- Email: [email protected]
Influence of brain catalase on ethanol-induced loss of righting reflex in mice
Drug and Alcohol Dependence 65 (2001) 9 – 15 www.elsevier.com/locate/drugalcdep
Influence of brain catalase on ethanol-induced loss of righting reflex in mice M. Correa, C. Sanchis-Segura, C.M.G. Aragon * ` rea de Psicobiologia, Uni6ersitat Jaume I, Campus Crta. Borriol, Apartado 224, Castello´ 12080, Spain A Received 29 August 2000; accepted 9 February 2001
Abstract The effect of lead acetate and 3-amino-1, 2, 4-triazole (AT) on ethanol-induced loss of righting reflex (LORR) and brain catalase activity was studied in an attempt to confirm earlier observations on the involvement of catalase in ethanol-induced effects. Lead acetate (0 or 100 mg/kg) or AT (0 or 500 mg/kg) was injected (acutely) into mice 7 days or 5 h before testing. Other mice were exposed to drinking fluid containing 500 ppm lead acetate for 60 days. On the test day, mice received an intraperitoneal injection of ethanol (4.0 or 4.5 g/kg) and the duration of LORR was recorded. Acute lead-treated animals demonstrated a reduction in the duration of the LORR. However, both chronic administration of lead acetate and AT treatment increased the duration of ethanol-produced LORR. Furthermore, brain catalase activity in acute lead pretreated animals showed a significant induction, whereas it was reduced in chronic lead and AT treated mice. These results suggest that brain catalase activity, and by implication centrally formed acetaldehyde, may modulate ethanol-induced LORR. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ethanol; Catalase; Loss of righting reflex; Lead acetate; 3-Amino-1, 2, 4-triazole; Acetaldehyde
1. Introduction Ethanol acts on the central nervous system to produce many behavioural effects in rodents. For example, acute intraperitoneal administration of small doses of ethanol results in enhanced locomotion and open field behaviour in mice (Draski and Deitrich, 1993). Conversely, medium to large doses of ethanol induce locomotor depression, motor incoordination and the loss of righting reflex or narcosis (Draski and Deitrich, 1993). At present, it is still unclear whether these different effects are mediated by the same mechanisms or not. However, a role of brain catalase for ethanol-induced changes in locomotor activity has been clearly established. Indeed, it has been demonstrated that rodents treated with several catalase inhibitors such as 3-amino1,2,4-triazole (AT), sodium azide, cyanamide, and chronic lead acetate, have lower ethanol-induced loco* Corresponding author. Tel.: + 34-964-729337; fax: +34-964729350. E-mail address: [email protected] (C.M.G. Aragon).
motion than control animals (Aragon and Amit, 1993; Correa et al., 1999b; Sanchis-Segura et al., 1999a,b; Escarabajal et al., 2000). Conversely, acute lead or chronic cyanamide administration enhances brain catalase activity and increases ethanol-induced locomotor activity (Correa et al., 1999a, 2000; Sanchis-Segura et al., 1999c). The changes observed in the behavioural effects of ethanol after modification of brain catalase activity are bi-directional and locomotor output is directly related to the amount of brain catalase activity. This is shown by the significant correlations between these variables (Correa et al., 1999a, 2000; Sanchis-Segura et al., 1999b,c; Escarabajal, et al., 2000). The results of these studies are also consistent with previous data obtained from genetically acatalasemic mice that show lower ethanol-induced locomotor activity as compared with normal mice (Aragon et al., 1992; Aragon and Amit, 1993). Therefore, it seems that there is a clear similarity between the directional changes that several substances have on brain catalase activity and on ethanol-induced locomotion. Thus, these studies lend support for the proposed role of brain catalase in ethanol-induced locomotion on mice.
0376-8716/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 6 - 8 7 1 6 ( 0 1 ) 0 0 1 4 2 - 9
10
M. Correa et al. / Drug and Alcohol Dependence 65 (2001) 9–15
On the other hand, acatalasemic mice have a longer duration of sleep time following ethanol administration than normal mice (Aragon and Amit, 1993). Likewise, after an injection of ethanol C57BL/6 mice display less locomotor activity and longer sleeping time as well as lower brain catalase activity than mice of the DBA/2 strain (Kiianmaa et al., 1983; Aragon and Amit, 1987; Gill et al., 1996). Moreover, acute lead administration, that enhances brain catalase activity (Correa et al., 1999a, 2000), has also been shown to increase the latency and to reduce the duration of the loss of righting reflex after an acute ethanol injection (Swartzwelder, 1984). Taken together these data suggest that the duration of loss of righting reflex after an acute ethanol challenge could also be related to brain catalase activity. Consequently, the aim of the present study was to assess the role of the enzyme catalase in mediating the psychopharmacological effects of narcotic doses of ethanol in mice. In this study, brain catalase activity was manipulated by means of: (1) the administration of the catalase inhibitor AT (Escarabajal, et al., 2000); (2) acute lead acetate administration that results in an enhancement of catalase activity (Correa et al., 1999a, 2000); and (3) chronic lead acetate exposure, which reduces catalase activity in different organisms and tissues (Sandhir et al., 1994; Correa et al., 1999b).
2. Materials and methods
2.1. Subjects Male Swiss–Webster mice purchased from HarlanInterfauna Ibe´ rica S.A. (Barcelona, Spain) were housed in groups of four in Plexiglass cages (26.6× 20.5 × 13.5 cm), with standard laboratory rodent chow and tap water available ad libitum. Subjects were maintained 7 days prior to experimentation at 2291°C with lights on from 08:00 to 20:00 h. All experimental procedures complied with the European Community Council directive (86/609/ECC) for the use of laboratory animal subjects.
2.2. Drugs Lead acetate (Sigma Aldrich S.A., Spain) dissolved in saline solution was prepared at a concentration of 50 mg/10 ml for the acute studies. 3-Amino-1, 2, 4-triazole (AT) (Sigma Aldrich S.A., Spain) was obtained from a standard solution of 500 mg/10 ml saline solution. Ethanol solutions (20% v/v) were prepared from 96% ethanol (Panreac Quı´mica S.A., Spain) dissolved in saline. The doses of ethanol used were the smallest that produced LORR in similar studies (Miquel et al., 1999a; Sanchis-Segura et al., 2000).
Chronically administered solutions were prepared dissolving lead acetate or sodium acetate (Panreac Quı´mica S.A., Spain) in distilled water. The concentration of the solutions was 500 ppm. Control animals received water-containing sodium acetate to equalize acetate exposure for the two groups.
2.3. General procedure Following 7 days of habituation to laboratory conditions, animals were randomly assigned to different groups. AT (0 or 500 mg/kg) was injected (IP) in mice 5 h prior to testing. For the acute treatment, lead acetate (0 or 100 mg/kg) was injected (IP) 7 days before testing. During chronic lead acetate treatment, animals were maintained for 60 days with lead acetate or sodium acetate solutions (500 ppm) as the only fluid available. These times and doses were chosen following previous studies (Correa et al., 1999a, 2000; Escarabajal et al., 2000).
2.4. Test of latency and duration of loss of the righting reflex (LORR) After the lead acetate and AT treatments, ethanol (4.0 or 4.5 g/kg) was injected intraperitoneally and individual mice were placed immediately in a Plexiglass cage. The time between alcohol injection and the LORR was assessed, at 2 min intervals, for 10 min. The latency was defined as the time elapsed between ethanol injection and LORR. After the mice lost the righting reflex, they were put on their backs in a V-shaped bed. The duration of LORR was defined as the time from the loss of the righting reflex to that at which it was regained. Recovery was determined when mice could right themselves twice in 1 min after being placed on their backs (Miquel et al., 1999b). All the animals recovered the righting reflex. The behavioural room was illuminated with a soft light and external noise was attenuated.
