Neuropharmacology 85 (2014) 36e44

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Induction of brain cytochrome P450 2E1 boosts the locomotorstimulating effects of ethanol in mice Juan Carlos Ledesma a, Marta Miquel a, María Pascual b, Consuelo Guerri b, Carlos M.G. Aragon a, * a b



n, Spain Area de Psicobiologia, Universitat Jaume I, Avda. Vicente Sos Baynat s/n, 12071 Castello
n Príncipe Felipe, Departamento de Patología Celular, C/ Eduardo Primo Yúfera 3, 46012 Valencia, Spain Centro de Investigacio

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 December 2013 Received in revised form 14 April 2014 Accepted 13 May 2014 Available online 24 May 2014

In the central nervous system ethanol (EtOH) is metabolized into acetaldehyde by different enzymes. Brain catalase accounts for 60% of the total production of EtOH-derived acetaldehyde, whereas cerebral cytochrome P450 2E1 (CYP 2E1) produces 20% of this metabolite. Acetaldehyde formed by the activity of central catalase has been implicated in some of the neurobehavioral properties of EtOH, yet the contribution of CYP 2E1 to the pharmacological actions of this drug has not been investigated. Here we assessed the possible participation of CYP 2E1 in the behavioral effects of EtOH. Thus, we induced CYP 2E1 activity and expression by exposing mice to chronic acetone intake (1% v/v for 10 days) and examined its consequences on the stimulating and uncoordinating effects of EtOH (0e3.2 g/kg) injected intraperitoneally. Our data showed that 24 h after withdrawal of acetone brain expression and activity of CYP 2E1 was induced. Furthermore, the locomotion produced by EtOH was boosted over the same interval of time. Locomotor stimulation produced by amphetamine or tert-butanol was unchanged by previous treatment with acetone. EtOH-induced motor impairment as evaluated in a Rota-Rod apparatus was unaffected by the preceding exposure to acetone. These results indicate that cerebral CYP 2E1 activity could contribute to the locomotor-stimulating effects of EtOH, and therefore we suggest that centrally produced acetaldehyde might be a possible mediator of some EtOH-induced pharmacological effects. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Ethanol Cytochrome P450 2E1 Locomotor stimulation Catalase-H2O2 system Acetaldehyde Mice

1. Introduction Acetaldehyde, the first product formed after the metabolism of ethyl alcohol (ethanol, EtOH) is a highly reactive molecule with psychoactive properties, and it has been shown to mimic a broad range of behavioral and neurochemical effects induced by EtOH in rodents when it is administered centrally (Rodd-Henricks et al., 2002; Foddai et al., 2004; Arizzi-LaFrance et al., 2006; Diana
n et al., 2009; Sirca et al., 2008; Enrico et al., 2009; S
anchez-Catala et al., 2011). For this reason, it has been suggested that this metabolite could participate in the mediation of some of the psychopharmacological actions of EtOH. Like the rest of the psychoactive drugs, acetaldehyde needs to interact with the central nervous system (CNS) to elicit its neurobehavioral actions. Nevertheless, under normal physiological conditions EtOH-derived

* Corresponding author. Tel.: þ34 964 729835; fax: þ34 964 729867. E-mail addresses: [email protected], [email protected] (C.M.G. Aragon). http://dx.doi.org/10.1016/j.neuropharm.2014.05.018 0028-3908/© 2014 Elsevier Ltd. All rights reserved.

acetaldehyde formed in the liver reaches the brain with difficulty because it is rapidly converted into acetic acid by the activity of hepatic aldehyde dehydrogenase (ALDH), and residual acetaldehyde that escapes this enzyme into the bloodstream is eliminated by the metabolic activity presented by ALDH located in the brainblood barrier (Hunt, 1996; Zimatkin and Deitrich, 1997). Thus, it was firstly concluded that only very low acetaldehyde concentrations may be achieved within the brain under a situation of normal alcohol consumption, and it was therefore postulated that this metabolite is unlikely to play a significant role in the psychopharmacological effects of EtOH (Lindros and Hillbom, 1979; Petersen and Tabakoff, 1979; Stowell et al., 1980). Nonetheless, in the brain there exist the same enzymatic pathways that metabolize EtOH into acetaldehyde than in the liver, but most of its production (about 60% of the total amount of acetaldehyde formed) occurs through the activity of the catalase-H2O2 system (Compound I) (Zimatkin et al., 2006). Thus, it has been demonstrated that brain homogenates and neural tissue cultures incubated with EtOH are able to produce acetaldehyde, and brain

