Interaction Between Ethanol and Diazepam in Mice: Chronobiological Aspects
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ORIGINAL ARTICLES Department of Pharmacodynamics, Medical University Ło´dz´, Poland Interaction between ethanol and diazepam in mice: chronobiological aspects B. Pietrzak, E. Czarnecka Received July 19, 2004, accepted November 25, 2004 Dr. Boguslawa Pietrzak, Department of Pharmacodynamics, Medical University, Muszyn´skiego 1, 90-151 Ło´dz´, Poland [email protected] Pharmazie 60: 761–764 (2005) The mechanism of action of benzodiazepines and ethanol demonstrates that these agents can syner- gistically affect the central nervous system (CNS). The effects of both ethanol and diazepam are likely to depend on the time of the day when they were administered. Diazepam influence on ethanol-induced sleeping and hypothermic activity in mice as well as the influence of combined administration of these agents on spontaneous locomotor activity and coordination in mice (rota-rod) were investigated. Ex- periments were carried out in the light phase (10:00–12:00 h) and the dark phase (22:00–24:00 h). It was shown that ethanol-induced sleeping time was longer in the dark phase than the light phase, and that ethanol increased spontaneous locomotor activity both in the light and the dark. Ethanol-induced hypothermia was lower in the dark than in the light. Diazepam decreased locomotor activity more strongly in the dark phase than by day. It impaired the hypothermic action of ethanol in the light phase, but did not have such an effect in the dark phase. Diazepam prolonged ethanol-induced sleep in the light phase, enhanced its action on locomotor coordination and decreased the stimulating effect of ethanol on spontaneous locomotor activity in mice. The chronobiological effect of the interaction between diazepam and ethanol seems to be of practical importance (sleep and motor coordination). 1. Introduction study was to observe interactions between ethanol and dia- zepam during the day/night cycle (light and dark phase). One of the new pharmacology specialities is chronophar- macology. It deals with examination of the relationship 2. Investigations and results between timing of drug administration and drug efficacy. Basic concepts of chronopharmacology involve chron- 2.1. Ethanol sleeping time esthesy (circadian changeability of receptor sensitivity to Ethanol administered to mice in a dose of 4 g kgÀ1 pro- drugs) and chronokinetics (circadian rhythms of drug duced sleep lasting about 12 min in a light phase. In a availability, biotransformation and excretion processes). dark phase sleep was longer and lasted on average 21 min. Circadian variations in drug efficacy (chronergy) are the Diazepam prolonged ethanol-induced sleep in the light consequence of chronesthesy and chronokinetics. phase only (Fig. 1). Ethanol has a broad range of actions on many neurotrans- mitter systems. The depressant actions of ethanol in the brain are related in part to facilitation of gamma-aminobu- 35 tiric acid (GABA) neurotransmission via its interaction Light Dark with the benzodiazepine/GABA receptor complex. 30 The benzodiazepines are a family of anxiolytic and hypno- a 25 tic drugs. When taken concurrently with ethanol, a phar- macodynamic interaction may occur, potentiating the cen- 20 tral nervous system depression produced by either drug. c Diazepam is a popular and frequently used benzodiazepine, 15 also indicated in the alcohol abstinence syndrome. The Sleep time [min] 10 GABA-ergic mechanism associated with the action of both benzodiazepines and ethanol can lead to their synergistic ac- 5 tion. The synergistic effect of benzodiazepines and ethanol 0 has been shown in experimental (Van-Steveninck et al. 1996; Et Et+Dz Et Et+Dz Vanover et al. 1999; Vanover 1999; Storustovu and Ebert 2003; Voss et al. 2003) and clinical research (Eves and Lader Fig. 1: The effect of treatment with diazepam on ethanol sleeping time in À1 1989; Hrbek et al. 1989; Van-Steveninck et al. 1996). mice. Et –– ethanol, Dz –– diazepam 0.5 mg kg . Ethanol was given at a dose of 4 g kgÀ1 i.p. ten minutes after diazepam. The Chronobiological aspects of ethanol and diazepam action data are shown as means Æ SEM; a p < 0.05 versus ethanol group, may have essential practical importance. The aim of the cp< 0.05 light versus dark Pharmazie 60 (2005) 10 761 ORIGINAL ARTICLES 4 Light Dark 3 b 2 Time [min] b,c 1 b,d a,b,d 0 Et Et+Dz Dz Sal Et Et+DzDz Sal Fig. 2: The effect of treatment with diazepam on ethanol impairs motor Fig. 4: The effect of treatment with diazepam on ethanol-induced hy- coordination (rota-rod) in mice. Mean time of mice performance on pothermia in mice. Et –– ethanol (2.5 g Á kgÀ1), Dz –– diazepam the rod after treatment with: Et – ethanol (1.5 g Á