2.5. Catalase acti6ity determination Brain catalase activity was measured in mice treated (IP) with lead acetate (100 mg/kg), AT (500 mg/kg) or saline. The brains were collected 7 days or 5 h after these treatments, respectively. For the chronic lead acetate administration, mice were treated with sodium acetate or lead acetate (500 ppm) for 60 days and then brains were collected. All mice were perfused with 50 ml of heparinized (1000 U/l) isotonic saline. The whole brain was removed and homogenized in a phosphate buffer (50 mmol/l; pH 7.0) with digitonin (0.01%). Brain homogenates were centrifuged at 10000 rpm for 10 min in an Eppendorf microcentrifuge. Supernatant aliquots were used to determine brain catalase levels.
M. Correa et al. / Drug and Alcohol Dependence 65 (2001) 9–15
Catalase activity was assayed spectrophotometrically by measuring the decrease in absorbance of H2O2 at 240 nm (Aebi, 1984). Protein levels were determined from supernatants (Bradford, 1976).
2.6. Blood ethanol assay Following the lead acetate (0 or 100 mg/kg) and AT (0 or 500 mg/kg) treatments, ethanol (4.5 g/kg) was injected into mice (n =5). Trunk blood was collected either at 60 or 120 min after ethanol administration. For the chronic lead acetate administration, mice were treated with either sodium acetate or lead acetate (500 ppm) for 60 days (n=4 per group). On day 60, ethanol (4.0 g/kg) was administered intraperitoneally. In this case, trunk blood was collected 75 min after the ethanol administration. Animals were sacrificed by decapitation under ether anaesthesia. Each sample of blood was collected in heparinized microcentrifuge tubes and immediately placed in an Eppendorf centrifuge where the samples were spun down for 5 min at 5000 rpm. A
Fig. 1. Effect of an acute IP injection of lead acetate on the latency (A) and duration (B) of LORR induced by ethanol administration. Mean9S.E.M. minutes for all treatment groups (n= 15 per group). Lead acetate (0 or 100 mg/kg) were injected IP to mice 7 days prior to an ethanol IP injection (4.0 or 4.5 g/kg) (** PB 0.01, * P B0.05 significantly different between groups in the same ethanol dose). ( c PB 0.01 significantly different from 4.0 g/kg ethanol in the same lead dose).
11
micropipette was then used to extract 160 ml of plasma and to add it to 1.44 ml of TCA (20%). The mixture was spun down again (5 min at 5000 rpm) to obtain a clear, protein-free supernatant. This protein-free serum was then placed in cuvettes with optical properties suitable for use with a spectrophotometer set at 340 nm. Blood ethanol content was determined enzymatically (Jones et al., 1970) with an Alcohol Diagnostic Kit from Sigma Aldrich S.A., Spain.
2.7. Statistical analyses Data were analysed by means of analyses of variance (ANOVA). Post hoc comparisons were undertaken if a significant main effect or interaction was found at P5 0.05. These comparisons were made using Fisher’s least significant difference tests (LSD). A computerized statistical program (STATISTICA 4.1) was used.
Fig. 2. Effect of an acute IP injection of AT on the latency (A) and duration (B) of LORR induced by ethanol administration. Mean 9 S.E.M. minutes for all treatment groups (n = 13 per group). AT (0 or 500 mg/kg) were injected IP to mice 5 h prior to an ethanol IP injection (4.0 or 4.5 g/kg). (* PB 0.05 significantly different between groups in the same ethanol dose). ( c c P B0.01 significantly different from 4.0 g/kg ethanol in the same AT dose).
M. Correa et al. / Drug and Alcohol Dependence 65 (2001) 9–15
12
Table 1 Mean9S.E.M. of brain catalase activity of lead acetate or AT treated mice
Table 2 Mean 9 S.E.M. of blood ethanol levels after an acute IP injection of 4.5 g/kg of ethanola
Treatment
Treatment
Brain catalase activity (mmol H2O2/min per mg protein)
Blood ethanol levels (mg/dl) 60 min
Saline Lead Acetate AT a
n=8 n=8 n=6
0.99 90.06 1.33 90.04a 0.58 90.19a
Saline Lead acetate AT
n=7 n =7 n =7
120 min 396.37 395.89 369.33
N =8 N =5 N =8
331.04 364.45 347.77
PB0.01 compared to saline group. a
3. Results Fig. 1A and B depicts the effect of acutely administrated lead acetate (0 or 100 mg/kg) on the latency and duration of LORR induced by several doses of ethanol. A two-way factorial ANOVA on the latency data demonstrated an effect of the main factor: lead acetate dose [F(1,56)=16.84, P B0.01], but neither the factor ethanol dose, nor the interaction was significant. These data demonstrate that lead acetate increases the time elapsed between the administration of ethanol and its narcotic effect, independently of the dose of ethanol. On the other hand, the ANOVA on the duration data (Fig. 1B) showed a significant effect of both lead acetate dose [F(1,48) =6.62, P B 0.01)] and ethanol dose [F(1,48)=34.94, PB0.01]. The interaction was not significant. The LSD test indicated that only the lead acetate reduced the duration of LORR significantly when the dose of ethanol was 4.5 g/kg (PB 0.05). The duration of LORR produced after this dose of ethanol was higher than that observed after 4.0 g/kg of ethanol administration and this was not affected by the previous treatment (saline or lead acetate). In order to evaluate the effect of AT (0 or 500 mg/kg) on the latency and duration of LORR induced by ethanol (4.0 or 4.5 g/kg), this alcohol was administered 5 h after AT pretreatment to mice. Fig. 2A and B represents these results. A two-way ANOVA on the latency data showed no statistical difference for either the main factors (AT doses and ethanol doses) or their interaction. These data indicate that both ethanol doses were equally effective on the start point in producing the loss of the reflex and that AT did not modify this property of ethanol. However, when the duration of LORR data were analysed by two-way ANOVA, a significant effect of both main factors, ethanol dose [F(1,48)= 37.05, PB0.01] and AT dose [F(1,48)= 5.89, PB 0.01] appeared, but there was no interaction between them. The post hoc test (LSD) showed a significant difference between saline or AT (500 mg/kg) when the animals where injected with the highest dose of ethanol. There was also a significant difference between ethanol doses in both groups of AT treated mice (P B 0.01).
Animals were treated with lead acetate (100 mg/kg, 7 days before ethanol injection) or AT (500 mg/kg, 5 h before ethanol). Trunk blood was collected 60 or 120 min after ethanol administration.
Table 1 shows brain catalase activity of mice treated with saline, lead acetate and AT. A one-way ANOVA for the factor treatment displayed a significant effect [F(2,19)= 42.77, PB 0.01]. The LSD post hoc test demonstrated that lead acetate as well as AT were different from the control group. Lead acetate potentiated brain catalase activity by 34%, while AT reduced that activity by 41%. Table 2 summarises the effects of saline, acute lead acetate and AT on ethanol blood levels 60 and 120 min after a single administration of ethanol (4.5 g/kg). A
Fig. 3. Effect of chronic administration of sodium or lead acetate on the latency (A) and duration (B) of the LORR induced by ethanol administration. Mean 9S.E.M. minutes for all treatment groups (n =13 per group). Treatments (500 ppm) were administered to mice for 60 days prior an ethanol IP injection (4.0 g/kg) (* PB 0.05 significantly different from sodium acetate treated group).