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homogenates of rats previously treated with catalase inhibitors have a significantly lower acetaldehyde formation than non-treated rats (Aragon et al., 1992b; Gill et al., 1992; Hamby-Mason et al., 1997). Moreover, the addition of H2O2 generating agents to the same in vitro preparations causes an increment in the activity of Compound I and results in an enhanced production of this metabolite (Aragon et al., 1992a; Gill et al., 1992). Furthermore, it has also been shown that treatments that impaired brain catalase activity reduced the amount of acetaldehyde detected in vivo as measured by microdialysis in the striatum of freely moving rats injected intraperitoneally with EtOH (Jamal et al., 2007). In behavioral studies, it has been observed that the same manipulations that modulate brain EtOH metabolism by the activity of Compound I modify a large number of EtOH-related behaviors in rodents (Koechling and Amit, 1994; Escarabajal et al., 2000; Correa et al., 2001b; Manrique et al., 2005; Quintanilla et al., 2012). Besides, it has also been proven that administration of acetaldehyde sequestering agents block many EtOH-induced outcomes (Font et al., 2005, 2006a, 2006b; Martí-Prats et al., 2010; Peana et al., 2010; Pautassi et al., 2010). As a result, it has been extensively proposed that centrally formed acetaldehyde plays a key role in the neurobehavioral effects of alcohol (Hunt, 1996; Smith et al., 1997;
lito et al., 2007; Deitrich, 2004; Quertemont et al., 2005; Hipo Deng and Deitrich, 2008). In addition to Compound I, the P450 2E1 subfamily of microsomal cytochromes (CYP 2E1) also metabolizes EtOH within the brain. Thus, it has been shown that the CYP 2E1 inhibitors diallyl sulfide (2 mM) and beta-phenethyl isothiocyanate (0.1 mM) significantly lowered (20% approximately) the accumulation of EtOH-derived acetaldehyde in rat brain homogenates preincubated with these substances (Zimatkin et al., 2006). However, data assessing the possible role of CYP 2E1 in the behavioral effects of EtOH are scarce. To our knowledge, there are only two reports that evaluate the role of this enzyme in EtOH-elicited behaviors in alcohol-naïve mice. A study by Vasiliou and colleagues (Vasiliou et al., 2006) demonstrated that transgenic CYP 2E1/ and double mutant CYP 2E1//Cs/Cs knockout (KO) mice exhibit a longer sleep time evoked by EtOH when compared with their respective wildtype counterparts. On the other hand, Correa et al. (2009) showed that both EtOH-induced locomotor stimulation and sensitization, and also voluntary alcohol intake were not substantially different between CYP 2E1 KO mice and their control mates. The failure to find any differences in EtOH-induced behaviors between CYP 2E1 KO and normal mice in the latter study could be due to the fact that, given that CYP 2E1 only generates above 20% of the acetaldehyde that is formed totally in the CNS (Zimatkin et al., 1998, 2006), the hypothetically lower (about 20%) production of acetaldehyde in CYP 2E1 KO mice would not be enough to observe significant differences in EtOH-elicited behaviors when compared to wild-type subjects. The activity and expression of CYP 2E1 could be enhanced sixfold by chronic alcohol ingestion or by the administration of different compounds such as solvents, ketones, anesthetics, and muscle relaxants in both rats and humans (Anandatheerthavarada et al., 1993; Tindberg and Ingelman-Sundberg, 1996; Lieber, 1997;
nchez-Catala
n et al., 2008). Hence, it may be Yadav et al., 2006; Sa possible that under some circumstances cerebral CYP 2E1 could make a greater contribution to the metabolism of EtOH, such as by pharmacologically increasing its activity. An enhanced activity of brain CPY 2E1 would augment the formation of acetaldehyde after EtOH administration, which in turn could have some behavioral consequences. Therefore, given the lack of psychopharmacological studies assessing the role of this enzyme in the behavioral effects of alcohol, the aim of the present research was to investigate the involvement of CYP 2E1 in acute EtOH-induced locomotion. For

