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Ethanol-induced motor activity in normal and acatalasemic mice
Alcohol, Vol. 9, pp. 207-211, 1992
0741-8329/92$5.00 + .00 Copyright©1992PergamonPress Ltd.
Printedin the U.S.A. All rightsreserved.
Ethanol-Induced Motor Activity in Normal and Acatalasemic Mice C. M. G. A R A G O N , C. N. P E S O L D A N D Z. A M I T 1
Center for Studies in Behavioral Neurobioiogy, Department o f Psychology, Concordia University, Montreal, Quebec, Canada H3G 1MS Received 24 O c t o b e r 1991; Accepted 8 N o v e m b e r 1991 ARAGON, C. M. G., C. N. PESOLD AND Z. AMIT. Ethanol-induced motor activity in normal and acatalasemic mice. ALCOHOL 9(3) 207-211, 1992.-The role of brain catalase in modulating the psychopharmacological effects of ethanol was investigated by examining ethanol induced motor activity in normal, C3H-N, and a corresponding group of acataiasemic C3H-A, mice. Following administration of one of three doses of ethanol (0.8, 1.6, and 3.2 g/kg) or saline, mice were placed in open field chambers and locomotor and rearing activity was measured during a 10-min testing period. A significant increase in locomotor activity was recorded in both groups of mice at lower doses of ethanol, while the higher dose produced a marked depression. Normal mice demonstrated more locomotor activity than acataiasemic mice at all ethanol doses. No differences between both groups of mice were observed in rearing activity. Also, no differences in blood ethanol levels were observed between the two substrains. Brain and liver residual catalase activity in the acataiasemic mice was found to be 40~/0 and 50~0, respectively, of normal mice. Furthermore, evidence for possible involvement of the peroxidatic activity in ethanol-induced motor activity is presented. These results suggest a role for centrally formed acetaldehyde as a factor mediating some of ethanol's psychopharmacologicai effects. Ethanol
Acateldehyde
Motor activity
Catalase
Ethanol metabolism
RESULTS from several studies in different laboratories have suggested a role for brain catalase in the mediation of ethanol's psychopharmacological effects. For example, animals pretreated with 3-amino-l,2,4-triazole (AT), a catalase blocker, and therefore, functionally devoid of brain catalase activity, exhibited a significant attenuation or blockade of several known ethanol effects. It has been reported that when rats pretreated with 3-amino-l,2,4-triazole (AT), were compared with untreated controls, the former displayed periods of shorter narcosis (26), less motor depression (3), less lethality (26), less ethanol-induced corticosterone release (1), and finally, a complete blockade of ethanol-induced conditioned taste aversion (2). These AT effects appeared to be specific to ethanol, because AT did not attenuate the behavioral effects induced by other drugs such as morphine, lithium chloride, or phenobarbital (2,26). AT administration inhibited both brain and liver catalase in rats (14,16,20). However, AT did not affect plasma levels of ethanol measured at different times following intraperitoneal (i.p.) injections of a wide range of ethanol doses (2,3,16,27). It was suggested, therefore, that the alterations in ethanol's induced behaviors by AT must be due to some direct interference, at a central level, possibly by brain catalase activity. Moreover, it has he~n reported that AT failed to alter ethanol effects in rats whose brain catalase inhibition has been prevented by prior administration of ethanol
(26). In addition to these findings, a direct relationship between brain catalase activity and voluntary ethanol consumption in rats has also been reported (4). Although these studies suggest an involvement of brain catalase in ethanol's behavioral effects, some caution must nevertheless be exercised in the interpretation of some of these studies. AT has been reported to inhibit cytochrome P-450dependent activities in liver after acute administration (11) and to inhibit fatty acid synthesis in liver, as well as produce alterations in liver structures after chronic administration (18). Even though these effects have not been shown in brain, at present, one cannot totally exclude other explanations concerning the nature of AT's effects on the behavioral consequences of ethanol administration. To rule out the possible complications of the use of AT as a tool to manipulate catalase activities in vivo, this study was designed to further investigate brain catalase as a significant factor in regulating some of ethanors central effects. Specifically, the role of brain catalase was assessed by comparing ethanors effects on locomotor activity and rearing in normal and acatalasemic mice. Genetically acatalasemic mice produced by x-ray irradiation from normal mice (strain C3H) (9, 10) differing in the activity of this enzyme, wiU be used in this investigation. This autosomally inherited acatalasemia results in loss of catalatic activity in liver, blood, and brain (7,10).
Requests for reprints should be addressed to Z. Amit, Center for Studies in Behavioral Neurobiology, Department of Psychology, 1455 de Maisonneuve Boulevard West, Montreal, Quebec Canada H3G 1MS. 207
208
ARAGON, PESOLD AND AMIT METHOD
iO0-
Subjects
BO"
Wild type mice C3H-N (normal) and a corresponding colony of acatalasemic mice, C3H-A (acatalasemic), produced by x-ray irradiation (9,10) were supplied by Oak Ridge Laboratory (Oak Ridge, TN). This colony was originally established by Dr. Feinstein at the Argonne National Laboratory. Male mice of about 32 g were used in the present experiments.
Locomotor and rearing activities were measured in an open-field apparatus which consisted of a circular glass container divided into four quadrants by black markings on the floor of the container. A single locomotor activity count was considered each time the mouse crossed over from one quadrant to another with all four paws. Rearing was measured each time the mouse raised both forepaws off the floor.
