Acta path. microbiol. scand. Sect. A. 86: I 11-1 16, 1978.
EFFECT OF LONG-TERM ADMINISTRATION OF VARIOUS ALCOHOLIC BEVERAGES ON THE IN V I T . 0 INCORPORATION OF 'H-LEUCINE INTO PROTEINS IN RAT CEREBRAL CORTEX, CEREBELLUM AND LIVER JAN JARLSTEDT, LARS JORDb and ROLF OLSSON Departments of Neurobiology and Oto-rhino-laryngology, University of Goteborg and Medical Clinic 11, Sahlgren's Hospital, Goteborg. Sweden
Jarlstedt, J.. Jordo. L. & Olsson. R. Effect of long-term administration of various alcoholic beverages on the in vifro incorporation of 3H-Leucine into proteins in rat cerebral cortex, cerebellum and liver. Acta path. microbiol. scand. Sect. A. 86: I 1 1 - 1 16. 1978. The incorporation of H3 - leucine into protein from anterior and posterior cerebellum, cerebral cortex and liver was studied in rats given 50% of calories as ethanol, brandy, whisky, gin, red wine or isocaloric amounts of glucose together with diets with moderate or low protein-vitamin content for 8-9 months. Higher incorporation rates were usually observed with higher protein-vitamin administration. Red wine and brandy rats usually had the highest, ethanol and gin rats usually the lowest incorporation rates. The incorporation rate thus increased with amount of congeners present. Key words: Alcoholic beverages; amino acid incorporation; cerebellum; cerebrum; liver; rat; malnutrition. Jan Jarlstedt, bronkliniken, Sahlgrenska sjukhuset. 41 3 45 Goteborg, Sweden.
Accepted 23.ix.77
It has been estimated that alcohol often provides about half the caloric requirement in a chronic alcoholic (19). This obviously creates a risk of malnutrition with deficient intake of all important food constituents, notably proteins and vitamins. The combined effects of ethanol and a poor diet may in the long run induce degenerative changes in the central nervous system, resulting in, for instance, atrophy of the cerebral cortex and cerebellar degeneration ( I , 7, I 8). Apart from ethanol, the consumed alcoholic beverages contain small and variable amounts of other substances known as congeners. These are methanol, furfural, fuse1 alcohols, etc. ( I 3, 19). The possible adverse effects on nervous system functions of these congeners have long been disputed.
Most investigators tend to regard ethanol per se as the only harmful factor in alcoholic beverages (for review, see Wallgren and Barry 191, while results presented by other authors suggest that the toxicity varies between different spirits (4, 9). As far as we know, there has been no previous study of protein synthesis in the nervous system where rats have been fed diets containing various alcoholic beverages for a prolonged period. In the present study, rats were fed 50% of the total calories as ethanol, brandy, gin, whisky or red wine. together with either a protein and vitamin sufficient or a low protein. low vitamin diet for 8-9 months. The incorporation rate of radioactive leucine into cerebral and cerebellar proteins was measured at sacrifice. Separate investigations were performed on anterior and posterior parts of the cerebellum, since
previous studies have demonstrated differences in sensitivity to ethanol between these two regions ( I , 6). The incorporation into liver proteins was also determined in order to provide an evaluation of whether possible changes were brain-specific or not.