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that purpose, we used a strategy to increase the expression and activity of this isoenzyme in the brain, which consists in exposing animals to chronic acetone ingestion (1% v/v, 10 days) (Yadav et al., 2006). We decided to use acetone to increase CYP 2E1 activity/ expression in the present study instead of chronic EtOH intake in order to avoid related neuroadaptations that can be found after a repeated treatment with alcohol, such as sensitization or tolerance, which could alter the acute drug-induced behavioral effects of EtOH and therefore confound the effects of CYP 2E1 induction in these experiments. Parallel experiments were conducted to demonstrate that the expression and activity of CYP 2E1 are induced in the brain by acetone treatment. Our hypothesis predicted that the induction of CYP 2E1 by acetone will potentiates the locomotor-stimulating effects of EtOH, presumably by increasing the cerebral production of acetaldehyde. 2. Materials and methods 2.1. Subjects Four-week old male Swiss (IOPS Orl) albino mice were purchased from CERJJanvier (Barcelona, Spain). Mice were housed four per cage and were maintained in a humidity- (50%) and temperature-controlled (22 ± 1  C) environment on a reversed 12 h light/dark cycle with lights on at 1 p.m. All experimental procedures took place within the first 4 h of the light phase. Animals were acclimated to the colony room for at least 2 weeks prior to experimentation. All behavioral procedures began after handling the mice for one week. Food (Panlab S.L., Spain) and tap water were available ad libitum, except during the acetone-drinking period for subjects exposed to acetone. All experimental procedures followed the European Community Council Directive (86/609/ECC) for the use of laboratory animal subjects and Real Decreto 1201/2005. 2.2. Drugs and treatments Acetone was prepared for drinking in tap water at a concentration of 1% (v/v) and was offered to the experimental mice as their only fluid for 10 days. This treatment had been demonstrated to induce liver (Koop, 1986; Koop and Coon, 1986)
nchez-Catala
n et al., 2008; Hipo
lito and brain CYP 2E1 expression and activity (Sa et al., 2009). At the end of this period the bottles of acetone were removed and replaced with water bottles. The rest of the animals (control subjects) were always exposed to tap water. Both the daily volume of fluid ingested and the weight of the animals were measured, the results showing that the drinking and weight values were not significantly different between groups (7.2 ± 0.13 ml, 35.2 ± 0.29 g for the H2O control subjects; and 6.9 ± 0.15 ml, 35.05 ± 0.21 g for the acetone exposed individuals). Finally, 24, 48 or 76 h after acetone withdrawal animals were sacrificed by decapitation under anesthesia (ketamine 150 mg/kg; 3 ml from 1 ml/10 ml solution) for the biochemical assays or tested in the behavioral experiments. EtOH and tert-butanol (Panreac S.A., Spain) were diluted to 20% and 10% (v/v), respectively, in physiological saline (0.9% w/v; Sal) from a 96% (v/v) stock. EtOH was injected intraperitoneally (IP) at doses of 0.8, 1, 1.6, 2.4, or 3.2 g/kg, and tert-butanol was administered IP at a dose of 0.5 g/kg. Amphetamine was obtained from SigmaeAldrich Química S.A. (Spain) and injected IP at doses of 3 and 6 mg/kg. All drugs were freshly prepared in Sal. 2.3. Experiment 1: effect of chronic acetone intake on the expression of CYP 2E1 in the brain To test the effect of acetone ingestion on the expression of CYP 2E1 in the CNS, cerebral cortex and cerebellum tissue from animals (n ¼ 4 per group) exposed to acetone (1% v/v) or tap water during 10 days were homogenized in lysis buffer (phosphate-buffered saline containing 1% Nonidet P-40; 0.5% sodium deoxicholate; 0.1% sodium dodecyl sulfate, SDS; 1.25 mM phenylmethylsulfonyl fluoride, PMSF; 40 mM leupeptin; 10 g/ml aprotinin; and 1 mM sodium orthovanadate), centrifuged at 10,000g, and the supernatants were used for the Western blotting analysis, after protein determination with the Bio-Rad protein assay kit (Bio-Rad, Madrid, Spain). These samples from the supernatant were then mixed with an equal volume of SDS buffer (0.125 M TriseHC1, pH 6.8, 2% SDS, 0.5% (v/v) 2-mercaptoethanol, 1% bromophenol blue and 19% glycerol) and boiled for 5 min. Proteins were separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to a nitrocellulose membrane (Schleicher & Schuell, Barcelona, Spain). Membranes were blocked with 5% dried milk in TBS-T (Tris-buffered saline containing 0.05% Tween-20) and incubated overnight with the following antibodies: anti-CYP P450 2E1 (1:1000, Oxford BM, Michigan, USA) and anti-glyceraldehyde 3-phosphate dehydrogenase as the loading control (GAPDH; 1:5000, Chemicon, Hampshire, UK). Membranes were washed in TBS-T and then incubated for 1 h with alkaline phosphatase (AP)-conjugated goat anti-mouse IgG (1:1000). Proteins were visualized with alkaline phosphatase conjugate (Promega, Barcelona, Spain), and the intensity of the bands was quantified with SigmaGel version 1.0 image analysis software (Jandel Scientific,