Procedure Immediately before testing, mice received an i.p. injection of one of three doses of ethanol: 0.8 (5%), 1.6 (10%), or 3.2 g/kg (200?0) or saline. The mice were placed after injection into the open-field apparatus where locomotor activity and rearing were recorded in l-rain blocks for a total of l0 consecutive min. Preliminary data increasing the time interval between injection and testing, with or without acclimation of the animals to the open field chamber, suggest that these factors do not influence the observed results. Administration of low doses of ethanol has been repeatedly shown to result in behavioral activation (13,22). For each dose and mouse strain =
8.
Blood Ethanol Determinations An additional 36 male C 3 H - N mice and 36 male C 3 H - A mice were used to determine whether there are differences in plasma ethanol levels between the two strains during the time of behavioral testing. Both strains of mice were tested at three doses of ethanol (0.8, 1.6, and 3.2 g/kg). Mice were injected i.p. with ethanol as just described. Seven and 14 min later they were killed by decapitation under ether anesthesia. Trunk blood was collected and later assayed for ethanol levels by
300
5 o u
40-
0.0
O.S
I .6
32
ETHANOL DOSE (g/kg)
FIG. 2. Mean rearing activity (counts/10 min) for normal and acatalasemic mice as a function of ethanol dose (g/kg).
head-space gas chromatography with a flame-ionized detector (24). Six mice were tested at each dose and time.
Catalase Activity Determination An additional five male C3H-N mice and five male C3HA mice were used to determine brain and liver catalatic activity. Mice were placed in an ether-inhalation chamber l rain prior to being killed by decapitation. Brains and livers were removed and assayed for catalase activity. Ten percent brain homogenates were prepared with 0.1°70 Triton X-100 in 10 mmol/1 potassium phosphate buffer, pH 7.0. The liver homogenate (250?o) was prepared with 0.25 mol/l sucrose-0.1 mmol/l EDTA, p H 7.5. All homogenates were stored at 0°C and were assayed for catalase activity the same day. Catalase activity was measured using a Yellow Springs oxygen monitor equipped with a Clark style oxygen electrode (6). The reaction cell was temperature controlled and maintained at 25°C. A 0.01 mmol/1 potassium phosphate buffer, pH 7.0 (1.7 ml) was deoxygenated with a stream of nitrogen. Hydrogen peroxide (7.6 t~mol in 10 #l) was added to the deoxygenated buffer at time zero, and the baseline 02 formation rate was recorded. At 1 rain a 25 #l aliquot of brain or liver homogenate was added. The difference between the rate of 02 formation before and after the addition of tissue homogenate was taken as the actual reaction rate. Catalase activity is expressed in units of nanomoles of 02 formed per minute per microgram of protein. Protein was determined using the Lowry method with bovine serum albumin as the standard (15).
Ethanol Oxidation by Brain Homogenates 200 C3H-N
- ¢ ~
-~O---C3H-N .....t - - - - C 3 H - A
20~
Apparatus
n
60-
C3H-A
I00-
I 0.0
--I 0.8
I
~
1.6
3.2
ETHANOL DOSE ( g / k g )
FIG. 1. Mean locomotor activity (counts/10 min) for normal and acatalasemic mice as a function of ethanol dose (g/kg).
Five hours following 3-amino-l,2,4-triazole (AT) (0.5 g/ kg), a catalase inhibitor, or saline injections, mice were killed. Brains of C3H-N and C 3 H - A male mice were excised and 100?0 homogenates were prepared with 0.10?0 Triton X-100 in 0.1 m m o l / L potassium phosphate buffer (pH 7.6) at 4°C. All homogenates were stored at 0°C and were assayed the same day. Aliquots of these homogenates equivalent to 25 nag of wet tissue were incubated at 37°C for 1 hour in sealed clear 6-ml Hypo-vials with 90 mmol/l potassium phosphate buffer (pH 7.6), 10 mmol/l glucose, and ethanol (50 retool/l) in the presence or absence of sodium azide (5 retool/l). The acetaldehyde content of the gaseous phase of each vial was measured by head-space gas chromatography (24). The flasks were incu-
ETHANOL-INDUCED MOTOR ACTIVITY IN MICE
209
TABLE 1 BLOOD ETHANOL LEVELS IN NORMAL AND ACATALASEMICMICE 7 AND 14 MIN FOLLOWINGETHANOL I.P. ADMINISTRATION. DATA ARE EXPRESSED IN GRAMS ETHANOL/100 ml PLASMA 7 Min
EthanolDose(g/kg) .8
1.6 3.2
14 Min
C3H-N Strain
C3H-AStrain
C3H-NStrain
C3H-AStrain
.509 + .052 1.648 _+ .102 3.538 ± .412
.467 + .041 1.793 _+ .187 3.624 ± .528
.910 + .072 2.248 + .201 4.596 + .432
1.042 ± .085 2.206 ± .232 4.807 _+ .617
bated at 65°C for 25 minutes, and 2 ml of the head-space was injected into a Varian Model 1400 gas chromatograph with flame ionization detectors. A 180 cm x 2 mm column of Chromosorb 101 mesh 80/100 was used with inlet and detector temperatures of 140°C and 180°C, respectively, and a nitrogen flow rate of about 20 ml/min. Under these conditions, the retention time was 1.8 minutes for acetaldehyde and 3.4 minutes for ethanol. Relative peak heights were determined by comparison with standards prepared by the addition of known amounts of acetaldehyde to zero-time controls. Blanks with boiled homogenates were employed in each experiment. For each treatment and mouse strain n = 8. RESULTS Figure 1 represents the mean number of locomotor activity counts in 10-min blocks at each of the four treatment doses for both strains of mice. Ethanol-induced biphasic effects on locomotor activity in both strains of mice where moderated doses (0.8 and 1.6 g/kg) produced excitation and high doses of ethanol (3.2 g/kg) produced depression. A two-way ANOVA (strain × dose) completely randomized design yielded a significant interaction F(3, 63) = 2.953, p < 0.05; a significant strain effect, F(1, 63) = 26.262, p < 0.01; a significant treatment effect, F(3, 63) = 35.358, p < 0.01. Pairwise comparisons with Tukey tests revealed a significant difference in locomotor activity between the two strains of mice only when tested with an ethanol dose of 0.8 or 1.6 g/kg. For the normal strain of mice, Tukey tests revealed significant differences in locomotor activity between all the doses tested q(1, 63) = 69.913, p < 0.01, except between saline and 3.2 g/kg ethanol-tested animals, q(1, 63) = 59.025, p > 0.05. Acatalasemic mice only revealed significant differences in locomotor activity when tested with 1.6 g/kg and compared with mice treated with saline or 3.2 g/kg ethanol, q(l, 63) = 69.913, p < 0.01.