group received kocaloric amounts of glucose instead of alcohol. Each subgroup initially consisted of 10 animals. After 8-9 months the animals were killed by exsanguination through aortic puncture under ether anesthesia. During 24 hours before death the alcohol groups were reared on the diet of the glucose-control group. The rats in the present series were also subjected to other biochemical and morphological studies that have been presented elsewhere (10, 11, 12). Because of the time-consuming analyses which had to be performed immediately post mortem the period of sacrifice had to be extended to one month. The sacrifice of the animals in each subgroup was evenly distributed over this period. The serum ethanol concentration was determined in IS animals from different subgroups after 8 months of the 50% alcohol diets by aid of gas chromatography. The tail-vein punctures were performed at different times of the day. At sacrifice, the brain was rapidly removed. Pieces of cerebral cortex, anterior and posterior cerebellum and liver were rapidly removed, sliced with a MacIlwain tissue chopper, set at 0.4 mm and transferred directly into the incubation vessels. The vessels were gassed for 60 sec with 100% oxygen. The incubation medium contained: 35 mM Tris-HCI, pH 7.6; 5 mM Naphosphate buffer, pH 7.6; 5 mM KCI; 120 mM NaCl; 2.5 mM MgC12; 5 mM glucose; 2.5 mM ATP and 3Hleucine 40 ul/ml medium (4.5-3H-leucine. spec. act. 22 Ci/mM, The Radiochemical Centre, Amersham). All media were freshly prepared and millipore (0.22 u)filtered. The slices were incubated for 30 minutes, and the incubation was stopped by transferring the incubation vessels to ice. The medium was filtered off, and the
MATERIAL AND METHODS Male Sprague-Dawley rats, weighing 175-183 g at the start of the experiments were used for the study. Five animals were housed in every cage. Diets: The rats were given semi-liquid diets as the sole source of food and water. Two different diets were used in which the calories derived from fat, protein, carbohydrate and ethanol were as follows (in % of total calories):
Diet A
Diet B
6 14 30
20 6.5 23.5
50
50
Fat Protein Carbohydrate Ethanol
Details of the composition and preparation of the diets have been published earlier ( 1 0). Alcohol was isocalorically given as ethanol, whisky, brandy, gin and red wine of cheap qualities. A control
TABLE I . Summary of Statistically Signgcant Dgferences in HI-leucine Incorporation between Different Alcoholgroups in Different Investigated Tissues.
Number of animals
Ant. cerebellum
Post. cerebellum
Cerebral Cortex
E
E
A 5 B S
B
A 5 B 9
E G
E G
A 7 B 6
G
G
GC,G
W
G
A 3 B 5
RW
A 5 B S
E GC,G,W.B,E
A 6
E
Gc
B 8
E E G
Liver
E E
E E
E
From this table it is evident that, for example. brandy B rats had significantly higher incorporation into protein in anterior and posterior cerebellum than gin rats given the B diet, and higher incorporation into liver protein than ethanol rats given the B diet. For explanation of abbreviations, see text of Fig. I .
I12
tissue samples were homogenized in excess volumes (5 ml/ 100 mg tissue) ice-cold 5 % TCA. After centrifugation of the homogenate, aliquots were taken from the TCA supernatant for determination of TCA soluble activity and calculated as CPM per mg protein in the homogenate. The TCA insoluble material was washed three times with 5 % TCA, the last time after heating the samples in boiling waterbath for 20 min. After cooling on ice the pellet obtained after centrifugaion was lipidextracted with ether-absolute ethanol ( I / I ) and subsequently with pure ether. The lipid-extracted material was centrifuged, and the protein content in the pellet was determined according to Lowry et a/. (14). The radioactivity was determined by liquid scintillation counting and expressed as CPM per mg protein. Counting efficiency was 26 % ,and the mean background activity was 19 CPM. Studies using identical labelling techniques have shown that the in v i m incorporation of 3H-leucine into
ANTERIOR CPM/
2ooo
brain protein is linear with time from 10-60 min (3, 5). Haglid and Hamberger ( 5 ) showed that the in vitro incorporation rate of 3H-leucine into brain protein is not changed by adding I , 3 or 5 times the plasma level of a standard amino axid mixture to the avove-mentioned incubation medium. Fisher’s non-parametric permutation test was used for the statistical comparisons. The level of significance was 5%.
RESULTS General Remarks
The mean daily consumption initially was about 380 kcal/kg and decreased to about 160 kcal/kg towards the end of the experiments. The mean daily alcohol consumption over the whole period was about 20 g/kg. Alcohol rats except red wine rats in
CEREBELLUM
mg
1
T
1000
looo GC
E
G
POSTERIOR CPM/ ‘mg 2000
W
I
1:
G C E
B R W
G
W
n
B R W
CEREBELLUM
1000
1000
G C E
G
W B R W
GC
E
G
W
B R W
Fig. 1. 3H-leucine incorporation (CPM per mg protein) into protein from anterior cerebellum. posterior cerebellum. or red wine (RW) cerebral cortex and liver in rats fed 50 % of calories as ethanol (E).whisky (W), brandy (B).gin (G) together with diets with moderate (A-white) or low (B-black)protein and vitamin content. In glucose control groups (GC)the alcohol calories were replaced by glucose. indicates statistically significant difference between A and B groups. A summary of statistical differences between groups given the same diet but different alcoholic beverages or glucose is given in Table I .