M. Correa et al. / Drug and Alcohol Dependence 65 (2001) 9–15
13
Table 3 Mean9 S.E.M. of brain catalase activity and blood ethanol levels of mice treated (60 days, 500 ppm) with sodium acetate (control) or lead acetatea Treatment (500 ppm)
Brain catalase activity (mmol H2O2/min per mg protein)
Sodium acetate Lead Acetate
(n=6) (n= 6)
a b
1.77 9 0.09 0.97 9 0.19b
Blood ethanol levels (mg/dl) (n =5) (n =5)
586.42 975.34 582.65 9 46.39
Blood ethanol levels were determined 75 min after an acute IP injection of 4.0 g/kg of ethanol to mice. PB0.01 compared to sodium acetate group.
two-way ANOVA (treatment× time) showed no significant effect for the main factor treatment or for the interaction. However, the factor time was significant [F(1,37)= 3.93, PB 0.05]. Ethanol levels were smaller after 120 min than after 60 min. Two separate t-tests for independent samples were performed to analyse the effect of chronic exposure to lead acetate on latency and duration of ethanol-produced LORR (Fig. 3). These statistical tests showed no significant effect of lead exposure on the latency to lose the righting reflex. Conversely, the duration of the ethanol produced LORR was statistically longer in the group treated with lead acetate than in the control group [t(11)= 2.16, P B0.05)]. The blood ethanol levels and brain catalase activity of chronically-exposed mice are presented in Table 3. Seventy-five minutes after an acute administration of 4.0 g/kg ethanol, blood ethanol levels were not significantly different between sodium and lead acetate groups. However, chronic exposure to lead acetate decreased the activity of brain catalase in brain mice homogenates compared to sodium acetate exposed animals [t(10)=3.61, PB 0.01)].
4. Discussion The findings of the present study revealed opposite effects of acute lead acetate and AT or chronic lead exposure on the ethanol-induced loss of righting reflex and brain catalase activity in mice. Acute lead acetate administration shortened, while AT or chronic lead exposure lengthened, the duration of ethanol-produced LORR. Moreover, in accordance with previous studies (Correa et al., 1999a,b, 2000; Sanchis-Segura et al., 1999b; Escarabajal, et al., 2000), acute lead acetate increased, whereas AT or chronic lead exposure decreased, the cerebral activity of this enzyme. These treatments changed the duration of sleeping time without altering blood ethanol levels. The absence of pharmacokinetic changes agrees with previous reports showing that these treatments do not modify peak blood ethanol levels in rodents (Correa et al., 1999a,b, 2000; Escarabajal, et al., 2000). This suggests that the observed changes in behaviour are due to actions of
these substances on a central target, rather than upon a peripheral one. The duration of LORR seems to be inversely related to the activity of brain catalase. Thus, in comparison with control animals, acute lead-treated mice demonstrated an increase in the onset and a shorter duration of the loss of the righting reflex (16% less than control), as well as a significant induction of total brain catalase activity (45%). Conversely, chronic lead exposure or AT treatment potentiate the duration of LORR produced by ethanol (18 and 16%, respectively). These treatments also reduced brain catalase activity, 55% in the case of lead exposed animals and 56% in the case of AT treated mice. Therefore, more catalase activity corresponds to shorter LORR and a reduction of the activity of brain catalase is related to longer LORR. In fact, a significant Pearson correlation coefficient (r= 0.99; PB 0.001, data not shown) was found between the percentage changes produced by these treatments on the duration of LORR and brain catalase activity. The inverse relationship between brain catalase activity and the duration of the sleeping time is in agreement with previous reports using rodents genetically devoid of catalase activity (Aragon et al., 1992; Aragon and Amit, 1993). Thus, acatalasemic mice display a longer duration of ethanol-induced LORR than normal mice (Aragon and Amit, 1993). In the same way, C57BL/6 mice, which have low levels of brain catalase activity as compared with other inbred strains of mice such as DBA (Kiianmaa et al., 1983; Aragon and Amit, 1987; Gill et al., 1996) reveal longer LORR after ethanol administration. On the other hand, there are some differences in the relationship between brain catalase activity and the duration of ethanol-induced LORR as compared with the relationship between the activity of this enzyme and ethanol-induced locomotion in mice (Aragon and Amit, 1993; Correa et al., 1999a,b, 2000; Sanchis-Segura et al., 1999a,b,c; Escarabajal, et al., 2000). Firstly, although in both cases the correlations obtained between brain catalase activity and ethanol behavioural effects are high, they are opposite in sign. Thus, in the locomotor studies, an increase in brain catalase activity meant an increase in the observed stimulant effects of ethanol, whereas in the present study an inverse relationship
14
M. Correa et al. / Drug and Alcohol Dependence 65 (2001) 9–15
between the variables was found. Moreover, the difference in the size of the effect of these treatments on each behavioural effect of ethanol is also remarkable. Thus, although the changes in brain catalase activity produced by lead or AT are similar to those obtained previously (around a 40– 45% of change for lead and 55% for AT), their impact on ethanol-produced LORR is smaller (16–18%) than that on ethanol-induced locomotion (around 65– 70% for lead and 65% for AT) (Correa et al., 1999a,b, 2000; Escarabajal et al., 2000). Although the precise meaning of these differences has still to be elucidated, it can be suggested that they may reflect a differential role of brain catalase in the behavioural effects of ethanol in mice. Thus, the clear correlation between brain catalase activity and the behavioural output after an ethanol challenge in both cases shows that brain catalase is a mediator of ethanol’s psychopharmacological effects. However, the differences on the size of the effect reveal that its contribution to the different behavioural effects of ethanol can be different in each case. The role of brain catalase in the behavioural effects of ethanol has been linked to its ability to produce acetaldehyde in the brain. Thus, in several in vitro studies, brain homogenates of rats (Aragon et al., 1992; Gill et al., 1992; Zimatkin et al., 1998) and mice (Aragon et al., 1992b; Aragon and Amit, 1993) or neural tissue cultures (Reddy et al., 1995; Eysseric et al., 1997; Hamby-Mason et al., 1997) incubated in the presence of ethanol, oxidize ethanol to acetaldehyde via the peroxidatic activity of catalase. These and other data show that acetaldehyde can produce even more effectively some of the behavioural and physiological effects of ethanol (Amit et al., 1986; Reddy et al., 1995; Hunt, 1996; Smith et al., 1997) and suggest that centrally formed acetaldehyde, via catalase, could be responsible for some of the effects observed after acute ethanol administration. Therefore, it can be suggested that brain catalase and by implication, centrally formed acetaldehyde may play a major role in the stimulant effects of ethanol in mice (as the enhancement of locomotor activity observed after low or moderate doses of ethanol; Correa et al., 1999a,b, 2000; Escarabajal et al., 2000; Sanchis-Segura et al., 1999a,b,c), whereas it only has a modulatory effect on the depressant effects of higher doses of ethanol (i.e. LORR). Both the inverse relationship between brain catalase activity and ethanol-produced LORR and the different effect size for catalase manipulations of the different behavioural effects of ethanol can be understood as an opposing effect due to catalase’s dependent and non-dependent effects in the brain after ethanol administration. In summary, the present study reveals an inverse relationship between brain catalase activity and the duration of ethanol-produced LORR. These data
provide further support for the proposed role of brain catalase on the mechanisms of action of ethanol (for review: Amit et al., 1986; Hunt, 1996; Smith et al., 1997; Zimatkin and Deitrich, 1997). In addition, the comparison of the present data with other obtained in previous studies assessing the impact of the same catalase manipulations on ethanol-induced locomotor activity (Aragon and Amit, 1993; Correa et al., 1999a,b, 2000; Sanchis-Segura et al., 1999a,b,c; Escarabajal, et al., 2000) reveal a quantitatively different role of catalase and, by implication, of centrally formed acetaldehyde, on the different consequences observed after ethanol administration.