Figure 2 represents the mean number of rearing counts in 10-rain time blocks at each of the four doses of ethanol. A two-way analysis of variance (ANOVA) (strain x dose) completely randomized design yielded only a significant dose effect, F(3, 63) = 67.225, p < 0.01. The two strains of mice did not differ in their rearing activity at each of the ethanol doses, F(1, 63) = 0.0028, p > 0.05. Blood ethanol levels were analyzed to determine if there were differences in peripheral ethanol levels between the two strains of mice. Table 1 shows the plasma ethanol levels (g/ dl) of the two strains of mice killed at 7 and 14 min postinjection, and given doses of ethanol (0.8, 1.6, and 3.2 g/kg). A completely randomized three-way ANOVA (strain x dose x time) only revealed a significant dose, F(2, 60) = 2452.54, p < 0.01, and time effect, F(l, 60) = 313.78, p < 0.01. However, when the two strains of mice were tested at the same ethanol dose, no significant differences in their plasma ethanol levels were found F(2, 60) = 2.962, p > 0.05. Mean brain and liver catalatic activities for normal and acatalasemic mice are shown in Table 2. An independent t-test was performed on the catalatic activity of brain and liver of the two strains of mice. These analyses revealed a significant difference between the activities of the two strains of mice in brain, t = 11.273, p < 0.001, and liver, t = 9.4622, p < 0.001. Table 3 demonstrates a significant differential recovery of acetaldehyde following incubation of ethanol (50 mmol/1) in brain homogenates of both strains. Brains of mice treated i.p. with saline or AT (0.5 g/kg), 5 hours before sacrifice, were assayed for ethanol oxidation. A two-way ANOVA (strain x treatment) completely randomized design yielded a significant strain effect, F(1, 28) = 202.47, p < 0.01, and a significant treatment effect, F(1, 28) = 437.49, p < 0.01. The fact that administration of AT, a catalase inhibitor, proportionally decreases the amount of recovered acetaldehyde, provides further support for the aforementioned notion. DISCUSSION
TABLE 2 LEVELS OF CATALASEACTIVITYIN BRAIN AND LIVER FROM NORMAL AND ACATALASEMICMICE Nanomoles02 FormedPer min//zgProtein MouseStrain C3H-N C3H-A *p < 0.001.
Brain
Liver
1.07 + .05 0.47 ± .02*
198.09 + 8.94 104.42 ± 4.26*
The results of the present study reveal a significant difference in ethanol-induced locomotor activity between normal and acatalasemic mice. This difference was particularly significant in the excitation induced in these mice by low doses of ethanol. The two groups of mice did not differ significantly in their locomotor activity at the 3.2 g/kg ethanol dose. Also, no differences were observed between the mice tested at the 3.2 g/kg dose of ethanol and those tested with saline. These findings further support the notion of a role for brain catalase in mediating some of the psychopharmacological effects induced by ethanol. No differences were found in spontaneous locomotor activ-
210
ARAGON, PESOLD AND AMIT
TABLE 3 MEAN NANOMOLESOF ACETALDEHYDE/HOUR PER MILLIGRAM PROTEIN OBTAINEDAFTER INCUBATION OF BRAIN HOMOGENATESOF NORMAL (C3H-N) AND ACATALASEMIC(C3H-A) MICE WITH 50 mmol/l ETHANOL. MICE WERE PRETREATEDWITH THE CATALASEINHIBITORAT (0.5 G/KG, I.P.) OR SALINE Strain of Mice Treatment Saline AT (0.5 g/kg)
C3H-N
C3H-A
12,49 _+ 1.15 1.8 __+_1.27'
0,57 _+ 1.33" 0,20 + 0.06*
*p < 0.001.