1 I3
CEREBRAL CORTEX
CPM
100 50
GC
E
G
W
CEREBELLUM
CPM
GC
E
G
(I W
B R W
POSTERIOR CEREBELLUM
B R W
CPM
LIVER
CPM
2oo 150
Ii
ANTERIOR
1
200
1
150
100
100
50
50
G C E
G
W
B R W
GC
E
G
W
B R W
Fig. 2. TCA-soluble radioactivity in homogenates (CPM per mg total protein in the homogenate) from anterior cerebellum. posterior cerebellum. cerebral cortex and liver in the same groups as in Fig. I . The following significant differences were observed:
Anterior cerebellum. Posterior cerebellum: Liver.
Brandy B> Ethanol B Red Wine B>Ethanol B Brandy B>Gin B Whisky B>Gin B Whisky A>Cin A
No statistically significant differences were detected between groups given the same alcoholic beverage but different diets.
series A displayed a slightly higher body weight increase in relation to caloric intake than the alcohol rats in series B, whereas no such difference was noticed between the control groups. The serum ethanol concentration, measured in I5 animals at different times of the day during the last two months, varied between 55 and 270 mg/100 ml (mean 143 mg/100 ml). Details on growth of animals, food consumption, body and organ weights, etc. are given in another paper (10).
Biochemical Findings With the exception of the ethanol group, the rats given the moderate protein diet (A) usually had higher incorporation rates into cerebellar and cerebral proteins than the rats given the low protein diet (B) (Fig 1 and Table 1). This trend was not apparent when studying liver proteins. The different alcoholic beverages exerted different effects on the incorporation rate, but the pattern of influence was usually the same in the different
tissues studied. Thus, ethanol and gin rats usually showed the lowest incorporation rates, whereas red wine rats showed the highest. In general, the TCAsoluble radioactivity (reflecting the amino acid pool available for protein synthesis)was somewhat lower in nervous tissue from B rats than from A rats (Fig 2). Significant differences between different groups were rare, but the same pattern of influence as in the incorporation studies was discernible. DISCUSSION The results obtained in the control rats show that the administration of a diet rich in fat and low in protein causes a decreased incorporation of leucine into proteins in brain tissue. Since the uptake of the labelled amino acid (measured as TCA-soluble activity) was not affected, the decreased incorporation is due to a decreased capacity for protein synthesis. Similar results have been obtained in other experiments (8). A tentative explanation of these findings is that the low protein supply caused an adaption to a lower protein synthesis. The depressive effect of ethanol on amino acid incorporation into liver proteins, observed in the present study, is in agreement with previous observations. Thus, whereas acute ethanol intoxication (0.45 glkg, Ashworth et al. (2); 8 glkg, Rubin et al. (17) does not decrease the incorporation of radioactive amino acids into liver proteins, prolonged administration(36 % of calories for 24 days) causes a 25 % reduction (1 6). It seems likely that the decreased incorporation found in the tissue from ethanol-treatedA rats is due to an impaired protein synthesis, since ethanol did not significantly influence the uptake of the precursor. Compared to ethanol, brandy and red wine increased the amount of incorporated activity in all investigated brain areas. In fact, the rats given red wine sometimes showed higher incorporation than the controls. The higher amino acid incorporation into CNS protein could be related to an increased uptake of labelled acid reflected in a suggested but only exceptionally statistically significant higher TCA-soluble activity in these groups. A striking finding was the consistent pattern of beverage-related differences in leucine incorporation in all the tissues studied. This observation is of special interest when considering that the amount of congeners present in the different beverages increases in the same order, i.e. ethanol - gin - whisky brandy - red wine ( 1 3) as the leucine incorporation in the present series (Fig. I). It is thus obvious that a high amount of congeners does not impair, but rather increases,the over-all Fpacity for amino acid uptake and incorporation into protein in the tissues
studied. Unfortunately, rather little is known about the functional importance of such changes in cerebral protein metabolism as those observed in the present study. However, it is noticeable that the differences in effects between the different alcoholic beverages observed in brain tissue were clearly not specific for this tissue, since the same pattern of influence was observed in liver tissue. It is also noticeable that in a biochemical and histological study of the livers in the present series of animals, red wine and whisky appeared to have more deleterious influences on the liver than ethanol or gin (10). ~~~~~
~~
This work was supported with grants from the Swedish Medical Research Council.