Acknowledgements This research was supported by a grant from Bancaixa (P1B97-11), Spain. C. S-S. was supported by a fellowship from the Conselleria de Cultura, Educacio´ i Cie`ncia de la Generalitat Valenciana, Spain.
References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121 –126. Amit, Z., Aragon, C.M.G., Smith, B.R., 1986. Alcohol metabolizing enzymes as possible markers mediating voluntary consumption. Can. J. Public Health 77 (Suppl. 1), 15 – 20. Aragon, C.M.G., Amit, Z., 1987. Genetic variation in ethanol sensitivity in C57BL/6 and DBA/2: a further investigation of the differences in brain catalase activity. In: Rubin, E. (Ed.), Alcohol and the Cell. New York Academy of Science, New York, pp. 398 – 401. Aragon, C.M.G., Amit, Z., 1993. Differences in ethanol-induced behaviours in normal and acatalasemic mice: systematic examination using a behavioural approach. Pharmacol. Biochem. Behav. 44, 547 – 554. Aragon, C.M.G., Pesold, C.N., Amit, Z., 1992. Ethanol induced motor activity in normal and acatalasemic mice. Alcohol 9, 207 – 211. Aragon, C.M.G., Rogan, F., Amit, Z., 1992b. Ethanol metabolism in rat brain homogenates by a catalase-H2O2 system. Biochem. Pharmacol. 44, 93 – 98. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248 – 254. Correa, M., Miquel, M., Sanchis-Segura, C., Aragon, C.M.G., 1999a. Acute lead acetate administration potentiates ethanol-induced locomotor activity in mice: the role of brain catalase. Alcohol. Clin. Exp. Res. 23, 799 – 805. Correa, M., Miquel, M., Sanchis-Segura, C., Aragon, C.M.G., 1999b. Effects of chronic lead administration on ethanol-induced locomotor and brain catalase activity. Alcohol 19, 43 – 49. Correa, M., Miquel, M., Aragon, C.M.G., 2000. Lead acetate potentiates brain catalase activity and enhances ethanol-induced locomotion in mice. Pharmacol. Biochem. Behav. 66, 137 – 142. Draski, L.J., Deitrich, R.A., 1993. Initial effects of ethanol on the central nervous system. In: Deitrich, R.A., Erwin, V.G. (Eds.), Pharmacological Effects of Ethanol on the Nervous System. CRC Press, Boca Raton, pp. 227 – 249.
M. Correa et al. / Drug and Alcohol Dependence 65 (2001) 9–15 Escarabajal, M.D., Miquel, M., Aragon, C.M.G., 2000. A psychopharmacological study of the relationship between brain catalase activity and ethanol-induced locomotor activity in mice. J. Stud. Alcohol. 61, 493 –498. Eysseric, H., Gonthier, A., Soubeyran, G., Bessard, R., Saxod, R., Barret, L., 1997. Characterization of the production of acetaldehyde by astrocytes in culture after ethanol exposure. Alcohol. Clin. Exp. Res. 19, 1018 – 1023. Gill, K., Menez, J.F., Lucas, D., Deitrich, R.A., 1992. Enzymatic production of acetaldehyde from ethanol in rat brain tissue. Alcohol. Clin. Exp. Res. 16, 910 –915. Gill, K., Liu, Y., Deitrich, R.A., 1996. Voluntary alcohol consumption in BXD recombinant inbred mice: relationship to alcohol metabolism. Alcohol. Clin. Exp. Res. 20, 185 –190. Hamby-Mason, R., Chen, J.J., Schenker, S., Pe´ rez, A., Henderson, G.I., 1997. Catalase mediates acetaldehyde formation from ethanol in fetal and neonatal rat brain. Alcohol. Clin. Exp. Res. 21, 1063 – 1072. Hunt, W., 1996. Role of acetaldehyde in the actions of ethanol on the brain. A review. Alcohol 13, 147 –151. Jones, D., Gerber, L.P., Drell, W., 1970. A rapid enzymatic method for estimating ethanol in body fluids. Clin. Chem. 16, 402 – 407. Kiianmaa, K., Hoffman, P.L., Tabakoff, B., 1983. Antagonism of the behavioural effects of ethanol by naltrexone in BALB/c, C57BL/ 6, and DBA/2 mice. Psychopharmacology 79, 291 – 294. Miquel, M., Correa, M., Aragon, C.M.G., 1999a. Methionine potentiates alcohol induced loss of righting reflex in mice. Pharmacol. Biochem. Behav. 64, 83 – 89. Miquel, M., Correa, M., Aragon, C.M.G., 1999b. Methionine enhances alcohol-induced narcosis in mice. Pharmacol. Biochem. Behav. 64, 89 – 93.
15
Reddy, B.V., Boyadjieva, N., Sarkar, D.K., 1995. Effect of ethanol, propanol, butanol and catalase enzyme blockers on b-endorphin secretion from primary cultures of hypothalamic neurons: evidence for mediatory role of acetaldehyde in ethanol stimulation of b-endorphin release. Alcohol. Clin. Exp. Res. 19, 339 – 344. Sanchis-Segura, C., Miquel, M., Correa, M., Aragon, C.M.G., 1999a. The catalase inhibitor sodium azide reduces ethanol-induced locomotor activity. Alcohol 19, 37 – 42. Sanchis-Segura, C., Miquel, M., Correa, M., Aragon, C.M.G., 1999b. Cyanamide reduces brain catalase and ethanol-induced locomotor activity: is there a functional link? Psychopharmacology 144, 83 – 89. Sanchis-Segura, C., Miquel, M., Correa, M., Aragon, C.M.G., 1999c. Daily cyanamide injections enhance ethanol-induced locomotor and brain catalase activity. Behav. Pharmacol. 10, 459 –465. Sanchis-Segura, C., Correa, M., Aragon, C.M.G., 2000. Lesion on the hypothalamic arcuate nucleus by estradiol valerate results in a blockade of ethanol-induced locomotion. Behav. Brain Res. 114, 57 – 63. Sandhir, R., Julka, D., Gill, K.D., 1994. Lipidoperoxidative damage on lead exposure in rat brain and its implications on membrane bound enzymes. Pharmacol. Toxicol. 74, 66 – 71. Smith, B.R., Aragon, C.M.G., Amit, Z., 1997. Catalase and the production of acetaldehyde: a possible mediator of the psychopharmacological effects of ethanol. Addict. Biol. 2, 277 –289. Swartzwelder, H.S., 1984. Altered responsiveness to alcohol after exposure to organic lead. Alcohol 1, 181 – 183. Zimatkin, S.M., Deitrich, R.A., 1997. Ethanol metabolism in the brain. Addict. Biol. 2, 387 –399. Zimatkin, S.M., Liopo, A.V., Deitrich, R.A., 1998. Distribution and kinetics of ethanol metabolism in rat brain. Alcohol. Clin. Exp. Res. 22, 1623 – 1627.