ity in open field between the two strains of mice, and because these mice are known to differ phenotypically in their catalase activities, it follows that catalase does not seem to play a role in the spontaneous locomotor activity of these mice. Further support for this finding comes from the fact that AT administration alters catalase activity in rats without altering baseline activity in a wide range of behaviors (1-3, 26). In contrast to the locomotor activity, ethanol's effects on rearing activity are not biphasic. Ethanol produced a depression of this behavior in the two strains of mice at all doses of ethanol tested. This depression was dose dependent with higher rearing counts in animals tested with saline, and maximum depression of rearing behavior at higher doses of ethanol (3.2 g/kg). No significant differences, however, were observed among the two groups of mice at all doses. It is suggested that catalase does not seem to mediate ethanol's effects on rearing activity. It is important to note that several studies have reported that not all ethanol-induced behaviors are controlled through the same genetic mechanism (8). No significant differences were found in plasma ethanol levels at each dose and time tested, between the two groups of mice. Both strains of mice demonstrated similar ethanol levels during the time of behavioral testing. This finding supports the suggestion that the enzyme catalase is not an important factor in determining the rate of ethanol metabolism in the whole organism (16,27). Liver catalase can oxidize ethanol to acetaldehyde when hydrogen peroxide is available. AT inhibits liver catalase in vivo (16). However, studies using this cataiase inhibitor have shown that this compound does not slow ethanol oxidation in vivo (2,3,16,27), thus, it has been concluded that liver catalase probably does not play an important role in ethanol metabolism. Recently, other reports have presented data revealing high ethanol blood levels in rats pretreated with AT during the first hour following i.p. ethanol administration
in rats at doses higher than 2.7 kg (25). These authors suggested that hepatic catalase may play a role in liver first pass effect. However, in the present study, high doses of ethanol (3.2 g/kg) in mice did not result in different plasma ethanol levels. In conclusion, any differences in motor behavior observed in the two groups of mice cannot be attributed to differential levels of ethanol sufficient to produce central effects, because there were no differences between the two substrains in blood ethanol levels when mice were tested with the same dose of ethanol. Saline-tested animals depleted of brain catalase did not seem to be affected in their spontaneous locomotor activity in the open field. However, when ethanol was administered, differential levels of cerebral catalase activity could have been the factor responsible for the changes observed in ethanol-induced behavior. The data presented in Table 3 seem to suggest, in view of the differences in the acetaldehyde obtained in the two substrains, that differential peroxidatic activity of catalase may have been involved in the behavioral differences observed. These findings support the notion that brain catalase may play an important role in ethanol's effects and further confirm results from other studies using the catalase inhibitor aminotriazole (1-3,26). It has been suggested that the enzyme catalase, in conjunction with hydrogen peroxide, may metabolize ethanol directly in brain (5). Previous studies have shown that the peroxidatic activity of catalase, determined by the formation of dopaquinone from L-/3-3-4-dihydroxyphenylalanine, is completely normal in acatalasemic mice (23). These findings may suggest that the observed difference in ethanol-induced behavior between the two strains of mice is not due to differential ethanol oxidation in the brain via catalase. However, the apparent discrepancy between their findings and the results obtained in this study may be explained if the reaction of catalase with ethanol were kinetically and structurally different from the reaction of catalase with L-/3-3-4-dihydroxyphenylalanine(12, 19). Although the formation of compound II (catalase-h20 2hydrogen donor complex) is necessary for the oxidation of phenol groups, ethanol, like hydrogen peroxide, requires the formation of compound I (catalase-H202). It is, therefore, reasonable to be alert to the suggestion that the role of catalase in ethanol's effects may be through its putative ability to oxidize ethanol in the brain. Hydrogen peroxide, necessary for catalase oxidation of ethanol, has been demonstrated in the brain (6,17,20), and acetaldehyde, the first metabolite of this oxidation, has been implicated in ethanol's effects (14,21). ACKNOWLEDGEMENT This study was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (A0991) to C.M.G.A.
REFERENCES 1. Aragon, C. M. G.; Amit, Z. Genetic variation in ethanol sensitivity in C57BL/6 and DBA/2 mice: A further investigation of the differences in brain catalase activity. Ann. NY Acad. Sci. 492: 398-400; 1987. 2. Aragon, C. M. G.; Spivak, K.; Amit, Z. Blockade of ethanol induced conditioned taste aversion by 3-amino-l,2,4-triazole: Evidence for catalase-mediated synthesis of acetaldehyde in rat brain. Life Sci. 37:2077-2084; 1985. 3. Aragon, C. M. G.; Spivak, K.; Amit, Z. Effects of 3-amino1,2,4-triazole on ethanol induced open field activity: Evidence for brain catalas¢ mediation of ethanol's effects. Alcoholism: Clin. Exp. Res. 13:104-108; 1989.
4. 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. 5. Aragon, C. M. G.; Stotland, L. M.; Amit, Z. Studies on ethanolbrain catalas¢ interaction: Evidence for central ethanol oxidation. Alcoholism: Clin. Exp. Res. 15:165-169; 1991. 6. De Master, E. G,; Redfern, B.; Shirota, F. N.; Nagasawa, H. T. Deferential inhibition of rat tissue catalase by cyanamide. Biochem. Pharmacol. 35:2081-2085; 1986. 7. Eide, I.; Syversen, L. M. Uptake of elemental mercury and activity of catalase in rat, hamster, guinea pig, normal and acatalasemic mice. Acta Pharmacol. Toxicol. 51:371-376; 1982.
E T H A N O L - I N D U C E D M O T O R A C T I V I T Y IN M I C E 8. Eriksson, C. J. P.; Sarviharju, M. Motor impairment, narcosis and hypothermia by ethanol separate genetic mechanisms. Alcohol 1:59-62; 1984. 9. Feinstein, R. N.; Seaholm, J. E.; Howard, J. B.; Russell, W. L. Acatalasemic mice. Genetics 52:661-662; 1964. 10. Feinstein, R. N.; Howard, J. B.; Braun, J. T.; Seaholm, J. E. Acatalasemic and hypocatalasemic mouse mutants. Genetics 53: 923-933; 1966. 11. Handler, J. A.; Bradford, B. V.; Glassman, E.; Ladine, J. K.; Thurman, R. G. Catalase-dependent ethanol metabolism in vivo in deer mice lacking alcohol dehydrogenase. Biochem. Pharmacol. 35:4487-4492; 1986. 12. Keilin, D.; Nicholls, P. Reactions of catalase with hydrogen peroxide and hydrogen donors. Biochem. Biophys. Acta 29:302-307; 1958. 13. Liljequist, S.; Berjjren, U.; Engel, J. The effect of catechamine antagonists on ethanol-induced locomotor stimulation. J. Neurotransmission 50:57-67; 1981. 14. Lindros, K. O. Acetaldehyde, its metabolism and role in the action of ethanol. In: Isreal, Y.; Glaser, F. S.; Kalant, H.; Papham, R.; Smidt, W.; Smart, R. L. Brain advances in alcohol and drug problems, vol. 4. New York: Plenum Press; 1978:111-176. 15. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275; 1951. 16. Nelson, G. H.; Kinard, F. W.; Aull, J. C.; Hay, B. S. Effect of aminotriazole on alcohol metabolism and hepatic enzyme activities in several species. Q. J. Stud. Alc. 18:343-348; 1956. 17. Patole, M. S.; Swaroop, A.; Ramasarna, T. Generation of H202 in brain mitochondria. J. Neurochem. 47:1-8; 1986. 18. Reitze, H. L.; Seitz, K. A. Light and electron microscopical
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22. 23. 24. 25. 26. 27.