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Madrid, Spain). For the statistical analyses, after the densitometric determination of the immunoreactive bands, the values of the expression of CYP 2E1 obtained in both brain areas of mice exposed to acetone measured at different time intervals (24, 48 or 72 h) after acetone withdrawal, were converted as a percentage with respect to the CYP 2E1 expression displayed by the brains of control mice (subjects exposed to tap water). 2.4. Experiment 2: effect of chronic acetone intake on the activity of brain CYP 2E1 To evaluate the effect of acetone on the activity of cerebral CYP 2E1, we quantified the hydroxylation of p-nitrophenol to 4-nitrocathecol in the brain of mice (n ¼ 9 per group) exposed to water or acetone (1% v/v) ingestion for 10 days after 24, 48 or 72 h of acetone withdrawal. Because p-nitrophenol is a specific substrate for CYP 2E1, which is converted into 4-nitrocathecol mainly by the catalytic activity of this enzyme, this reaction could be used as an indirect measure of the activity of CYP 2E1 (Koop, 1986, 1992; Koop et al., 1989; Montoliu et al., 1995; Zerilli et al., 1997, € fgren et al., 2004). Thus, 1998; Kobayashi et al., 2002; Mikstacka et al., 2002; Lo animals were anesthetized with ketamine and perfused transcardially with ice-cold Tris buffer (100 mM, pH 7.4) prior to decapitation. Brains were removed and immediately stored at 80  C until microsomal fractions were prepared. Following the procedure described by Montoliu et al. (1995), for the preparation of the microsomal fraction the brains were pooled and homogenized in 9% volume of icecold 100 mM Tris HCL buffer, pH 7.4, with 1.15% w/v KCL, 0.2 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM dithiothreitol and 10% glycerol. The hydroxylation of p-nitrophenol to 4nitrocathecol was analyzed in a reaction mixture containing 100 mM of potassium phosphate buffer, pH 6.8, 100 mM of p-nitrophenol, 500 ml of the brain microsomal fraction from the whole brain and 1 mM of nicotinamide adenine dinucleotide phosphate (NADPH) for a final volume of 1 ml. The reaction was initiated with NADPH after preincubation for 3 min at 30  C and was terminated after 6 min with 0.5 of prechloric acid. 4-Nitrocathecol was determined spectrophotometrically in 1 ml of supernatant at 546 nm. As 4-nitrocathecol is a highly photosensitive compound, all assays were performed in a dark room. Protein levels were determined from 10 ml of the brain microsomal fraction in accordance with the method described by Bradford (1976). 2.5. General behavioral procedures Behavioral testing was always conducted in sound-attenuated rooms with dim illumination (20 W; regular white light). To assess the effect of acetone intake on the locomotor stimulation induced by EtOH, amphetamine, and tert-butanol animals were tested in open-field chambers that consisted of a clear glass cylinder 25 cm in diameter and 30 cm high. Locomotor activity was registered in the open-field apparatus by a computerized video-tracking system (SMART; Spontaneous Motor Activity Recording & Tracking, Letica S.A., Spain). Movement of the mouse inside the open-field chambers was registered and translated automatically by the SMART software to horizontal distance traveled in cm during 20 min. However, for the statistical analyses only the last 10 min were considered. This time interval was chosen to minimize a putative masking effect of our treatments provoked by unspecific activation derived from handling, the peritoneal irritation of an IP injection of EtOH, the exposure to a novel environment, and also to ensure the absorption and distribution of alcohol into the CNS after its IP injection (Kelley, 1993; Dudek and Tritto, 1994; Yim and Gonzales, 2000; Quertemont et al., 2003). Motor impairment produced by an EtOH injection was tested on a Rota-Rod apparatus (Ugo Basile, Comerio, Italy). First, mice were injected with Sal and trained in the Rota-Rod apparatus for one trial. After the first trial each mouse was immediately injected with Sal or EtOH (1 g/kg) and 10 min later its performance in the Rota-Rod was assessed and registered as time running on the wheel at a fixed speed of 40 rpm for another trial. 2.5.1. Experiment 3: effect of several doses of EtOH on locomotion after chronic acetone intake This study assessed the consequences of acetone (1% v/v) ingestion for 10 days on the locomotor activity of mice treated with different doses of EtOH. Thus, 24 h after withdrawing acetone, animals (n ¼ 12e17 per group) were injected with EtOH (0, 0.8, 1.6, 2.4 and 3.2 g/kg) just before placing them in the open field, where their horizontal locomotion was measured for 20 min. 2.5.2. Experiment 4: effect of chronic acetone intake on EtOH-induced locomotor stimulation measured at different temporal intervals In order to investigate the time pattern of the effect of drinking acetone (1% v/v) for 10 days on the locomotor stimulation produced by an EtOH challenge, in the present experiment, 24, 48 or 72 h following acetone withdrawal, animals (n ¼ 11e14 per group) were injected with EtOH (2.4 g/kg) and immediately afterward their locomotion was measured in the open field. 2.5.3. Experiment 5: effect of chronic acetone intake on the locomotor stimulation induced by amphetamine and tert-butanol The present experiment analyzed whether the effect of 10 days of acetone (1% v/ v) consumption is selective in modulating EtOH-induced locomotion, or is shared by