Influence of brain catalase on ethanol-induced loss of righting reflex in mice M. Correa, C. Sanchis-Segura, C.M.G. Aragon * ` rea de Psicobiologia, Uni6ersitat Jaume I, Campus Crta. Borriol, Apartado 224, Castello´ 12080, Spain A Received 29 August 2000; accepted 9 February 2001
Abstract The effect of lead acetate and 3-amino-1, 2, 4-triazole (AT) on ethanol-induced loss of righting reflex (LORR) and brain catalase activity was studied in an attempt to confirm earlier observations on the involvement of catalase in ethanol-induced effects. Lead acetate (0 or 100 mg/kg) or AT (0 or 500 mg/kg) was injected (acutely) into mice 7 days or 5 h before testing. Other mice were exposed to drinking fluid containing 500 ppm lead acetate for 60 days. On the test day, mice received an intraperitoneal injection of ethanol (4.0 or 4.5 g/kg) and the duration of LORR was recorded. Acute lead-treated animals demonstrated a reduction in the duration of the LORR. However, both chronic administration of lead acetate and AT treatment increased the duration of ethanol-produced LORR. Furthermore, brain catalase activity in acute lead pretreated animals showed a significant induction, whereas it was reduced in chronic lead and AT treated mice. These results suggest that brain catalase activity, and by implication centrally formed acetaldehyde, may modulate ethanol-induced LORR. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ethanol; Catalase; Loss of righting reflex; Lead acetate; 3-Amino-1, 2, 4-triazole; Acetaldehyde
1. Introduction Ethanol acts on the central nervous system to produce many behavioural effects in rodents. For example, acute intraperitoneal administration of small doses of ethanol results in enhanced locomotion and open field behaviour in mice (Draski and Deitrich, 1993). Conversely, medium to large doses of ethanol induce locomotor depression, motor incoordination and the loss of righting reflex or narcosis (Draski and Deitrich, 1993). At present, it is still unclear whether these different effects are mediated by the same mechanisms or not. However, a role of brain catalase for ethanol-induced changes in locomotor activity has been clearly established. Indeed, it has been demonstrated that rodents treated with several catalase inhibitors such as 3-amino1,2,4-triazole (AT), sodium azide, cyanamide, and chronic lead acetate, have lower ethanol-induced loco* Corresponding author. Tel.: + 34-964-729337; fax: +34-964729350. E-mail address: [email protected] (C.M.G. Aragon).
motion than control animals (Aragon and Amit, 1993; Correa et al., 1999b; Sanchis-Segura et al., 1999a,b; Escarabajal et al., 2000). Conversely, acute lead or chronic cyanamide administration enhances brain catalase activity and increases ethanol-induced locomotor activity (Correa et al., 1999a, 2000; Sanchis-Segura et al., 1999c). The changes observed in the behavioural effects of ethanol after modification of brain catalase activity are bi-directional and locomotor output is directly related to the amount of brain catalase activity. This is shown by the significant correlations between these variables (Correa et al., 1999a, 2000; Sanchis-Segura et al., 1999b,c; Escarabajal, et al., 2000). The results of these studies are also consistent with previous data obtained from genetically acatalasemic mice that show lower ethanol-induced locomotor activity as compared with normal mice (Aragon et al., 1992; Aragon and Amit, 1993). Therefore, it seems that there is a clear similarity between the directional changes that several substances have on brain catalase activity and on ethanol-induced locomotion. Thus, these studies lend support for the proposed role of brain catalase in ethanol-induced locomotion on mice.
0376-8716/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 6 - 8 7 1 6 ( 0 1 ) 0 0 1 4 2 - 9
10
M. Correa et al. / Drug and Alcohol Dependence 65 (2001) 9–15
On the other hand, acatalasemic mice have a longer duration of sleep time following ethanol administration than normal mice (Aragon and Amit, 1993). Likewise, after an injection of ethanol C57BL/6 mice display less locomotor activity and longer sleeping time as well as lower brain catalase activity than mice of the DBA/2 strain (Kiianmaa et al., 1983; Aragon and Amit, 1987; Gill et al., 1996). Moreover, acute lead administration, that enhances brain catalase activity (Correa et al., 1999a, 2000), has also been shown to increase the latency and to reduce the duration of the loss of righting reflex after an acute ethanol injection (Swartzwelder, 1984). Taken together these data suggest that the duration of loss of righting reflex after an acute ethanol challenge could also be related to brain catalase activity. Consequently, the aim of the present study was to assess the role of the enzyme catalase in mediating the psychopharmacological effects of narcotic doses of ethanol in mice. In this study, brain catalase activity was manipulated by means of: (1) the administration of the catalase inhibitor AT (Escarabajal, et al., 2000); (2) acute lead acetate administration that results in an enhancement of catalase activity (Correa et al., 1999a, 2000); and (3) chronic lead acetate exposure, which reduces catalase activity in different organisms and tissues (Sandhir et al., 1994; Correa et al., 1999b).
2. Materials and methods
2.1. Subjects Male Swiss–Webster mice purchased from HarlanInterfauna Ibe´ rica S.A. (Barcelona, Spain) were housed in groups of four in Plexiglass cages (26.6× 20.5 × 13.5 cm), with standard laboratory rodent chow and tap water available ad libitum. Subjects were maintained 7 days prior to experimentation at 2291°C with lights on from 08:00 to 20:00 h. All experimental procedures complied with the European Community Council directive (86/609/ECC) for the use of laboratory animal subjects.
2.2. Drugs Lead acetate (Sigma Aldrich S.A., Spain) dissolved in saline solution was prepared at a concentration of 50 mg/10 ml for the acute studies. 3-Amino-1, 2, 4-triazole (AT) (Sigma Aldrich S.A., Spain) was obtained from a standard solution of 500 mg/10 ml saline solution. Ethanol solutions (20% v/v) were prepared from 96% ethanol (Panreac Quı´mica S.A., Spain) dissolved in saline. The doses of ethanol used were the smallest that produced LORR in similar studies (Miquel et al., 1999a; Sanchis-Segura et al., 2000).
Chronically administered solutions were prepared dissolving lead acetate or sodium acetate (Panreac Quı´mica S.A., Spain) in distilled water. The concentration of the solutions was 500 ppm. Control animals received water-containing sodium acetate to equalize acetate exposure for the two groups.
2.3. General procedure Following 7 days of habituation to laboratory conditions, animals were randomly assigned to different groups. AT (0 or 500 mg/kg) was injected (IP) in mice 5 h prior to testing. For the acute treatment, lead acetate (0 or 100 mg/kg) was injected (IP) 7 days before testing. During chronic lead acetate treatment, animals were maintained for 60 days with lead acetate or sodium acetate solutions (500 ppm) as the only fluid available. These times and doses were chosen following previous studies (Correa et al., 1999a, 2000; Escarabajal et al., 2000).
2.4. Test of latency and duration of loss of the righting reflex (LORR) After the lead acetate and AT treatments, ethanol (4.0 or 4.5 g/kg) was injected intraperitoneally and individual mice were placed immediately in a Plexiglass cage. The time between alcohol injection and the LORR was assessed, at 2 min intervals, for 10 min. The latency was defined as the time elapsed between ethanol injection and LORR. After the mice lost the righting reflex, they were put on their backs in a V-shaped bed. The duration of LORR was defined as the time from the loss of the righting reflex to that at which it was regained. Recovery was determined when mice could right themselves twice in 1 min after being placed on their backs (Miquel et al., 1999b). All the animals recovered the righting reflex. The behavioural room was illuminated with a soft light and external noise was attenuated.
