Psychopharmacology
Psychopharmacology 52, 111 - 113 (1977)
9 by Springer-Verlag 1977
Brain Monoamine Oxidase Activity after Chronic Ethanol Treatment of Rats ~SA WIBERG, GORAN WAHLSTROM, and LARS ORELAND Department of Pharmacology, University of Umefi, S-901 87 Umegt, Sweden
Abstract. R a t s were given c h r o n i c t r e a t m e n t s with e t h a n o l in two ways: by offering 10 % e t h a n o l in water as the only liquid supply for 34 weeks a n d by exposing the rats to e t h a n o l v a p o u r d u r i n g 5 h daily for 7 weeks. I n this way b l o o d e t h a n o l levels of 1 . 4 - 2 . 3 a n d 5 . 5 - 6 . 9 m g / m l , respectively, were accomplished. I n neither of the cases was b r a i n m o n o a m i n e oxidase activity changed. T h e result supports a n hypothesis previously a d v a n c e d that the lowered m o n o a m i n e oxidase activity f o u n d in the b r a i n of a suicide p a t i e n t was n o t due to a direct effect of e t h a n o l o n alcoholics in the suicide group, b u t that the low e n z y m e activity reflected a low m o n o a m i n e r g i c activity in the b r a i n s of the patients in the suicide group. Key words: M o n o a m i n e oxidase C h r o n i c t r e a t m e n t with ethanol.
-
Ethanol
-
W e have recently f o u n d a low m o n o a m i n e oxidase ( M A O ) (EC 1.4.3.4) activity in the b r a i n s of suicides as c o m p a r e d to c o n t r o l s (Gottfries et al., 1975). The decrease was m o s t m a r k e d in the suicide cases with a previous history o f alcohol abuse. A low M A O activity in the platelets o f alcoholics has also been f o u n d ( W i b e r g et al., 1977). These results as well as the findings o f low c o n c e n t r a t i o n s of 5-hydroxyindolacetic acid in c e r e b r o s p i n a l fluid of patients with suicidal b e h a v i o r ( h s b e r g et al., 1976), have m a d e us suggest that low platelet a n d b r a i n M A O activities reflect c o n s t i t u t i o n a l l y " w e a k " m o n o a m i n e r g i c systems. These " w e a k " systems might in t u r n cause a n increased v u l n e r a b i l i t y to, e.g., e t h a n o l abuse a n d / o r suicidal b e h a v i o r (Wiberg et al., 1977). Before p u t t i n g to great a n emphasis o n this hypothesis, however, the effect of c h r o n i c alcohol a d m i n i s t r a t i o n o n M A O activity should be investigated.
MATERIALS AND METHODS Chronic Ethanol Treatment of the Rats. Male Sprague-Dawley rats (Nih/Han/Mol) with an initial body weight of 300 g were used. Two kinds of ethanol treatments were given: 1. In the oral treatment the rats had access to a drinking fluid consisting of ethanol (10 % w/v) for only 2 periods of I h each day (Wahlstr6m, 1972). This ethanol treatment lasted for 34 weeks. During the treatment the average weekly consumption of ethanol increased from 2.1 g/kg/day to 5.8 g/kg/day. Ethanol concentrations in the blood were determined by gas chromatography (Curry et al., 1966; Wahlstr6m and Widerl6f, 1971) after 24-30 weeks of treatment. Blood levels between 1.4-2.3mg/ml were recorded after the first hour of consumption. Similar treated rats had at conclusion of the treatment clear increases in a hexobarbital threshold with a maximum of 117 • 4 (n = 18) % of a pre-experimental average on the 8th day of the abstinence, indicating an induced cross tolerance to hexobarbital (Wahlstr6m, unpublished). In the present experiment the rats were kilied 24 h after the last ethanol exposure. 2. In the second experiment ethanol was given as vapor. Every day for 7 weeks the rats were exposed to ethanol vapor for 5 h. Air containing 120 mg of ethanol per litre was blown through the chamber in which the rats were kept at 200 1/h. Blood ethanol concentrations between 5.5-6.9 mg/ml were reached. Similar treatments gave increases in a hexobarbital threshold with a maximum (124 • 6%, n = 9) on the first day of the abstinence indicating similar cross tolerance as in the oral treatment. In the present experimentthe rats were killed 18 h after the last exposure to ethanol. Preparation of Rat Brain Synaptosomes and Extrasynaptosomal Mitochondria. After the rats were killed by decapitation, the whole brain, with the cerebellum cut off, was immediately removed and synaptosomes and extrasynaptosomal mitochondria were prepared essentially according to Gray and Whittaker (1962). Monoamine Oxidase. The monoamine oxidase activity was estimated with five different substrates. With benzylamine as substrate a slight modification of the spectrophotometric method described by McEwen and Cohen (1963) was used. With serotonin and tyramine as substrates an oxygen polarographic method was used as described earlier (Oreland and Ekstedt, 1972). With a third method the enzyme activity was estimated with labeled substrates according to Wurtman and Axelrod (1963) as applied by Gottfries et al. (1975). The substrates used were phenylethylamine (/%phenylethylamine-l-lgc-hydrochloride,7 mC/mmole, New England Nuclear, Boston, Mass.), tryptamine (tryptamine-214C-bisuccinate, 47 mC/mmole, New England Nuclear, Boston, Mass.) and serotonin (5-hydroxytryptamine binoxalate 2-14C,
112
Psychopharmacology 52 (1977)
Table I.