other drugs that are also able to increase locomotion. For this reason, we explored here the consequence of this treatment on the locomotor-stimulating actions of amphetamine and tert-butanol. The rationale for the use of tert-butanol in addition to amphetamine in this experiment was as follows: Tert-butanol, as occurs with acute EtOH injection, is also able to increase locomotion in mice (Sanchis-Segura et al., 2004). However, tert-butanol is a tertiary alcohol (branched chain alcohol) that is not converted into acetaldehyde by the activity of CYP 2E1. It is largely metabolized by oxidation via 2emethyl-1,2-propanediol into 2-hydroxyisobutyrate, and also into acetic acid at high doses (McGregor, 2010). For this reason, to rule out an unspecific effect of the induction of CYP 2E1 on the locomotor-stimulating properties of other alcohols, in this experiment we treated animals with tertbutanol in order to test whether chronic acetone intake could modulate the psycho-locomotor effects of this substance. Hence, following 24 h of acetone withdrawal, subjects (n ¼ 8e12 per group) were injected with amphetamine (0, 3 or 6 mg/kg; experiment 5a) or tert-butanol (0 or 0.5 g/kg; experiment 5b) and tested as in the rest of the behavioral experiments. 2.5.4. Experiment 6: effect of chronic acetone intake on the motor impairment induced by EtOH This experiment was performed to rule out the possibility that the increase in the locomotor-stimulating effects of EtOH observed in acetone-drinking mice might be a consequence of a reduction in the ataxic properties of alcohol by this treatment. That is, it could be possible that the enhancement of CYP 2E1 activity by acetone consumption maybe potentiates EtOH-induced locomotion by reducing its uncoordinating effects. Thus, in an attempt to confirm this possibility we evaluated whether acetone (1% v/v) exposure for 10 days affected the motor impairment elicited by an acute injection of EtOH (1 g/kg) in mice (n ¼ 6 per group) tested in the Rota-Rod apparatus 24 h after acetone withdrawal, as described above (see Section 2.5). We administered 1 g/kg because there are no reports using lower doses of alcohol in this behavioral protocol in mice. Moreover, pilot studies from our laboratory revealed that the smallest dose of this drug that is able to alter performance in ~o this paradigm in mice is close to 1 g/kg (unpublished data, please also see Balin et al., 2012). 2.6. Experiment 7: effect of chronic acetone intake on blood EtOH levels Given that alcohol is absorbed within the first 30 min after its IP injection in
n et al., 2003), the current mice (Bonthius et al., 2002; Livy et al., 2003; Tarrago experiment was performed to rule out the possibility that acetone (1% v/v) consumption during 10 days could produce any change in the absorption of EtOH. For that purpose, additional mice were used to investigate whether this treatment influenced blood alcohol levels under our experimental conditions. Twenty-four hours after the removal of acetone, animals (n ¼ 6 per group) were injected with EtOH (2.4 g/kg) and 15 or 30 min later trunk blood samples were collected to measure plasma EtOH concentrations using the Alcohol Diagnostic Kit from Sigma Aldrich Química (Spain). 2.7. Statistical analyses One- or two-way ANOVAs following post-hoc comparisons with Duncan's test were applied when appropriate. The alpha level was set at p < 0.05 for all analyses. The Statistica 6.1 (StatSoft, Tulsa, OK) software package was used.