REFERENCES I . Allsop, J. and Turner, B.: Cerebellar degeneration associated with chronic alcoholism. J. Neurol. Sci. 3: 238-249. 1966. 2. Ashworth, C. T..Johnson, C. F. and Wrightsman, F. J.: Biochemical and morphological correlations of hepatic protein synthesis in acure ethanol intoxication in rats. Amer. J. Path. 64:757-774, 1965. 3. Blomstrand. C . and Hamberger. H.: Amino acid incorporation in vitro into protein of neuronal and glial-cell enriched fractions. J. Neurochem. 17: 1187-1 195, 1970. 4. Haggard, H. W . , Greenberg,L. A. and Cohen. L. H.: The influence of congeners of distilled spirits upon the physiological action of alcohol. Quart. J. Stud. Alc. 4: 3-57, 1943. 5. Haglid, K. G. and Hamberger. A,: Cellular and subcellular distribution of protein-bound radioactivity in the rat brain during maturation after incorporation of 3H-leucine in vitro. Brain Res. 52: 277-287, 1973. 6. Hultborn, R . and Jarlstedt. J.: Effect of ethanol on the oxygen consumption of cerebral cortex. cerebellar cortex and liver homogenates. J. Neuropath. Exp. Neurol. 33: 107-1 12. 1974. 7. Jarlstedt, J.: Experimental alcoholism in rats. Protein synthesis in subcellular fractions from cerebellum, cerebral cortex and liver after long-term treatment. J. Neurochem. f9: 603-608, 1972. 8. Jarlsredt, J.: Effect of alcohol and diet on 3H-leucine incorporation into brain and liver protein. 1. Acute intoxication and vitamin deficiency in rats. J. Stud. Alc. 37: 1178-1 187, 1976. 9. Josfroy. a. and Servaux, R.: Nouveau pro& de menstruation de la toxicite des liquides. Med. Exp. 7: 569-588, 1895. 10. Jordo, L. and Olsson. R.: Effect of long-term administration of different hard liquors and red wine on the rat liver - A histological and biochemical study. Acta path. microbiol. scand. Sect. A. 83: 345-354. 1975. 1 I . Jordo, L., Olsson, R . and Scherstkn, T.: Effect of long-term administration of different alcoholic
12.
13.
14. 15.
I16
beverages to rats on liver lipid metabolism and bile acid conjugation. Digestion 11: 183-193, 1974. Jordo, L., Olsson, R. and Zettergren, L.: Effects of long-term administration of various alcoholic beverages on the kidney, heart, skeletal muscle and pancreas of rats. A histological study. J. Stud. A.c. 36: I I3 1-1 138, 1975. Leake. C. D. and Silverman, M.:The chemistry of alcoholic beverages. In: The biology of alcoholism. Vol. I : Biochemistry. Pp 575-6 12. Eds. Kissin, B. and Begleiter, H. Plenum Press New York-London. 1971. Lowry. 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R . J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 195I . Porta, E. H. and Gomez-Dumm. C. L. A.: A new experimental approach in the study of chronic alcoholism. I. Effects of high alcohol intake in rats
fed a commercial laboratory diet. Lab. Invest. 18: 352-364. 1968. 16. Rubin, E., Beattie, D. S.and Lieber, C. S.:Effects of ethanol on the biogenesis of mitochondria1 membranes and associated mitochondrial functions. Lab. Invest. 23: 620-627, 1970. 17. Rubin. E., Jindrak. K.,Toth. A.. Beattie, D. S.and Lieber, C. S.: Effects of ethanol on hepatic mitochondria1 protein synthesis. J. Clin. Invest. 49: 83a, 1970. 18. Victor, M..Adams, R. D. and Mancall. E. L.: A restricted form of cerebellar cortical degeneration occurring in alcoholic patients. Arch-Neur. I: 579688, 1959. 19. Wallgren, H. and Barry, H.: Actions of alcohol. Ch. 2 and 9. Elsevier publishing company, Amsterdam I9 70.
