ethanol tolerance development. Alcohol 8:279-282; 1991. 62. Vallee, B. L.; Bazzone, T. J. Isozymes of human liver alcohol dehydrogenase. In: Rattazzi, M. C.; Scandalio, J. G.; Whirr, G. S., eds. lsozymes, current topics in biological and medical research. Vol. 8. New York: Alan R. Liss; 1983:219-244. 63. Waller, M. B.; McBride, W. J.; Lumeng, L.; Li, T.-K. Effects of intravenous ethanol and of 4-methylpyrazole on alcohol drinking in alcohol-preferring rats. Pharmacol. Biochem. Behav. 17:763768; 1982. 64. Warner, M.; Gustafsson, J.-.~. Effect of ethanol on cytochrome P450 in rat brain. Proc. Natl. Acad. Sci. USA 91:1019-1023; 1994. 65. Warner, M.; Str6mstedt, M.; Wyss, A.; Gustafsson, J.-A. Regulation of cytochrome P450 in the central nervous system. J. Steroid Biochem. Mol. Biol. 47:191-194; 1993. 66. Weiss, F.; Lorang, M. T.; Bloom, F. E.; Koob, G. F. Oral alcohol self-administration stimulates dopamine release in the rat nucleus accumbens: Genetic and motivational determinants. J. Pharmacol. Exp. Ther. 267:250-258, 1993.