3. Results 3.1. Experiment 1: effect of chronic acetone intake on the expression of CYP 2E1 in the brain Fig. 1 shows the expression of CYP 2E1 of the cerebral cortex (Fig. 1a) and cerebellum (Fig. 1b) of mice exposed to tap water or acetone (1% v/v, for 10 days) measured after different time intervals following acetone withdrawal. Western blotting analyses of lysates were used to quantify the levels of this enzyme, which results in a single band of z50 kDa (Montoliu et al., 1995). Results from a oneway ANOVA indicated significant differences between groups (control, 24, 48 and 72 h post-acetone withdrawal) in the expression of CYP 2E1 in both the cerebral cortex [F (3, 12) ¼ 30.22; p < 0.01] and the cerebellum [F (3, 12) ¼ 11.75; p < 0.01]. Post-hoc Duncan's comparisons revealed that 10 days of acetone (1% v/v) consumption as the only fluid ingested produced a clear induction of CYP 2E1 expression when measured 24 h after acetone withdrawal in both the cerebral cortex and the cerebellum compared to H2O-drinking control mice (p < 0.01). In contrast, this enhancement in the expression of CYP 2E1 was not found following 48 and 72 h of acetone withdrawal, because the expression of this enzyme in the

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Fig. 2. Effect of chronic acetone intake on the activity of brain CYP 2E1. The activity of CYP 2E1 was determined by the hydroxylation of p-nitrophenol to 4-nitrocathecol (see Section 2.4) in H2O-drinking control subjects or 24, 48 and 72 h following acetone withdrawal in mice exposed to acetone (1% v/v) as the only fluid for 10 days. Bars represent mean ± SEM of nmol of 4-nitrocathecol formed per minute in mg of protein for each experimental group (**p < 0.01 significantly different from the H2O-24 h group; ##p < 0.01 significantly different from the Acetone-24 h group).

differ from the activity of the H2O-drinking control groups (p > 0.05) when it was measured at these time intervals, and it was significantly lower than the levels found 24 h after withdrawing acetone (p < 0.01). 3.3. Experiment 3: effect of several doses of EtOH on locomotion after chronic acetone intake

Fig. 1. Effect of chronic acetone intake on the expression of CYP 2E1 in the brain. The expression of cerebral CYP 2E1 was quantified after H2O consumption (Ctrl) or 24, 48 and 72 h after the withdrawal of acetone in the cerebral cortex (Fig. 1a) and the cerebellum (Fig. 1b) of mice exposed to acetone (1% v/v) as the only drinking fluid for 10 days. Bars depict mean ± SEM of the densitometric values of the immunoreactive bands for each experimental group (Ctrl, 24 h, 48 h and 72 h) (**p < 0.01 significantly different from its corresponding Ctrl group). At the top of the figure a representative Western blot is presented for each experiment.

cerebral cortex and the cerebellum 48 and 72 h after the withdrawal of acetone did not differ from that of the H2O-drinking control subjects in the two structures (p > 0.05).