EFFECT OF LONG-TERM ADMINISTRATION OF VARIOUS ALCOHOLIC BEVERAGES ON THE IN V I T . 0 INCORPORATION OF 'H-LEUCINE INTO PROTEINS IN RAT CEREBRAL CORTEX, CEREBELLUM AND LIVER JAN JARLSTEDT, LARS JORDb and ROLF OLSSON Departments of Neurobiology and Oto-rhino-laryngology, University of Goteborg and Medical Clinic 11, Sahlgren's Hospital, Goteborg. Sweden
Jarlstedt, J.. Jordo. L. & Olsson. R. Effect of long-term administration of various alcoholic beverages on the in vifro incorporation of 3H-Leucine into proteins in rat cerebral cortex, cerebellum and liver. Acta path. microbiol. scand. Sect. A. 86: I 1 1 - 1 16. 1978. The incorporation of H3 - leucine into protein from anterior and posterior cerebellum, cerebral cortex and liver was studied in rats given 50% of calories as ethanol, brandy, whisky, gin, red wine or isocaloric amounts of glucose together with diets with moderate or low protein-vitamin content for 8-9 months. Higher incorporation rates were usually observed with higher protein-vitamin administration. Red wine and brandy rats usually had the highest, ethanol and gin rats usually the lowest incorporation rates. The incorporation rate thus increased with amount of congeners present. Key words: Alcoholic beverages; amino acid incorporation; cerebellum; cerebrum; liver; rat; malnutrition. Jan Jarlstedt, bronkliniken, Sahlgrenska sjukhuset. 41 3 45 Goteborg, Sweden.
Accepted 23.ix.77
It has been estimated that alcohol often provides about half the caloric requirement in a chronic alcoholic (19). This obviously creates a risk of malnutrition with deficient intake of all important food constituents, notably proteins and vitamins. The combined effects of ethanol and a poor diet may in the long run induce degenerative changes in the central nervous system, resulting in, for instance, atrophy of the cerebral cortex and cerebellar degeneration ( I , 7, I 8). Apart from ethanol, the consumed alcoholic beverages contain small and variable amounts of other substances known as congeners. These are methanol, furfural, fuse1 alcohols, etc. ( I 3, 19). The possible adverse effects on nervous system functions of these congeners have long been disputed.
Most investigators tend to regard ethanol per se as the only harmful factor in alcoholic beverages (for review, see Wallgren and Barry 191, while results presented by other authors suggest that the toxicity varies between different spirits (4, 9). As far as we know, there has been no previous study of protein synthesis in the nervous system where rats have been fed diets containing various alcoholic beverages for a prolonged period. In the present study, rats were fed 50% of the total calories as ethanol, brandy, gin, whisky or red wine. together with either a protein and vitamin sufficient or a low protein. low vitamin diet for 8-9 months. The incorporation rate of radioactive leucine into cerebral and cerebellar proteins was measured at sacrifice. Separate investigations were performed on anterior and posterior parts of the cerebellum, since
previous studies have demonstrated differences in sensitivity to ethanol between these two regions ( I , 6). The incorporation into liver proteins was also determined in order to provide an evaluation of whether possible changes were brain-specific or not.