kgÀ1), Dz –– dia- 0.5 mg kgÀ1, Sal –– saline (0.9% NaCl). Results are presented as zepam 0.5 mg kgÀ1, Sal –– saline (0.9% NaCl). The data are shown means, a p < 0.05 versus ethanol group, b p < 0.05 versus saline as means Æ SEM, a p < 0.05 versus ethanol group, b p < 0.05 group, c p < 0.05 light versus dark versus saline group, c p < 0.05 light versus dark, d p < 0.05 versus diazepam group (Fig. 4). In the dark, hypothermia 90 and 120 min after 2.2. Rota-rod performance ethanol administration was statistically significantly lower than in the light phase. Diazepam given before ethanol in Ethanol impairs motor coordination in mice both in the light the light phase decreased the hypothermic action of etha- and the dark phase, however, it acted more obviously in the nol, whereas it did not exert such an influence in the dark light phase. Diazepam administered in the dark phase before phase (Fig. 4). ethanol resulted in stronger impairment of motor coordina- Diazepam alone decreased mouse body temperature in the tion than was observed after ethanol given alone (Fig. 2). range from 1.4 C to 0.4 C in a similar way in both light and dark phases. 2.3. Spontaneous locomotor activity Ethanol injected at a dose of 2 g Á kgÀ1 increased sponta- 3. Discussion neous locomotor activity in mice both in the light and the The present study has shown that some actions of ethanol dark phase. Diazepam alone clearly decreased mouse are associated with circadian rhythms. The sleeping action spontaneous locomotor activity in the dark. Diazepam ad- has been found to be stronger in the dark than in the light ministered before ethanol significantly decreased mouse phase, however, during the light phase ethanol impairs lo- spontaneous locomotor activity. This activity was apparent comotor coordination to a greater extent and its hypother- in both the light and dark phases (Fig. 3). mic action is more evident. Ethanol affects different neurotransmitters, specific ion 2.4. Ethanol induced hypothermia channel and receptors. It enhances GABA-ergic inhibition increasing the action of g-aminobutyric acid (GABA) on Ethanol administered at a dose of 2.5 g kgÀ1 produced a Á GABA-A receptors (similarly to benzodiazepines) (Korpi marked decrease in mouse body temperature. Mean hy- 1994). This mechanism of action leads to intensification pothermia at 30 min following administration was 4.3 C of the CNS depression produced by ethanol and by benzo- and at 120 min 3 C below the baseline value in the light diazepines. Benzodiazepines give a marked enhancement phase, and in the dark phase 3.2 and 1.2 C, respectively of ethanol induced motor impairment and increased etha- nol sleep time in animals (Vanover et al. 1999; Voss et al. 2003). The chronobiological aspect of ethanol action has a dou- 90 Light Dark ble signifance. The first is the influence of single-dose b 75 b ethanol taken at different times of day and night. The sec- ond problem concerns the effect of chronically adminis- 60 tered ethanol on biological rhythms. c c Investigations on the chronobiological effect of ethanol ac- 45 b tion have been mostly performed in animals and more rarely in humans. Reinberg (1992) defined circadian motility time [%] 30 b changes in the psychological results of ethanol action. a,b,d They were the greatest at 23:00 h and the least at 11:00 h, 15 a,b,d a,b,d a,b,d however, blood pressure values were not associated with 30 min 0 day and night rhythms. Yap et al. (1993) determined ethan- Et Et+Dz Dz Sal Et Et+Dz Dz Sal 60 min ol concentration in blood and air breathed out by 10 healthy volunteers. Ethanol reached its highest blood con- Fig. 3: The influence of diazepam and ethanol on motility in mice. Et – centration at 9:00 h. À1 À1 ethanol (2.0 g kg ), Dz –– diazepam 0.5 mg kg , Sal –– saline Experiments on rats revealed that the pharmacokinetics of (0.9% NaCl). Results are presented as means Æ SEM, a p < 0.05 versus ethanol group, b p < 0.05 versus saline group, c p < 0.05 low but not high ethanol doses are associated with the light versus dark, d p < 0.05 versus diazepam group circadian rhythm. Maximum elimination was observed in 762 Pharmazie 60 (2005) 10 ORIGINAL ARTICLES the dark, i.e. the rats’ active phase (Piekoszewski 1997). It 4. Experimental was also stated that the highest activity of alcohol dehy- 4.1. Animals and treatment drogenase and hepatic microsomal enzymes (MEOS) oc- Treatment experiments were carried out on BALBc male mice (28–35 g). curs just at the time of highest activity of animals (Sturte- The mice were housed in group cages under normal laboratory conditions: vant and Garber 1980). Williams et al. (1993) evaluated temperature of 20–21 C, light/dark cycle (light phase 6.00–18.00, dark the development of tolerance to the hypothermic activity phase 18.00–6.00).