2.5. Catalase acti6ity determination Brain catalase activity was measured in mice treated (IP) with lead acetate (100 mg/kg), AT (500 mg/kg) or saline. The brains were collected 7 days or 5 h after these treatments, respectively. For the chronic lead acetate administration, mice were treated with sodium acetate or lead acetate (500 ppm) for 60 days and then brains were collected. All mice were perfused with 50 ml of heparinized (1000 U/l) isotonic saline. The whole brain was removed and homogenized in a phosphate buffer (50 mmol/l; pH 7.0) with digitonin (0.01%). Brain homogenates were centrifuged at 10000 rpm for 10 min in an Eppendorf microcentrifuge. Supernatant aliquots were used to determine brain catalase levels.
M. Correa et al. / Drug and Alcohol Dependence 65 (2001) 9–15
Catalase activity was assayed spectrophotometrically by measuring the decrease in absorbance of H2O2 at 240 nm (Aebi, 1984). Protein levels were determined from supernatants (Bradford, 1976).
2.6. Blood ethanol assay Following the lead acetate (0 or 100 mg/kg) and AT (0 or 500 mg/kg) treatments, ethanol (4.5 g/kg) was injected into mice (n =5). Trunk blood was collected either at 60 or 120 min after ethanol administration. For the chronic lead acetate administration, mice were treated with either sodium acetate or lead acetate (500 ppm) for 60 days (n=4 per group). On day 60, ethanol (4.0 g/kg) was administered intraperitoneally. In this case, trunk blood was collected 75 min after the ethanol administration. Animals were sacrificed by decapitation under ether anaesthesia. Each sample of blood was collected in heparinized microcentrifuge tubes and immediately placed in an Eppendorf centrifuge where the samples were spun down for 5 min at 5000 rpm. A
Fig. 1. Effect of an acute IP injection of lead acetate on the latency (A) and duration (B) of LORR induced by ethanol administration. Mean9S.E.M. minutes for all treatment groups (n= 15 per group). Lead acetate (0 or 100 mg/kg) were injected IP to mice 7 days prior to an ethanol IP injection (4.0 or 4.5 g/kg) (** PB 0.01, * P B0.05 significantly different between groups in the same ethanol dose). ( c PB 0.01 significantly different from 4.0 g/kg ethanol in the same lead dose).
11
micropipette was then used to extract 160 ml of plasma and to add it to 1.44 ml of TCA (20%). The mixture was spun down again (5 min at 5000 rpm) to obtain a clear, protein-free supernatant. This protein-free serum was then placed in cuvettes with optical properties suitable for use with a spectrophotometer set at 340 nm. Blood ethanol content was determined enzymatically (Jones et al., 1970) with an Alcohol Diagnostic Kit from Sigma Aldrich S.A., Spain.
2.7. Statistical analyses Data were analysed by means of analyses of variance (ANOVA). Post hoc comparisons were undertaken if a significant main effect or interaction was found at P5 0.05. These comparisons were made using Fisher’s least significant difference tests (LSD). A computerized statistical program (STATISTICA 4.1) was used.
Fig. 2. Effect of an acute IP injection of AT on the latency (A) and duration (B) of LORR induced by ethanol administration. Mean 9 S.E.M. minutes for all treatment groups (n = 13 per group). AT (0 or 500 mg/kg) were injected IP to mice 5 h prior to an ethanol IP injection (4.0 or 4.5 g/kg). (* PB 0.05 significantly different between groups in the same ethanol dose). ( c c P B0.01 significantly different from 4.0 g/kg ethanol in the same AT dose).
M. Correa et al. / Drug and Alcohol Dependence 65 (2001) 9–15
12
Table 1 Mean9S.E.M. of brain catalase activity of lead acetate or AT treated mice
Table 2 Mean 9 S.E.M. of blood ethanol levels after an acute IP injection of 4.5 g/kg of ethanola
Treatment
Treatment
Brain catalase activity (mmol H2O2/min per mg protein)
Blood ethanol levels (mg/dl) 60 min
Saline Lead Acetate AT a
n=8 n=8 n=6
0.99 90.06 1.33 90.04a 0.58 90.19a
Saline Lead acetate AT
n=7 n =7 n =7
120 min 396.37 395.89 369.33
N =8 N =5 N =8
331.04 364.45 347.77
PB0.01 compared to saline group. a
3. Results Fig. 1A and B depicts the effect of acutely administrated lead acetate (0 or 100 mg/kg) on the latency and duration of LORR induced by several doses of ethanol. A two-way factorial ANOVA on the latency data demonstrated an effect of the main factor: lead acetate dose [F(1,56)=16.84, P B0.01], but neither the factor ethanol dose, nor the interaction was significant. These data demonstrate that lead acetate increases the time elapsed between the administration of ethanol and its narcotic effect, independently of the dose of ethanol. On the other hand, the ANOVA on the duration data (Fig. 1B) showed a significant effect of both lead acetate dose [F(1,48) =6.62, P B 0.01)] and ethanol dose [F(1,48)=34.94, PB0.01]. The interaction was not significant. The LSD test indicated that only the lead acetate reduced the duration of LORR significantly when the dose of ethanol was 4.5 g/kg (PB 0.05). The duration of LORR produced after this dose of ethanol was higher than that observed after 4.0 g/kg of ethanol administration and this was not affected by the previous treatment (saline or lead acetate). In order to evaluate the effect of AT (0 or 500 mg/kg) on the latency and duration of LORR induced by ethanol (4.0 or 4.5 g/kg), this alcohol was administered 5 h after AT pretreatment to mice. Fig. 2A and B represents these results. A two-way ANOVA on the latency data showed no statistical difference for either the main factors (AT doses and ethanol doses) or their interaction. These data indicate that both ethanol doses were equally effective on the start point in producing the loss of the reflex and that AT did not modify this property of ethanol. However, when the duration of LORR data were analysed by two-way ANOVA, a significant effect of both main factors, ethanol dose [F(1,48)= 37.05, PB0.01] and AT dose [F(1,48)= 5.89, PB 0.01] appeared, but there was no interaction between them. The post hoc test (LSD) showed a significant difference between saline or AT (500 mg/kg) when the animals where injected with the highest dose of ethanol. There was also a significant difference between ethanol doses in both groups of AT treated mice (P B 0.01).
Animals were treated with lead acetate (100 mg/kg, 7 days before ethanol injection) or AT (500 mg/kg, 5 h before ethanol). Trunk blood was collected 60 or 120 min after ethanol administration.
Table 1 shows brain catalase activity of mice treated with saline, lead acetate and AT. A one-way ANOVA for the factor treatment displayed a significant effect [F(2,19)= 42.77, PB 0.01]. The LSD post hoc test demonstrated that lead acetate as well as AT were different from the control group. Lead acetate potentiated brain catalase activity by 34%, while AT reduced that activity by 41%. Table 2 summarises the effects of saline, acute lead acetate and AT on ethanol blood levels 60 and 120 min after a single administration of ethanol (4.5 g/kg). A
Fig. 3. Effect of chronic administration of sodium or lead acetate on the latency (A) and duration (B) of the LORR induced by ethanol administration. Mean 9S.E.M. minutes for all treatment groups (n =13 per group). Treatments (500 ppm) were administered to mice for 60 days prior an ethanol IP injection (4.0 g/kg) (* PB 0.05 significantly different from sodium acetate treated group).