group received kocaloric amounts of glucose instead of alcohol. Each subgroup initially consisted of 10 animals. After 8-9 months the animals were killed by exsanguination through aortic puncture under ether anesthesia. During 24 hours before death the alcohol groups were reared on the diet of the glucose-control group. The rats in the present series were also subjected to other biochemical and morphological studies that have been presented elsewhere (10, 11, 12). Because of the time-consuming analyses which had to be performed immediately post mortem the period of sacrifice had to be extended to one month. The sacrifice of the animals in each subgroup was evenly distributed over this period. The serum ethanol concentration was determined in IS animals from different subgroups after 8 months of the 50% alcohol diets by aid of gas chromatography. The tail-vein punctures were performed at different times of the day. At sacrifice, the brain was rapidly removed. Pieces of cerebral cortex, anterior and posterior cerebellum and liver were rapidly removed, sliced with a MacIlwain tissue chopper, set at 0.4 mm and transferred directly into the incubation vessels. The vessels were gassed for 60 sec with 100% oxygen. The incubation medium contained: 35 mM Tris-HCI, pH 7.6; 5 mM Naphosphate buffer, pH 7.6; 5 mM KCI; 120 mM NaCl; 2.5 mM MgC12; 5 mM glucose; 2.5 mM ATP and 3Hleucine 40 ul/ml medium (4.5-3H-leucine. spec. act. 22 Ci/mM, The Radiochemical Centre, Amersham). All media were freshly prepared and millipore (0.22 u)filtered. The slices were incubated for 30 minutes, and the incubation was stopped by transferring the incubation vessels to ice. The medium was filtered off, and the
MATERIAL AND METHODS Male Sprague-Dawley rats, weighing 175-183 g at the start of the experiments were used for the study. Five animals were housed in every cage. Diets: The rats were given semi-liquid diets as the sole source of food and water. Two different diets were used in which the calories derived from fat, protein, carbohydrate and ethanol were as follows (in % of total calories):
Diet A
Diet B
6 14 30
20 6.5 23.5
50
50
Fat Protein Carbohydrate Ethanol
Details of the composition and preparation of the diets have been published earlier ( 1 0). Alcohol was isocalorically given as ethanol, whisky, brandy, gin and red wine of cheap qualities. A control
TABLE I . Summary of Statistically Signgcant Dgferences in HI-leucine Incorporation between Different Alcoholgroups in Different Investigated Tissues.
Number of animals
Ant. cerebellum
Post. cerebellum
Cerebral Cortex
E
E
A 5 B S
B
A 5 B 9
E G
E G
A 7 B 6
G
G
GC,G
W
G
A 3 B 5
RW
A 5 B S
E GC,G,W.B,E
A 6
E
Gc
B 8
E E G
Liver
E E
E E
E
From this table it is evident that, for example. brandy B rats had significantly higher incorporation into protein in anterior and posterior cerebellum than gin rats given the B diet, and higher incorporation into liver protein than ethanol rats given the B diet. For explanation of abbreviations, see text of Fig. I .
I12
tissue samples were homogenized in excess volumes (5 ml/ 100 mg tissue) ice-cold 5 % TCA. After centrifugation of the homogenate, aliquots were taken from the TCA supernatant for determination of TCA soluble activity and calculated as CPM per mg protein in the homogenate. The TCA insoluble material was washed three times with 5 % TCA, the last time after heating the samples in boiling waterbath for 20 min. After cooling on ice the pellet obtained after centrifugaion was lipidextracted with ether-absolute ethanol ( I / I ) and subsequently with pure ether. The lipid-extracted material was centrifuged, and the protein content in the pellet was determined according to Lowry et a/. (14). The radioactivity was determined by liquid scintillation counting and expressed as CPM per mg protein. Counting efficiency was 26 % ,and the mean background activity was 19 CPM. Studies using identical labelling techniques have shown that the in v i m incorporation of 3H-leucine into
ANTERIOR CPM/
2ooo
brain protein is linear with time from 10-60 min (3, 5). Haglid and Hamberger ( 5 ) showed that the in vitro incorporation rate of 3H-leucine into brain protein is not changed by adding I , 3 or 5 times the plasma level of a standard amino axid mixture to the avove-mentioned incubation medium. Fisher’s non-parametric permutation test was used for the statistical comparisons. The level of significance was 5%.
RESULTS General Remarks
The mean daily consumption initially was about 380 kcal/kg and decreased to about 160 kcal/kg towards the end of the experiments. The mean daily alcohol consumption over the whole period was about 20 g/kg. Alcohol rats except red wine rats in
CEREBELLUM
mg
1
T
1000
looo GC
E
G
POSTERIOR CPM/ ‘mg 2000
W
I
1:
G C E
B R W
G
W
n
B R W
CEREBELLUM
1000
1000
G C E
G
W B R W
GC
E
G
W
B R W
Fig. 1. 3H-leucine incorporation (CPM per mg protein) into protein from anterior cerebellum. posterior cerebellum. or red wine (RW) cerebral cortex and liver in rats fed 50 % of calories as ethanol (E).whisky (W), brandy (B).gin (G) together with diets with moderate (A-white) or low (B-black)protein and vitamin content. In glucose control groups (GC)the alcohol calories were replaced by glucose. indicates statistically significant difference between A and B groups. A summary of statistical differences between groups given the same diet but different alcoholic beverages or glucose is given in Table I .