changes in the liver of mice following treatment with aminotriazole. Exp. Pathol. 27:17-31; 1985. Schonbaum, G. R.; Chance, B. Catalase. In: Boyer, P. D . e d . The enzymes, vol. 13. New York: Academic Press; 1975:363-408. Sinet, P. M.; Heikkila, R. E.; Cohen, G. Hydrogen peroxide production by rat brain in vivo. J. Neurochem. 34:1421-1428; 1980. Smith, B. R.; Amit, Z.; Aragon, C. M. G. Socaransky, S. Neurobiological correlates of ethanol self-administration: The role of acetaldehyde. In: Naranjo, C. A.; Seller, E. M. eds. Research advances in new psychopharmacological treatments for alcoholism. New York: Elsevier; 1985: 45-63. Smoothy, R.; Berry, M. S. Time course of the locomotor stimulant and depressant effects of a single low dose of ethanol. Psychopharmacology 85:57-61; 1985. Srivastava, S. K., Ansari, N. H. The peroxidatic and catalactic activity of catalase in normal and acatalasemic mice. Biochem. Biophys. Acta 633:317-322; 1980. Stowell, A. R. An improved method for determination of acetaldehyde in human blood with minimal ethanol interference. Clin. Chem. Acta 98:201-205; 1979. Tampier, L.; Mardones, J. Effect of 3-amino-l,2,4-triazole pretreatment on ethanol blood levels after intrapei'itoneal administration. Alcohol 3:181-183; 1986. Tampier, L.; Quintanilla, M. E.; Letelier, C.; Mardones, J. Effect of 3-amino-l,2,4-triazole on narcosis time and lethality of ethanol in UChA rats. Alcohol 5:5-8; 1988. Teschke, R.; Hasmura, Y.; Lieber, C. S. Hepatic ethanol metabolism: Respective role of alcohol dehydrogenase, the microsomal ethanol oxidizing system, and catalase. Arch. Biochem. Biophys. 175:635-643; 1976.
0741-8329/92$5.00 + .00 Copyright©1992PergamonPress Ltd.
Printedin the U.S.A. All rightsreserved.
Ethanol-Induced Motor Activity in Normal and Acatalasemic Mice C. M. G. A R A G O N , C. N. P E S O L D A N D Z. A M I T 1
Center for Studies in Behavioral Neurobioiogy, Department o f Psychology, Concordia University, Montreal, Quebec, Canada H3G 1MS Received 24 O c t o b e r 1991; Accepted 8 N o v e m b e r 1991 ARAGON, C. M. G., C. N. PESOLD AND Z. AMIT. Ethanol-induced motor activity in normal and acatalasemic mice. ALCOHOL 9(3) 207-211, 1992.-The role of brain catalase in modulating the psychopharmacological effects of ethanol was investigated by examining ethanol induced motor activity in normal, C3H-N, and a corresponding group of acataiasemic C3H-A, mice. Following administration of one of three doses of ethanol (0.8, 1.6, and 3.2 g/kg) or saline, mice were placed in open field chambers and locomotor and rearing activity was measured during a 10-min testing period. A significant increase in locomotor activity was recorded in both groups of mice at lower doses of ethanol, while the higher dose produced a marked depression. Normal mice demonstrated more locomotor activity than acataiasemic mice at all ethanol doses. No differences between both groups of mice were observed in rearing activity. Also, no differences in blood ethanol levels were observed between the two substrains. Brain and liver residual catalase activity in the acataiasemic mice was found to be 40~/0 and 50~0, respectively, of normal mice. Furthermore, evidence for possible involvement of the peroxidatic activity in ethanol-induced motor activity is presented. These results suggest a role for centrally formed acetaldehyde as a factor mediating some of ethanol's psychopharmacologicai effects. Ethanol
Acateldehyde
Motor activity
Catalase
Ethanol metabolism
RESULTS from several studies in different laboratories have suggested a role for brain catalase in the mediation of ethanol's psychopharmacological effects. For example, animals pretreated with 3-amino-l,2,4-triazole (AT), a catalase blocker, and therefore, functionally devoid of brain catalase activity, exhibited a significant attenuation or blockade of several known ethanol effects. It has been reported that when rats pretreated with 3-amino-l,2,4-triazole (AT), were compared with untreated controls, the former displayed periods of shorter narcosis (26), less motor depression (3), less lethality (26), less ethanol-induced corticosterone release (1), and finally, a complete blockade of ethanol-induced conditioned taste aversion (2). These AT effects appeared to be specific to ethanol, because AT did not attenuate the behavioral effects induced by other drugs such as morphine, lithium chloride, or phenobarbital (2,26). AT administration inhibited both brain and liver catalase in rats (14,16,20). However, AT did not affect plasma levels of ethanol measured at different times following intraperitoneal (i.p.) injections of a wide range of ethanol doses (2,3,16,27). It was suggested, therefore, that the alterations in ethanol's induced behaviors by AT must be due to some direct interference, at a central level, possibly by brain catalase activity. Moreover, it has he~n reported that AT failed to alter ethanol effects in rats whose brain catalase inhibition has been prevented by prior administration of ethanol
(26). In addition to these findings, a direct relationship between brain catalase activity and voluntary ethanol consumption in rats has also been reported (4). Although these studies suggest an involvement of brain catalase in ethanol's behavioral effects, some caution must nevertheless be exercised in the interpretation of some of these studies. AT has been reported to inhibit cytochrome P-450dependent activities in liver after acute administration (11) and to inhibit fatty acid synthesis in liver, as well as produce alterations in liver structures after chronic administration (18). Even though these effects have not been shown in brain, at present, one cannot totally exclude other explanations concerning the nature of AT's effects on the behavioral consequences of ethanol administration. To rule out the possible complications of the use of AT as a tool to manipulate catalase activities in vivo, this study was designed to further investigate brain catalase as a significant factor in regulating some of ethanors central effects. Specifically, the role of brain catalase was assessed by comparing ethanors effects on locomotor activity and rearing in normal and acatalasemic mice. Genetically acatalasemic mice produced by x-ray irradiation from normal mice (strain C3H) (9, 10) differing in the activity of this enzyme, wiU be used in this investigation. This autosomally inherited acatalasemia results in loss of catalatic activity in liver, blood, and brain (7,10).