M. Correa et al. / Drug and Alcohol Dependence 65 (2001) 9–15
13
Table 3 Mean9 S.E.M. of brain catalase activity and blood ethanol levels of mice treated (60 days, 500 ppm) with sodium acetate (control) or lead acetatea Treatment (500 ppm)
Brain catalase activity (mmol H2O2/min per mg protein)
Sodium acetate Lead Acetate
(n=6) (n= 6)
a b
1.77 9 0.09 0.97 9 0.19b
Blood ethanol levels (mg/dl) (n =5) (n =5)
586.42 975.34 582.65 9 46.39
Blood ethanol levels were determined 75 min after an acute IP injection of 4.0 g/kg of ethanol to mice. PB0.01 compared to sodium acetate group.
two-way ANOVA (treatment× time) showed no significant effect for the main factor treatment or for the interaction. However, the factor time was significant [F(1,37)= 3.93, PB 0.05]. Ethanol levels were smaller after 120 min than after 60 min. Two separate t-tests for independent samples were performed to analyse the effect of chronic exposure to lead acetate on latency and duration of ethanol-produced LORR (Fig. 3). These statistical tests showed no significant effect of lead exposure on the latency to lose the righting reflex. Conversely, the duration of the ethanol produced LORR was statistically longer in the group treated with lead acetate than in the control group [t(11)= 2.16, P B0.05)]. The blood ethanol levels and brain catalase activity of chronically-exposed mice are presented in Table 3. Seventy-five minutes after an acute administration of 4.0 g/kg ethanol, blood ethanol levels were not significantly different between sodium and lead acetate groups. However, chronic exposure to lead acetate decreased the activity of brain catalase in brain mice homogenates compared to sodium acetate exposed animals [t(10)=3.61, PB 0.01)].
4. Discussion The findings of the present study revealed opposite effects of acute lead acetate and AT or chronic lead exposure on the ethanol-induced loss of righting reflex and brain catalase activity in mice. Acute lead acetate administration shortened, while AT or chronic lead exposure lengthened, the duration of ethanol-produced LORR. Moreover, in accordance with previous studies (Correa et al., 1999a,b, 2000; Sanchis-Segura et al., 1999b; Escarabajal, et al., 2000), acute lead acetate increased, whereas AT or chronic lead exposure decreased, the cerebral activity of this enzyme. These treatments changed the duration of sleeping time without altering blood ethanol levels. The absence of pharmacokinetic changes agrees with previous reports showing that these treatments do not modify peak blood ethanol levels in rodents (Correa et al., 1999a,b, 2000; Escarabajal, et al., 2000). This suggests that the observed changes in behaviour are due to actions of
these substances on a central target, rather than upon a peripheral one. The duration of LORR seems to be inversely related to the activity of brain catalase. Thus, in comparison with control animals, acute lead-treated mice demonstrated an increase in the onset and a shorter duration of the loss of the righting reflex (16% less than control), as well as a significant induction of total brain catalase activity (45%). Conversely, chronic lead exposure or AT treatment potentiate the duration of LORR produced by ethanol (18 and 16%, respectively). These treatments also reduced brain catalase activity, 55% in the case of lead exposed animals and 56% in the case of AT treated mice. Therefore, more catalase activity corresponds to shorter LORR and a reduction of the activity of brain catalase is related to longer LORR. In fact, a significant Pearson correlation coefficient (r= 0.99; PB 0.001, data not shown) was found between the percentage changes produced by these treatments on the duration of LORR and brain catalase activity. The inverse relationship between brain catalase activity and the duration of the sleeping time is in agreement with previous reports using rodents genetically devoid of catalase activity (Aragon et al., 1992; Aragon and Amit, 1993). Thus, acatalasemic mice display a longer duration of ethanol-induced LORR than normal mice (Aragon and Amit, 1993). In the same way, C57BL/6 mice, which have low levels of brain catalase activity as compared with other inbred strains of mice such as DBA (Kiianmaa et al., 1983; Aragon and Amit, 1987; Gill et al., 1996) reveal longer LORR after ethanol administration. On the other hand, there are some differences in the relationship between brain catalase activity and the duration of ethanol-induced LORR as compared with the relationship between the activity of this enzyme and ethanol-induced locomotion in mice (Aragon and Amit, 1993; Correa et al., 1999a,b, 2000; Sanchis-Segura et al., 1999a,b,c; Escarabajal, et al., 2000). Firstly, although in both cases the correlations obtained between brain catalase activity and ethanol behavioural effects are high, they are opposite in sign. Thus, in the locomotor studies, an increase in brain catalase activity meant an increase in the observed stimulant effects of ethanol, whereas in the present study an inverse relationship
14
M. Correa et al. / Drug and Alcohol Dependence 65 (2001) 9–15
between the variables was found. Moreover, the difference in the size of the effect of these treatments on each behavioural effect of ethanol is also remarkable. Thus, although the changes in brain catalase activity produced by lead or AT are similar to those obtained previously (around a 40– 45% of change for lead and 55% for AT), their impact on ethanol-produced LORR is smaller (16–18%) than that on ethanol-induced locomotion (around 65– 70% for lead and 65% for AT) (Correa et al., 1999a,b, 2000; Escarabajal et al., 2000). Although the precise meaning of these differences has still to be elucidated, it can be suggested that they may reflect a differential role of brain catalase in the behavioural effects of ethanol in mice. Thus, the clear correlation between brain catalase activity and the behavioural output after an ethanol challenge in both cases shows that brain catalase is a mediator of ethanol’s psychopharmacological effects. However, the differences on the size of the effect reveal that its contribution to the different behavioural effects of ethanol can be different in each case. The role of brain catalase in the behavioural effects of ethanol has been linked to its ability to produce acetaldehyde in the brain. Thus, in several in vitro studies, brain homogenates of rats (Aragon et al., 1992; Gill et al., 1992; Zimatkin et al., 1998) and mice (Aragon et al., 1992b; Aragon and Amit, 1993) or neural tissue cultures (Reddy et al., 1995; Eysseric et al., 1997; Hamby-Mason et al., 1997) incubated in the presence of ethanol, oxidize ethanol to acetaldehyde via the peroxidatic activity of catalase. These and other data show that acetaldehyde can produce even more effectively some of the behavioural and physiological effects of ethanol (Amit et al., 1986; Reddy et al., 1995; Hunt, 1996; Smith et al., 1997) and suggest that centrally formed acetaldehyde, via catalase, could be responsible for some of the effects observed after acute ethanol administration. Therefore, it can be suggested that brain catalase and by implication, centrally formed acetaldehyde may play a major role in the stimulant effects of ethanol in mice (as the enhancement of locomotor activity observed after low or moderate doses of ethanol; Correa et al., 1999a,b, 2000; Escarabajal et al., 2000; Sanchis-Segura et al., 1999a,b,c), whereas it only has a modulatory effect on the depressant effects of higher doses of ethanol (i.e. LORR). Both the inverse relationship between brain catalase activity and ethanol-produced LORR and the different effect size for catalase manipulations of the different behavioural effects of ethanol can be understood as an opposing effect due to catalase’s dependent and non-dependent effects in the brain after ethanol administration. In summary, the present study reveals an inverse relationship between brain catalase activity and the duration of ethanol-produced LORR. These data
provide further support for the proposed role of brain catalase on the mechanisms of action of ethanol (for review: Amit et al., 1986; Hunt, 1996; Smith et al., 1997; Zimatkin and Deitrich, 1997). In addition, the comparison of the present data with other obtained in previous studies assessing the impact of the same catalase manipulations on ethanol-induced locomotor activity (Aragon and Amit, 1993; Correa et al., 1999a,b, 2000; Sanchis-Segura et al., 1999a,b,c; Escarabajal, et al., 2000) reveal a quantitatively different role of catalase and, by implication, of centrally formed acetaldehyde, on the different consequences observed after ethanol administration.