1 I3
CEREBRAL CORTEX
CPM
100 50
GC
E
G
W
CEREBELLUM
CPM
GC
E
G
(I W
B R W
POSTERIOR CEREBELLUM
B R W
CPM
LIVER
CPM
2oo 150
Ii
ANTERIOR
1
200
1
150
100
100
50
50
G C E
G
W
B R W
GC
E
G
W
B R W
Fig. 2. TCA-soluble radioactivity in homogenates (CPM per mg total protein in the homogenate) from anterior cerebellum. posterior cerebellum. cerebral cortex and liver in the same groups as in Fig. I . The following significant differences were observed:
Anterior cerebellum. Posterior cerebellum: Liver.
Brandy B> Ethanol B Red Wine B>Ethanol B Brandy B>Gin B Whisky B>Gin B Whisky A>Cin A
No statistically significant differences were detected between groups given the same alcoholic beverage but different diets.
series A displayed a slightly higher body weight increase in relation to caloric intake than the alcohol rats in series B, whereas no such difference was noticed between the control groups. The serum ethanol concentration, measured in I5 animals at different times of the day during the last two months, varied between 55 and 270 mg/100 ml (mean 143 mg/100 ml). Details on growth of animals, food consumption, body and organ weights, etc. are given in another paper (10).
Biochemical Findings With the exception of the ethanol group, the rats given the moderate protein diet (A) usually had higher incorporation rates into cerebellar and cerebral proteins than the rats given the low protein diet (B) (Fig 1 and Table 1). This trend was not apparent when studying liver proteins. The different alcoholic beverages exerted different effects on the incorporation rate, but the pattern of influence was usually the same in the different
tissues studied. Thus, ethanol and gin rats usually showed the lowest incorporation rates, whereas red wine rats showed the highest. In general, the TCAsoluble radioactivity (reflecting the amino acid pool available for protein synthesis)was somewhat lower in nervous tissue from B rats than from A rats (Fig 2). Significant differences between different groups were rare, but the same pattern of influence as in the incorporation studies was discernible. DISCUSSION The results obtained in the control rats show that the administration of a diet rich in fat and low in protein causes a decreased incorporation of leucine into proteins in brain tissue. Since the uptake of the labelled amino acid (measured as TCA-soluble activity) was not affected, the decreased incorporation is due to a decreased capacity for protein synthesis. Similar results have been obtained in other experiments (8). A tentative explanation of these findings is that the low protein supply caused an adaption to a lower protein synthesis. The depressive effect of ethanol on amino acid incorporation into liver proteins, observed in the present study, is in agreement with previous observations. Thus, whereas acute ethanol intoxication (0.45 glkg, Ashworth et al. (2); 8 glkg, Rubin et al. (17) does not decrease the incorporation of radioactive amino acids into liver proteins, prolonged administration(36 % of calories for 24 days) causes a 25 % reduction (1 6). It seems likely that the decreased incorporation found in the tissue from ethanol-treatedA rats is due to an impaired protein synthesis, since ethanol did not significantly influence the uptake of the precursor. Compared to ethanol, brandy and red wine increased the amount of incorporated activity in all investigated brain areas. In fact, the rats given red wine sometimes showed higher incorporation than the controls. The higher amino acid incorporation into CNS protein could be related to an increased uptake of labelled acid reflected in a suggested but only exceptionally statistically significant higher TCA-soluble activity in these groups. A striking finding was the consistent pattern of beverage-related differences in leucine incorporation in all the tissues studied. This observation is of special interest when considering that the amount of congeners present in the different beverages increases in the same order, i.e. ethanol - gin - whisky brandy - red wine ( 1 3) as the leucine incorporation in the present series (Fig. I). It is thus obvious that a high amount of congeners does not impair, but rather increases,the over-all Fpacity for amino acid uptake and incorporation into protein in the tissues
studied. Unfortunately, rather little is known about the functional importance of such changes in cerebral protein metabolism as those observed in the present study. However, it is noticeable that the differences in effects between the different alcoholic beverages observed in brain tissue were clearly not specific for this tissue, since the same pattern of influence was observed in liver tissue. It is also noticeable that in a biochemical and histological study of the livers in the present series of animals, red wine and whisky appeared to have more deleterious influences on the liver than ethanol or gin (10). ~~~~~
~~
This work was supported with grants from the Swedish Medical Research Council.