Effect of chronic ethanol treatment on rat brain monoamine oxidase Extrasynaptosomal mitochondrial fraction
Synaptosomal fraction
Benzylamine Tyramine Serotonin 14C/%Phenylethylamine 14C-Tryptamine a4c-Serotonin * "
Inhalation of ethanol vapor (n = 6)
Enforced drinking of 10% ethanol (n = 4)
Inhalation of ethanol vapor (n = 6)
Enforced drinking of 10% ethanol (n = 4)
Mean % of controls a
Mean % of controls"
P*
Mean % of controls"
P*
Mean % of controls a
P*
93 100 131 84 97 99.6
n.s. n.s. n.s. n.s. n.s. n.s.
110 100 104 97 96 96
n.s. n.s. n.s. n.s. n.s. n.s.
96 91 106 104 106 100
n.s. n.s. n.s. n.s. n.s. n.s.
116.7 114 118 101 111 102
P*
< 0.05 n.s. < 0.05 n.s. n.s. n.s.
The statistics were analyzed with the help of Student's t test A corresponding number of similarly treated rats, however, without exposure to ethanol, were used as controls
48.5 mC/mmole, New England Nuclear, Boston, Mass.) in final concentrations of 6.7 gM, 16.7 [aM and 16.7 gM, respectively.
Protein. The protein content was measured according to Lowry et al. with human serum albumin as standard (1951).
RESULTS As shown in Table 1, there were no significant changes in the rat brain monoamine oxidase activity after 34 weeks of daily intake of ethanol. This was true both for synaptosomal and extrasynaptosomal monoamine oxidase, regardless whether the monoamine oxidase type A substrate serotonin, type B substrate benzylamine and fl-phenylethylamine, or the type A + B substrates tyramine and tryptamine (Neff and Yang, 1974) was used. Neither was there any decrease in monoamine oxidase activity in the synaptosomal or in the extrasynaptosomal mitochondrial fraction after administration of ethanol vapor.
DISCUSSION No decrease in MAO-activity was found after either of the two kinds of chronic ethanol treatment used in the present investigation. Both treatments were known to be able to induce other signs of chronic changes in the animals. In rats chronic exposure to ethanol as such or the changes in amine metabolism which might have been induced by the present treatments (Deitrich and Erwin, 1975) have thus not influenced the MAOactivity. Athee and Eriksson (1972), however, found that no changes occurred in the 5-HT and 5 HIAA content in the brain of non-alcoholic rats who had been forced to drink ethanol for 1 month. The present result makes it less likely that the low MAO-activity in the suicide material earlier reported (Gottfries et
al., 1975) was due to chronic effects of ethanol on the enzyme activity in the brains of the patients with a history of alcohol abuse. Other explanations must be looked for and the hypothesis then put forward (Wiberg et al., 1977) that predisposition to certain psychiatric diseases, e.g., ethanol abuse, is due to constitutional factors implying weak monoaminergic systems, and that such a weakness is also reflected in a low monoamine oxidase activity, has thus been supported by the present experiments. Acknowledgements. The skilful technical assistance of Mr. Alf Olsson, Mr. Roland Larsson, Mrs. Kerstin Wahlstr6m, Miss Anna Persson and Mrs. Ingrid Persson is gratefully acknowledged. This work was supported by Grants from the Swedish Medical Research Council (No. 3771 and 4145) and the Medical Faculty, University of Ume&
REFERENCES Ahtee, L., Eriksson, K. : 5-Hydroxytryptamine and 5-hydroxyindolylacetic acid content in brain of rat strains selected for their alcohol intake. Physiol. and Behav. 8, 123- 126 (1972) Curry, A. S., Walker, G.W., Simpson, G. S.: Determination of ethanol in blood by gas chromatography. Analyst 91,742-743 (1966) Deitrich, R., Erwin, G.: Involvement of biogenic amine metabolism in ethanol addiction. Fed. Proc. 34, 1962-1968 (1975) McEwen, C., Cohen, J. : An amine oxidase in normal human serum. J. Lab. clin. Med. 62, 766-776 (1963) Gottfries, C. G., Oreland, L., Wiberg, A., Winblad, B.: Lowered monoamine oxidase activity in brains from alcoholic suicides. J. Neurochem. 25, 667-673 (1975) Gray, E. G., Whittaker, W. P.: The isolation of nerve endings from brain: an electron microscopic study of cell fragments derived by homogenization and centrifugation. 'J. Anat. (Lond.) 96, 79 87 (1962) Lowry, O. H., Rosebrough, A., Farr, L., Randall, R. : Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275 (1951) Neff, N. H., Yang, H. Y.T.: Another look at the monoamine oxidases and the monoamine oxidase inhibitor drugs. Life Sci. 14, 2061 -- 2074 (I 974)