Fig. 3 shows the locomotor activity of mice exposed to H2O or acetone (1% v/v, for 10 days) and injected with different doses of EtOH 24 h after withdrawing acetone. The results of a two-way ANOVA of treatment (H2O or acetone)  EtOH dose (0, 0.8, 1.6, 2.4 and 3.2 g/kg) showed a significant effect for the treatment factor [F(1, 133) ¼ 22.3, p < 0.01], for the EtOH dose factor [F(4, 133) ¼ 29.11, p < 0.01], and for their interaction [F(4, 133) ¼ 5.27, p < 0.01]. Pairwise comparisons demonstrated that in the H2O control treatment group the dose of 2.4 g/kg of EtOH (H2OeEtOH 2.4 g/kg group) produced a significant induction of locomotion compared to the H2OeEtOH 0 g/kg group (p < 0.01). Furthermore, the locomotor stimulation induced by the dose of 2.4 g/kg of EtOH was significantly boosted when animals were previously exposed to 10 days of acetone (1% v/v) ingestion, as can be seen when the

3.2. Experiment 2: effect of chronic acetone intake on the activity of brain CYP 2E1 Fig. 2 displays the activity of cerebral CYP 2E1 24, 48 and 72 h after acetone withdrawal, as measured by the hydroxylation of pnitrophenol to 4-nitrocathecol in microsomes from the whole brain of mice exposed to tap water or acetone (1% v/v, for 10 days). A twoway ANOVA treatment (acetone or H2O)  time (24, 48 and 72 h after acetone withdrawal) shows a significant effect for the treatment factor [F(1, 48) ¼ 6.22; p < 0.05], for the time factor [F(2, 48) ¼ 3.7; p < 0.05], and a significant interaction was found between these two factors [F(2, 48) ¼ 5.09; p < 0.01]. Further analyses with Duncan's post-hoc test revealed that the activity of CYP 2E1 in animals exposed to acetone was modified in a time-dependent manner. Thus, the activity of this enzyme was significantly higher at 24 h after the withdrawal of acetone when compared to mice exposed to H2O (p < 0.01). Moreover, CYP 2E1 activity returned to basal levels 48 and 72 h after acetone withdrawal, because it did not

Fig. 3. Effect of several doses of EtOH on locomotion after chronic acetone intake. EtOH (0, 0.8, 1.6, 2.4, and 3.2 g/kg; IP) was injected 24 h after acetone withdrawal in mice exposed to H2O or acetone (1% v/v) as the only drinking fluid for 10 days. Bars depict mean ± SEM locomotor activity (cm/10 min) for all groups (**p < 0.01, significantly different from H2OeEtOH 0 g/kg group; ##p < 0.01 significantly different from H2OeEtOH 2.4 g/kg control group).

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AcetoneeEtOH 2.4 g/kg group is compared with the H2OeEtOH 2.4 g/kg group (p < 0.01). In addition, mice exposed to acetone and treated with a 1.6 g/kg dose of EtOH (AcetoneeEtOH 1.6 g/kg group) also displayed an induction of their locomotion when compared to the H2OeEtOH 2.4 g/kg group (p < 0.01). 3.4. Experiment 4: effect of chronic acetone intake on EtOH-induced locomotor stimulation measured at different temporal intervals Fig. 4 depicts the effect of acetone (1% v/v, for 10 days) on the locomotor stimulation elicited by EtOH (2.4 g/kg) administered 24, 48 or 72 h after acetone withdrawal. A two-way ANOVA treatment (H2O or acetone)  time (24, 48 and 72 h after acetone withdrawal) revealed a significant effect for the treatment factor [F(1, 72) ¼ 8.66, p < 0.01], a significant effect for the time factor [F(2, 72) ¼ 4.3, p < 0.05], and an interaction between these two factors [F(2, 72) ¼ 3.92, p < 0.05]. Duncan's post-hoc comparisons showed that, as occurs in experiment 3, the locomotor stimulation induced by the dose of 2.4 g/kg of EtOH is enhanced 24 h after acetone withdrawal, when the Acetone-24 h group is compared to its corresponding H2O control group (p < 0.01). This effect clearly disappeared 48 h after acetone withdrawal, because the locomotor stimulation caused by this dose of EtOH was not altered when comparing the Acetone-48 and 72 h groups with the H2O treated subjects (p > 0.05). Moreover, the locomotor activity of mice in the Acetone-48 and 72 h groups was significantly lower than that of the Acetone-24 h group, thus confirming that the enhancement of EtOH-induced locomotion only occurs 24 h after acetone withdrawal (p < 0.01). 3.5. Experiment 5: effect of chronic acetone intake on the locomotor stimulation induced by amphetamine and tert-butanol Fig. 5 presents the effect of 10 days of acetone (1% v/v) drinking on the locomotor stimulation caused by amphetamine (3 and 6 mg/ kg; Fig. 5a), and tert-butanol (0.5 g/kg; Fig. 5b) administered 24 h after its withdrawal. A two-way ANOVA treatment (H2O or acetone)  amphetamine (0, 3 and 6 mg/kg; experiment 5a) or tertbutanol (0 and 0.5 g/kg; experiment 5b) showed a significant effect for the amphetamine factor [F(2, 53) ¼ 16.89, p < 0.01], and a significant effect for the tert-butanol factor [F(1, 42) ¼ 4.58, p < 0.05]. No significant effect for the treatment factor [F(1, 53) ¼ 0.86, p > 0.05], experiment 5a; and [F(1, 42) ¼ 1.09, p > 0.05], experiment 5b; or a significant interaction between treatment and