REFERENCES I . Allsop, J. and Turner, B.: Cerebellar degeneration associated with chronic alcoholism. J. Neurol. Sci. 3: 238-249. 1966. 2. Ashworth, C. T..Johnson, C. F. and Wrightsman, F. J.: Biochemical and morphological correlations of hepatic protein synthesis in acure ethanol intoxication in rats. Amer. J. Path. 64:757-774, 1965. 3. Blomstrand. C . and Hamberger. H.: Amino acid incorporation in vitro into protein of neuronal and glial-cell enriched fractions. J. Neurochem. 17: 1187-1 195, 1970. 4. Haggard, H. W . , Greenberg,L. A. and Cohen. L. H.: The influence of congeners of distilled spirits upon the physiological action of alcohol. Quart. J. Stud. Alc. 4: 3-57, 1943. 5. Haglid, K. G. and Hamberger. A,: Cellular and subcellular distribution of protein-bound radioactivity in the rat brain during maturation after incorporation of 3H-leucine in vitro. Brain Res. 52: 277-287, 1973. 6. Hultborn, R . and Jarlstedt. J.: Effect of ethanol on the oxygen consumption of cerebral cortex. cerebellar cortex and liver homogenates. J. Neuropath. Exp. Neurol. 33: 107-1 12. 1974. 7. Jarlstedt, J.: Experimental alcoholism in rats. Protein synthesis in subcellular fractions from cerebellum, cerebral cortex and liver after long-term treatment. J. Neurochem. f9: 603-608, 1972. 8. Jarlsredt, J.: Effect of alcohol and diet on 3H-leucine incorporation into brain and liver protein. 1. Acute intoxication and vitamin deficiency in rats. J. Stud. Alc. 37: 1178-1 187, 1976. 9. Josfroy. a. and Servaux, R.: Nouveau pro& de menstruation de la toxicite des liquides. Med. Exp. 7: 569-588, 1895. 10. Jordo, L. and Olsson. R.: Effect of long-term administration of different hard liquors and red wine on the rat liver - A histological and biochemical study. Acta path. microbiol. scand. Sect. A. 83: 345-354. 1975. 1 I . Jordo, L., Olsson, R . and Scherstkn, T.: Effect of long-term administration of different alcoholic
12.
13.
14. 15.
I16
beverages to rats on liver lipid metabolism and bile acid conjugation. Digestion 11: 183-193, 1974. Jordo, L., Olsson, R. and Zettergren, L.: Effects of long-term administration of various alcoholic beverages on the kidney, heart, skeletal muscle and pancreas of rats. A histological study. J. Stud. A.c. 36: I I3 1-1 138, 1975. Leake. C. D. and Silverman, M.:The chemistry of alcoholic beverages. In: The biology of alcoholism. Vol. I : Biochemistry. Pp 575-6 12. Eds. Kissin, B. and Begleiter, H. Plenum Press New York-London. 1971. Lowry. 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R . J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 195I . Porta, E. H. and Gomez-Dumm. C. L. A.: A new experimental approach in the study of chronic alcoholism. I. Effects of high alcohol intake in rats
fed a commercial laboratory diet. Lab. Invest. 18: 352-364. 1968. 16. Rubin, E., Beattie, D. S.and Lieber, C. S.:Effects of ethanol on the biogenesis of mitochondria1 membranes and associated mitochondrial functions. Lab. Invest. 23: 620-627, 1970. 17. Rubin. E., Jindrak. K.,Toth. A.. Beattie, D. S.and Lieber, C. S.: Effects of ethanol on hepatic mitochondria1 protein synthesis. J. Clin. Invest. 49: 83a, 1970. 18. Victor, M..Adams, R. D. and Mancall. E. L.: A restricted form of cerebellar cortical degeneration occurring in alcoholic patients. Arch-Neur. I: 579688, 1959. 19. Wallgren, H. and Barry, H.: Actions of alcohol. Ch. 2 and 9. Elsevier publishing company, Amsterdam I9 70.