~.. Wiberg et al. : Ethanol and Monoamine Oxidase Activity Oreland, L., Ekstedt, B. : Soluble and membrane-bound pig liver mitochondrial monoamine oxidase: thermostability tryptic digestability and kinetic properties. Biochem. Pharmacol. 21, 2479 - 2488 (1972) Wahlstr6m, G, : Cross tolerance between hexobarbital and ethanol in rats induced by forced drinking of intoxicating amounts of ethanol. The Finnish foundation for alcoholic studies 20, 241 - 249 (1972) Wahlstr6m, G., Widerl6f, E. : Interaction and acute cross tolerance between ethanol and hexobarbitone in the rat. J. Pharm. Pharmacol. 23, 5 8 - 6 0 (1971)
113 Wiberg, h., Gottfries, C.G., Oreland, L.: Low platelet MAO activity in human alcoholics. Med. Biol. (in press, 1977) Wurtman, R. J., Axelrod, J. : A sensitive and specific assay for the estimation of monoamine oxidase. Biochem. Pharmacol. 12, 1439- i441 (1963) A.sberg, M., Triiskman, L., Thor6n, P. : 5-HIAA in the cerebrospinal fluid: a biochemical suicide predictor ? Arch. gen. Psychiat. (in press)
Received December I9, 1975; Final Version December 17, 1976
Psychopharmacology 52, 111 - 113 (1977)
9 by Springer-Verlag 1977
Brain Monoamine Oxidase Activity after Chronic Ethanol Treatment of Rats ~SA WIBERG, GORAN WAHLSTROM, and LARS ORELAND Department of Pharmacology, University of Umefi, S-901 87 Umegt, Sweden
Abstract. R a t s were given c h r o n i c t r e a t m e n t s with e t h a n o l in two ways: by offering 10 % e t h a n o l in water as the only liquid supply for 34 weeks a n d by exposing the rats to e t h a n o l v a p o u r d u r i n g 5 h daily for 7 weeks. I n this way b l o o d e t h a n o l levels of 1 . 4 - 2 . 3 a n d 5 . 5 - 6 . 9 m g / m l , respectively, were accomplished. I n neither of the cases was b r a i n m o n o a m i n e oxidase activity changed. T h e result supports a n hypothesis previously a d v a n c e d that the lowered m o n o a m i n e oxidase activity f o u n d in the b r a i n of a suicide p a t i e n t was n o t due to a direct effect of e t h a n o l o n alcoholics in the suicide group, b u t that the low e n z y m e activity reflected a low m o n o a m i n e r g i c activity in the b r a i n s of the patients in the suicide group. Key words: M o n o a m i n e oxidase C h r o n i c t r e a t m e n t with ethanol.
-
Ethanol
-
W e have recently f o u n d a low m o n o a m i n e oxidase ( M A O ) (EC 1.4.3.4) activity in the b r a i n s of suicides as c o m p a r e d to c o n t r o l s (Gottfries et al., 1975). The decrease was m o s t m a r k e d in the suicide cases with a previous history o f alcohol abuse. A low M A O activity in the platelets o f alcoholics has also been f o u n d ( W i b e r g et al., 1977). These results as well as the findings o f low c o n c e n t r a t i o n s of 5-hydroxyindolacetic acid in c e r e b r o s p i n a l fluid of patients with suicidal b e h a v i o r ( h s b e r g et al., 1976), have m a d e us suggest that low platelet a n d b r a i n M A O activities reflect c o n s t i t u t i o n a l l y " w e a k " m o n o a m i n e r g i c systems. These " w e a k " systems might in t u r n cause a n increased v u l n e r a b i l i t y to, e.g., e t h a n o l abuse a n d / o r suicidal b e h a v i o r (Wiberg et al., 1977). Before p u t t i n g to great a n emphasis o n this hypothesis, however, the effect of c h r o n i c alcohol a d m i n i s t r a t i o n o n M A O activity should be investigated.