Requests for reprints should be addressed to Z. Amit, Center for Studies in Behavioral Neurobiology, Department of Psychology, 1455 de Maisonneuve Boulevard West, Montreal, Quebec Canada H3G 1MS. 207
208
ARAGON, PESOLD AND AMIT METHOD
iO0-
Subjects
BO"
Wild type mice C3H-N (normal) and a corresponding colony of acatalasemic mice, C3H-A (acatalasemic), produced by x-ray irradiation (9,10) were supplied by Oak Ridge Laboratory (Oak Ridge, TN). This colony was originally established by Dr. Feinstein at the Argonne National Laboratory. Male mice of about 32 g were used in the present experiments.
Locomotor and rearing activities were measured in an open-field apparatus which consisted of a circular glass container divided into four quadrants by black markings on the floor of the container. A single locomotor activity count was considered each time the mouse crossed over from one quadrant to another with all four paws. Rearing was measured each time the mouse raised both forepaws off the floor.
Procedure Immediately before testing, mice received an i.p. injection of one of three doses of ethanol: 0.8 (5%), 1.6 (10%), or 3.2 g/kg (200?0) or saline. The mice were placed after injection into the open-field apparatus where locomotor activity and rearing were recorded in l-rain blocks for a total of l0 consecutive min. Preliminary data increasing the time interval between injection and testing, with or without acclimation of the animals to the open field chamber, suggest that these factors do not influence the observed results. Administration of low doses of ethanol has been repeatedly shown to result in behavioral activation (13,22). For each dose and mouse strain =
8.
Blood Ethanol Determinations An additional 36 male C 3 H - N mice and 36 male C 3 H - A mice were used to determine whether there are differences in plasma ethanol levels between the two strains during the time of behavioral testing. Both strains of mice were tested at three doses of ethanol (0.8, 1.6, and 3.2 g/kg). Mice were injected i.p. with ethanol as just described. Seven and 14 min later they were killed by decapitation under ether anesthesia. Trunk blood was collected and later assayed for ethanol levels by
300
5 o u
40-
0.0
O.S
I .6
32
ETHANOL DOSE (g/kg)
FIG. 2. Mean rearing activity (counts/10 min) for normal and acatalasemic mice as a function of ethanol dose (g/kg).
head-space gas chromatography with a flame-ionized detector (24). Six mice were tested at each dose and time.
Catalase Activity Determination An additional five male C3H-N mice and five male C3HA mice were used to determine brain and liver catalatic activity. Mice were placed in an ether-inhalation chamber l rain prior to being killed by decapitation. Brains and livers were removed and assayed for catalase activity. Ten percent brain homogenates were prepared with 0.1°70 Triton X-100 in 10 mmol/1 potassium phosphate buffer, pH 7.0. The liver homogenate (250?o) was prepared with 0.25 mol/l sucrose-0.1 mmol/l EDTA, p H 7.5. All homogenates were stored at 0°C and were assayed for catalase activity the same day. Catalase activity was measured using a Yellow Springs oxygen monitor equipped with a Clark style oxygen electrode (6). The reaction cell was temperature controlled and maintained at 25°C. A 0.01 mmol/1 potassium phosphate buffer, pH 7.0 (1.7 ml) was deoxygenated with a stream of nitrogen. Hydrogen peroxide (7.6 t~mol in 10 #l) was added to the deoxygenated buffer at time zero, and the baseline 02 formation rate was recorded. At 1 rain a 25 #l aliquot of brain or liver homogenate was added. The difference between the rate of 02 formation before and after the addition of tissue homogenate was taken as the actual reaction rate. Catalase activity is expressed in units of nanomoles of 02 formed per minute per microgram of protein. Protein was determined using the Lowry method with bovine serum albumin as the standard (15).
Ethanol Oxidation by Brain Homogenates 200 C3H-N
- ¢ ~
-~O---C3H-N .....t - - - - C 3 H - A
20~
Apparatus
n
60-
C3H-A
I00-
I 0.0
--I 0.8
I
~
1.6
3.2
ETHANOL DOSE ( g / k g )
FIG. 1. Mean locomotor activity (counts/10 min) for normal and acatalasemic mice as a function of ethanol dose (g/kg).