Acknowledgements This research was supported by a grant from Bancaixa (P1B97-11), Spain. C. S-S. was supported by a fellowship from the Conselleria de Cultura, Educacio´ i Cie`ncia de la Generalitat Valenciana, Spain.
References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121 –126. Amit, Z., Aragon, C.M.G., Smith, B.R., 1986. Alcohol metabolizing enzymes as possible markers mediating voluntary consumption. Can. J. Public Health 77 (Suppl. 1), 15 – 20. Aragon, C.M.G., Amit, Z., 1987. Genetic variation in ethanol sensitivity in C57BL/6 and DBA/2: a further investigation of the differences in brain catalase activity. In: Rubin, E. (Ed.), Alcohol and the Cell. New York Academy of Science, New York, pp. 398 – 401. Aragon, C.M.G., Amit, Z., 1993. Differences in ethanol-induced behaviours in normal and acatalasemic mice: systematic examination using a behavioural approach. Pharmacol. Biochem. Behav. 44, 547 – 554. Aragon, C.M.G., Pesold, C.N., Amit, Z., 1992. Ethanol induced motor activity in normal and acatalasemic mice. Alcohol 9, 207 – 211. Aragon, C.M.G., Rogan, F., Amit, Z., 1992b. Ethanol metabolism in rat brain homogenates by a catalase-H2O2 system. Biochem. Pharmacol. 44, 93 – 98. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248 – 254. Correa, M., Miquel, M., Sanchis-Segura, C., Aragon, C.M.G., 1999a. Acute lead acetate administration potentiates ethanol-induced locomotor activity in mice: the role of brain catalase. Alcohol. Clin. Exp. Res. 23, 799 – 805. Correa, M., Miquel, M., Sanchis-Segura, C., Aragon, C.M.G., 1999b. Effects of chronic lead administration on ethanol-induced locomotor and brain catalase activity. Alcohol 19, 43 – 49. Correa, M., Miquel, M., Aragon, C.M.G., 2000. Lead acetate potentiates brain catalase activity and enhances ethanol-induced locomotion in mice. Pharmacol. Biochem. Behav. 66, 137 – 142. Draski, L.J., Deitrich, R.A., 1993. Initial effects of ethanol on the central nervous system. In: Deitrich, R.A., Erwin, V.G. (Eds.), Pharmacological Effects of Ethanol on the Nervous System. CRC Press, Boca Raton, pp. 227 – 249.
M. Correa et al. / Drug and Alcohol Dependence 65 (2001) 9–15 Escarabajal, M.D., Miquel, M., Aragon, C.M.G., 2000. A psychopharmacological study of the relationship between brain catalase activity and ethanol-induced locomotor activity in mice. J. Stud. Alcohol. 61, 493 –498. Eysseric, H., Gonthier, A., Soubeyran, G., Bessard, R., Saxod, R., Barret, L., 1997. Characterization of the production of acetaldehyde by astrocytes in culture after ethanol exposure. Alcohol. Clin. Exp. Res. 19, 1018 – 1023. Gill, K., Menez, J.F., Lucas, D., Deitrich, R.A., 1992. Enzymatic production of acetaldehyde from ethanol in rat brain tissue. Alcohol. Clin. Exp. Res. 16, 910 –915. Gill, K., Liu, Y., Deitrich, R.A., 1996. Voluntary alcohol consumption in BXD recombinant inbred mice: relationship to alcohol metabolism. Alcohol. Clin. Exp. Res. 20, 185 –190. Hamby-Mason, R., Chen, J.J., Schenker, S., Pe´ rez, A., Henderson, G.I., 1997. Catalase mediates acetaldehyde formation from ethanol in fetal and neonatal rat brain. Alcohol. Clin. Exp. Res. 21, 1063 – 1072. Hunt, W., 1996. Role of acetaldehyde in the actions of ethanol on the brain. A review. Alcohol 13, 147 –151. Jones, D., Gerber, L.P., Drell, W., 1970. A rapid enzymatic method for estimating ethanol in body fluids. Clin. Chem. 16, 402 – 407. Kiianmaa, K., Hoffman, P.L., Tabakoff, B., 1983. Antagonism of the behavioural effects of ethanol by naltrexone in BALB/c, C57BL/ 6, and DBA/2 mice. Psychopharmacology 79, 291 – 294. Miquel, M., Correa, M., Aragon, C.M.G., 1999a. Methionine potentiates alcohol induced loss of righting reflex in mice. Pharmacol. Biochem. Behav. 64, 83 – 89. Miquel, M., Correa, M., Aragon, C.M.G., 1999b. Methionine enhances alcohol-induced narcosis in mice. Pharmacol. Biochem. Behav. 64, 89 – 93.
15
Reddy, B.V., Boyadjieva, N., Sarkar, D.K., 1995. Effect of ethanol, propanol, butanol and catalase enzyme blockers on b-endorphin secretion from primary cultures of hypothalamic neurons: evidence for mediatory role of acetaldehyde in ethanol stimulation of b-endorphin release. Alcohol. Clin. Exp. Res. 19, 339 – 344. Sanchis-Segura, C., Miquel, M., Correa, M., Aragon, C.M.G., 1999a. The catalase inhibitor sodium azide reduces ethanol-induced locomotor activity. Alcohol 19, 37 – 42. Sanchis-Segura, C., Miquel, M., Correa, M., Aragon, C.M.G., 1999b. Cyanamide reduces brain catalase and ethanol-induced locomotor activity: is there a functional link? Psychopharmacology 144, 83 – 89. Sanchis-Segura, C., Miquel, M., Correa, M., Aragon, C.M.G., 1999c. Daily cyanamide injections enhance ethanol-induced locomotor and brain catalase activity. Behav. Pharmacol. 10, 459 –465. Sanchis-Segura, C., Correa, M., Aragon, C.M.G., 2000. Lesion on the hypothalamic arcuate nucleus by estradiol valerate results in a blockade of ethanol-induced locomotion. Behav. Brain Res. 114, 57 – 63. Sandhir, R., Julka, D., Gill, K.D., 1994. Lipidoperoxidative damage on lead exposure in rat brain and its implications on membrane bound enzymes. Pharmacol. Toxicol. 74, 66 – 71. Smith, B.R., Aragon, C.M.G., Amit, Z., 1997. Catalase and the production of acetaldehyde: a possible mediator of the psychopharmacological effects of ethanol. Addict. Biol. 2, 277 –289. Swartzwelder, H.S., 1984. Altered responsiveness to alcohol after exposure to organic lead. Alcohol 1, 181 – 183. Zimatkin, S.M., Deitrich, R.A., 1997. Ethanol metabolism in the brain. Addict. Biol. 2, 387 –399. Zimatkin, S.M., Liopo, A.V., Deitrich, R.A., 1998. Distribution and kinetics of ethanol metabolism in rat brain. Alcohol. Clin. Exp. Res. 22, 1623 – 1627.