MATERIALS AND METHODS Chronic Ethanol Treatment of the Rats. Male Sprague-Dawley rats (Nih/Han/Mol) with an initial body weight of 300 g were used. Two kinds of ethanol treatments were given: 1. In the oral treatment the rats had access to a drinking fluid consisting of ethanol (10 % w/v) for only 2 periods of I h each day (Wahlstr6m, 1972). This ethanol treatment lasted for 34 weeks. During the treatment the average weekly consumption of ethanol increased from 2.1 g/kg/day to 5.8 g/kg/day. Ethanol concentrations in the blood were determined by gas chromatography (Curry et al., 1966; Wahlstr6m and Widerl6f, 1971) after 24-30 weeks of treatment. Blood levels between 1.4-2.3mg/ml were recorded after the first hour of consumption. Similar treated rats had at conclusion of the treatment clear increases in a hexobarbital threshold with a maximum of 117 • 4 (n = 18) % of a pre-experimental average on the 8th day of the abstinence, indicating an induced cross tolerance to hexobarbital (Wahlstr6m, unpublished). In the present experiment the rats were kilied 24 h after the last ethanol exposure. 2. In the second experiment ethanol was given as vapor. Every day for 7 weeks the rats were exposed to ethanol vapor for 5 h. Air containing 120 mg of ethanol per litre was blown through the chamber in which the rats were kept at 200 1/h. Blood ethanol concentrations between 5.5-6.9 mg/ml were reached. Similar treatments gave increases in a hexobarbital threshold with a maximum (124 • 6%, n = 9) on the first day of the abstinence indicating similar cross tolerance as in the oral treatment. In the present experimentthe rats were killed 18 h after the last exposure to ethanol. Preparation of Rat Brain Synaptosomes and Extrasynaptosomal Mitochondria. After the rats were killed by decapitation, the whole brain, with the cerebellum cut off, was immediately removed and synaptosomes and extrasynaptosomal mitochondria were prepared essentially according to Gray and Whittaker (1962). Monoamine Oxidase. The monoamine oxidase activity was estimated with five different substrates. With benzylamine as substrate a slight modification of the spectrophotometric method described by McEwen and Cohen (1963) was used. With serotonin and tyramine as substrates an oxygen polarographic method was used as described earlier (Oreland and Ekstedt, 1972). With a third method the enzyme activity was estimated with labeled substrates according to Wurtman and Axelrod (1963) as applied by Gottfries et al. (1975). The substrates used were phenylethylamine (/%phenylethylamine-l-lgc-hydrochloride,7 mC/mmole, New England Nuclear, Boston, Mass.), tryptamine (tryptamine-214C-bisuccinate, 47 mC/mmole, New England Nuclear, Boston, Mass.) and serotonin (5-hydroxytryptamine binoxalate 2-14C,
112
Psychopharmacology 52 (1977)
Table I.
Effect of chronic ethanol treatment on rat brain monoamine oxidase Extrasynaptosomal mitochondrial fraction
Synaptosomal fraction
Benzylamine Tyramine Serotonin 14C/%Phenylethylamine 14C-Tryptamine a4c-Serotonin * "
Inhalation of ethanol vapor (n = 6)
Enforced drinking of 10% ethanol (n = 4)
Inhalation of ethanol vapor (n = 6)
Enforced drinking of 10% ethanol (n = 4)
Mean % of controls a
Mean % of controls"
P*
Mean % of controls"
P*
Mean % of controls a
P*
93 100 131 84 97 99.6
n.s. n.s. n.s. n.s. n.s. n.s.
110 100 104 97 96 96
n.s. n.s. n.s. n.s. n.s. n.s.
96 91 106 104 106 100
n.s. n.s. n.s. n.s. n.s. n.s.
116.7 114 118 101 111 102
P*
< 0.05 n.s. < 0.05 n.s. n.s. n.s.
The statistics were analyzed with the help of Student's t test A corresponding number of similarly treated rats, however, without exposure to ethanol, were used as controls
48.5 mC/mmole, New England Nuclear, Boston, Mass.) in final concentrations of 6.7 gM, 16.7 [aM and 16.7 gM, respectively.
Protein. The protein content was measured according to Lowry et al. with human serum albumin as standard (1951).