Five hours following 3-amino-l,2,4-triazole (AT) (0.5 g/ kg), a catalase inhibitor, or saline injections, mice were killed. Brains of C3H-N and C 3 H - A male mice were excised and 100?0 homogenates were prepared with 0.10?0 Triton X-100 in 0.1 m m o l / L potassium phosphate buffer (pH 7.6) at 4°C. All homogenates were stored at 0°C and were assayed the same day. Aliquots of these homogenates equivalent to 25 nag of wet tissue were incubated at 37°C for 1 hour in sealed clear 6-ml Hypo-vials with 90 mmol/l potassium phosphate buffer (pH 7.6), 10 mmol/l glucose, and ethanol (50 retool/l) in the presence or absence of sodium azide (5 retool/l). The acetaldehyde content of the gaseous phase of each vial was measured by head-space gas chromatography (24). The flasks were incu-
ETHANOL-INDUCED MOTOR ACTIVITY IN MICE
209
TABLE 1 BLOOD ETHANOL LEVELS IN NORMAL AND ACATALASEMICMICE 7 AND 14 MIN FOLLOWINGETHANOL I.P. ADMINISTRATION. DATA ARE EXPRESSED IN GRAMS ETHANOL/100 ml PLASMA 7 Min
EthanolDose(g/kg) .8
1.6 3.2
14 Min
C3H-N Strain
C3H-AStrain
C3H-NStrain
C3H-AStrain
.509 + .052 1.648 _+ .102 3.538 ± .412
.467 + .041 1.793 _+ .187 3.624 ± .528
.910 + .072 2.248 + .201 4.596 + .432
1.042 ± .085 2.206 ± .232 4.807 _+ .617
bated at 65°C for 25 minutes, and 2 ml of the head-space was injected into a Varian Model 1400 gas chromatograph with flame ionization detectors. A 180 cm x 2 mm column of Chromosorb 101 mesh 80/100 was used with inlet and detector temperatures of 140°C and 180°C, respectively, and a nitrogen flow rate of about 20 ml/min. Under these conditions, the retention time was 1.8 minutes for acetaldehyde and 3.4 minutes for ethanol. Relative peak heights were determined by comparison with standards prepared by the addition of known amounts of acetaldehyde to zero-time controls. Blanks with boiled homogenates were employed in each experiment. For each treatment and mouse strain n = 8. RESULTS Figure 1 represents the mean number of locomotor activity counts in 10-min blocks at each of the four treatment doses for both strains of mice. Ethanol-induced biphasic effects on locomotor activity in both strains of mice where moderated doses (0.8 and 1.6 g/kg) produced excitation and high doses of ethanol (3.2 g/kg) produced depression. A two-way ANOVA (strain × dose) completely randomized design yielded a significant interaction F(3, 63) = 2.953, p < 0.05; a significant strain effect, F(1, 63) = 26.262, p < 0.01; a significant treatment effect, F(3, 63) = 35.358, p < 0.01. Pairwise comparisons with Tukey tests revealed a significant difference in locomotor activity between the two strains of mice only when tested with an ethanol dose of 0.8 or 1.6 g/kg. For the normal strain of mice, Tukey tests revealed significant differences in locomotor activity between all the doses tested q(1, 63) = 69.913, p < 0.01, except between saline and 3.2 g/kg ethanol-tested animals, q(1, 63) = 59.025, p > 0.05. Acatalasemic mice only revealed significant differences in locomotor activity when tested with 1.6 g/kg and compared with mice treated with saline or 3.2 g/kg ethanol, q(l, 63) = 69.913, p < 0.01.
Figure 2 represents the mean number of rearing counts in 10-rain time blocks at each of the four doses of ethanol. A two-way analysis of variance (ANOVA) (strain x dose) completely randomized design yielded only a significant dose effect, F(3, 63) = 67.225, p < 0.01. The two strains of mice did not differ in their rearing activity at each of the ethanol doses, F(1, 63) = 0.0028, p > 0.05. Blood ethanol levels were analyzed to determine if there were differences in peripheral ethanol levels between the two strains of mice. Table 1 shows the plasma ethanol levels (g/ dl) of the two strains of mice killed at 7 and 14 min postinjection, and given doses of ethanol (0.8, 1.6, and 3.2 g/kg). A completely randomized three-way ANOVA (strain x dose x time) only revealed a significant dose, F(2, 60) = 2452.54, p < 0.01, and time effect, F(l, 60) = 313.78, p < 0.01. However, when the two strains of mice were tested at the same ethanol dose, no significant differences in their plasma ethanol levels were found F(2, 60) = 2.962, p > 0.05. Mean brain and liver catalatic activities for normal and acatalasemic mice are shown in Table 2. An independent t-test was performed on the catalatic activity of brain and liver of the two strains of mice. These analyses revealed a significant difference between the activities of the two strains of mice in brain, t = 11.273, p < 0.001, and liver, t = 9.4622, p < 0.001. Table 3 demonstrates a significant differential recovery of acetaldehyde following incubation of ethanol (50 mmol/1) in brain homogenates of both strains. Brains of mice treated i.p. with saline or AT (0.5 g/kg), 5 hours before sacrifice, were assayed for ethanol oxidation. A two-way ANOVA (strain x treatment) completely randomized design yielded a significant strain effect, F(1, 28) = 202.47, p < 0.01, and a significant treatment effect, F(1, 28) = 437.49, p < 0.01. The fact that administration of AT, a catalase inhibitor, proportionally decreases the amount of recovered acetaldehyde, provides further support for the aforementioned notion. DISCUSSION
TABLE 2 LEVELS OF CATALASEACTIVITYIN BRAIN AND LIVER FROM NORMAL AND ACATALASEMICMICE Nanomoles02 FormedPer min//zgProtein MouseStrain C3H-N C3H-A *p < 0.001.
Brain
Liver
1.07 + .05 0.47 ± .02*
198.09 + 8.94 104.42 ± 4.26*
The results of the present study reveal a significant difference in ethanol-induced locomotor activity between normal and acatalasemic mice. This difference was particularly significant in the excitation induced in these mice by low doses of ethanol. The two groups of mice did not differ significantly in their locomotor activity at the 3.2 g/kg ethanol dose. Also, no differences were observed between the mice tested at the 3.2 g/kg dose of ethanol and those tested with saline. These findings further support the notion of a role for brain catalase in mediating some of the psychopharmacological effects induced by ethanol. No differences were found in spontaneous locomotor activ-
210
ARAGON, PESOLD AND AMIT
TABLE 3 MEAN NANOMOLESOF ACETALDEHYDE/HOUR PER MILLIGRAM PROTEIN OBTAINEDAFTER INCUBATION OF BRAIN HOMOGENATESOF NORMAL (C3H-N) AND ACATALASEMIC(C3H-A) MICE WITH 50 mmol/l ETHANOL. MICE WERE PRETREATEDWITH THE CATALASEINHIBITORAT (0.5 G/KG, I.P.) OR SALINE Strain of Mice Treatment Saline AT (0.5 g/kg)
C3H-N
C3H-A
12,49 _+ 1.15 1.8 __+_1.27'
0,57 _+ 1.33" 0,20 + 0.06*
*p < 0.001.
ity in open field between the two strains of mice, and because these mice are known to differ phenotypically in their catalase activities, it follows that catalase does not seem to play a role in the spontaneous locomotor activity of these mice. Further support for this finding comes from the fact that AT administration alters catalase activity in rats without altering baseline activity in a wide range of behaviors (1-3, 26). In contrast to the locomotor activity, ethanol's effects on rearing activity are not biphasic. Ethanol produced a depression of this behavior in the two strains of mice at all doses of ethanol tested. This depression was dose dependent with higher rearing counts in animals tested with saline, and maximum depression of rearing behavior at higher doses of ethanol (3.2 g/kg). No significant differences, however, were observed among the two groups of mice at all doses. It is suggested that catalase does not seem to mediate ethanol's effects on rearing activity. It is important to note that several studies have reported that not all ethanol-induced behaviors are controlled through the same genetic mechanism (8). No significant differences were found in plasma ethanol levels at each dose and time tested, between the two groups of mice. Both strains of mice demonstrated similar ethanol levels during the time of behavioral testing. This finding supports the suggestion that the enzyme catalase is not an important factor in determining the rate of ethanol metabolism in the whole organism (16,27). Liver catalase can oxidize ethanol to acetaldehyde when hydrogen peroxide is available. AT inhibits liver catalase in vivo (16). However, studies using this cataiase inhibitor have shown that this compound does not slow ethanol oxidation in vivo (2,3,16,27), thus, it has been concluded that liver catalase probably does not play an important role in ethanol metabolism. Recently, other reports have presented data revealing high ethanol blood levels in rats pretreated with AT during the first hour following i.p. ethanol administration
in rats at doses higher than 2.7 kg (25). These authors suggested that hepatic catalase may play a role in liver first pass effect. However, in the present study, high doses of ethanol (3.2 g/kg) in mice did not result in different plasma ethanol levels. In conclusion, any differences in motor behavior observed in the two groups of mice cannot be attributed to differential levels of ethanol sufficient to produce central effects, because there were no differences between the two substrains in blood ethanol levels when mice were tested with the same dose of ethanol. Saline-tested animals depleted of brain catalase did not seem to be affected in their spontaneous locomotor activity in the open field. However, when ethanol was administered, differential levels of cerebral catalase activity could have been the factor responsible for the changes observed in ethanol-induced behavior. The data presented in Table 3 seem to suggest, in view of the differences in the acetaldehyde obtained in the two substrains, that differential peroxidatic activity of catalase may have been involved in the behavioral differences observed. These findings support the notion that brain catalase may play an important role in ethanol's effects and further confirm results from other studies using the catalase inhibitor aminotriazole (1-3,26). It has been suggested that the enzyme catalase, in conjunction with hydrogen peroxide, may metabolize ethanol directly in brain (5). Previous studies have shown that the peroxidatic activity of catalase, determined by the formation of dopaquinone from L-/3-3-4-dihydroxyphenylalanine, is completely normal in acatalasemic mice (23). These findings may suggest that the observed difference in ethanol-induced behavior between the two strains of mice is not due to differential ethanol oxidation in the brain via catalase. However, the apparent discrepancy between their findings and the results obtained in this study may be explained if the reaction of catalase with ethanol were kinetically and structurally different from the reaction of catalase with L-/3-3-4-dihydroxyphenylalanine(12, 19). Although the formation of compound II (catalase-h20 2hydrogen donor complex) is necessary for the oxidation of phenol groups, ethanol, like hydrogen peroxide, requires the formation of compound I (catalase-H202). It is, therefore, reasonable to be alert to the suggestion that the role of catalase in ethanol's effects may be through its putative ability to oxidize ethanol in the brain. Hydrogen peroxide, necessary for catalase oxidation of ethanol, has been demonstrated in the brain (6,17,20), and acetaldehyde, the first metabolite of this oxidation, has been implicated in ethanol's effects (14,21). ACKNOWLEDGEMENT This study was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (A0991) to C.M.G.A.
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