RESULTS As shown in Table 1, there were no significant changes in the rat brain monoamine oxidase activity after 34 weeks of daily intake of ethanol. This was true both for synaptosomal and extrasynaptosomal monoamine oxidase, regardless whether the monoamine oxidase type A substrate serotonin, type B substrate benzylamine and fl-phenylethylamine, or the type A + B substrates tyramine and tryptamine (Neff and Yang, 1974) was used. Neither was there any decrease in monoamine oxidase activity in the synaptosomal or in the extrasynaptosomal mitochondrial fraction after administration of ethanol vapor.
DISCUSSION No decrease in MAO-activity was found after either of the two kinds of chronic ethanol treatment used in the present investigation. Both treatments were known to be able to induce other signs of chronic changes in the animals. In rats chronic exposure to ethanol as such or the changes in amine metabolism which might have been induced by the present treatments (Deitrich and Erwin, 1975) have thus not influenced the MAOactivity. Athee and Eriksson (1972), however, found that no changes occurred in the 5-HT and 5 HIAA content in the brain of non-alcoholic rats who had been forced to drink ethanol for 1 month. The present result makes it less likely that the low MAO-activity in the suicide material earlier reported (Gottfries et
al., 1975) was due to chronic effects of ethanol on the enzyme activity in the brains of the patients with a history of alcohol abuse. Other explanations must be looked for and the hypothesis then put forward (Wiberg et al., 1977) that predisposition to certain psychiatric diseases, e.g., ethanol abuse, is due to constitutional factors implying weak monoaminergic systems, and that such a weakness is also reflected in a low monoamine oxidase activity, has thus been supported by the present experiments. Acknowledgements. The skilful technical assistance of Mr. Alf Olsson, Mr. Roland Larsson, Mrs. Kerstin Wahlstr6m, Miss Anna Persson and Mrs. Ingrid Persson is gratefully acknowledged. This work was supported by Grants from the Swedish Medical Research Council (No. 3771 and 4145) and the Medical Faculty, University of Ume&
REFERENCES Ahtee, L., Eriksson, K. : 5-Hydroxytryptamine and 5-hydroxyindolylacetic acid content in brain of rat strains selected for their alcohol intake. Physiol. and Behav. 8, 123- 126 (1972) Curry, A. S., Walker, G.W., Simpson, G. S.: Determination of ethanol in blood by gas chromatography. Analyst 91,742-743 (1966) Deitrich, R., Erwin, G.: Involvement of biogenic amine metabolism in ethanol addiction. Fed. Proc. 34, 1962-1968 (1975) McEwen, C., Cohen, J. : An amine oxidase in normal human serum. J. Lab. clin. Med. 62, 766-776 (1963) Gottfries, C. G., Oreland, L., Wiberg, A., Winblad, B.: Lowered monoamine oxidase activity in brains from alcoholic suicides. J. Neurochem. 25, 667-673 (1975) Gray, E. G., Whittaker, W. P.: The isolation of nerve endings from brain: an electron microscopic study of cell fragments derived by homogenization and centrifugation. 'J. Anat. (Lond.) 96, 79 87 (1962) Lowry, O. H., Rosebrough, A., Farr, L., Randall, R. : Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275 (1951) Neff, N. H., Yang, H. Y.T.: Another look at the monoamine oxidases and the monoamine oxidase inhibitor drugs. Life Sci. 14, 2061 -- 2074 (I 974)
~.. Wiberg et al. : Ethanol and Monoamine Oxidase Activity Oreland, L., Ekstedt, B. : Soluble and membrane-bound pig liver mitochondrial monoamine oxidase: thermostability tryptic digestability and kinetic properties. Biochem. Pharmacol. 21, 2479 - 2488 (1972) Wahlstr6m, G, : Cross tolerance between hexobarbital and ethanol in rats induced by forced drinking of intoxicating amounts of ethanol. The Finnish foundation for alcoholic studies 20, 241 - 249 (1972) Wahlstr6m, G., Widerl6f, E. : Interaction and acute cross tolerance between ethanol and hexobarbitone in the rat. J. Pharm. Pharmacol. 23, 5 8 - 6 0 (1971)
113 Wiberg, h., Gottfries, C.G., Oreland, L.: Low platelet MAO activity in human alcoholics. Med. Biol. (in press, 1977) Wurtman, R. J., Axelrod, J. : A sensitive and specific assay for the estimation of monoamine oxidase. Biochem. Pharmacol. 12, 1439- i441 (1963) A.sberg, M., Triiskman, L., Thor6n, P. : 5-HIAA in the cerebrospinal fluid: a biochemical suicide predictor ? Arch. gen. Psychiat. (in press)
Received December I9, 1975; Final Version December 17, 1976