Fig. 5. Effect of chronic acetone intake on the locomotor stimulation induced by amphetamine and tert-butanol. Amphetamine (0, 3 and 6 mg/kg; Fig. 5a) and tertbutanol (0 and 0.5 g/kg; Fig. 5b) were injected after H2O exposure or 24 h after withdrawing acetone in mice exposed to acetone (1% v/v) as the only drinking fluid for 10 days. The locomotor stimulation produced by amphetamine (Fig. 5a) and tertbutanol (Fig. 5b) was unchanged by previous consumption of acetone (p > 0.05).

amphetamine [F(2, 53) ¼ 2.24, p > 0.05] or tert-butanol [F(1, 42) ¼ 0.01, p > 0.05] factors were found. Thus, the locomotor activity induced by these drugs was not changed by prior exposure to acetone in the present study. 3.6. Experiment 6: effect of chronic acetone intake on the motor impairment induced by EtOH Table 1 shows the effect of acetone exposure (1% v/v, for 10 days) on the time running on the wheel of the Rota-Rod apparatus in mice under Sal or EtOH (1 g/kg) injections, 24 h after acetone withdrawal. A two-way ANOVA treatment (H2O or acetone)  EtOH dose (0 or 1 g/kg) only found a significant effect for the EtOH dose factor [F(1, 19) ¼ 108.02, p < 0.01]. Therefore, acetone treatment did not change the motor impairment evoked by this dose of EtOH in this paradigm.

Fig. 4. Effect of chronic acetone intake on EtOH-induced locomotor stimulation measured at different temporal intervals. EtOH (2.4 g/kg) was injected after H2O exposure or 24, 48 and 72 h after acetone withdrawal in mice exposed to acetone (1% v/v) as the only drinking fluid for 10 days. Bars depict mean ± SEM locomotor activity (cm/10 min) for all groups (**p < 0.01, significantly different from H2O-24 h group; ## p < 0.01, significantly different from the Acetone-24 h group).

3.7. Experiment 7: effect of chronic acetone intake on blood EtOH levels As can be seen in Table 2, acetone consumption (1% v/v, for 10 days) did not alter blood alcohol concentrations in mice injected with the dose of 2.4 g/kg of EtOH following 24 h of acetone

J.C. Ledesma et al. / Neuropharmacology 85 (2014) 36e44 Table 1 Effect of chronic acetone intake on the motor impairment induced by EtOH. Animals (n ¼ 6 per group) exposed to H2O drinking or acetone (1% v/v) for 10 days were placed on the wheel of the Rota-Rod apparatus at a fixed speed of 40 rpm 24 h after acetone withdrawal, and their performance (mean ± SEM of seconds running on the wheel) was registered following saline or EtOH (1 g/kg) injections. Time on wheel (seconds) at a Fixed speed of 40 rpm

Saline EtOH (1 g/kg)

Tap water

Acetone (1% v/v)

254.16 ± 45.4 3.16 ± 0.65

283.50 ± 16.5 2.50 ± 0.34

withdrawal. This conclusion was reported by a two-way ANOVA treatment (H2O or acetone)  time after EtOH injection (15 or 30 min) which failed to reach statistical significance for the treatment factor [F(1, 20) ¼ 1.98, p > 0.05], for the time factor [F(1, 20) ¼ 0.91, p > 0.05] or for the interaction between these two factors [F(1, 20) ¼ 0.76, p > 0.05]. 4. Discussion In the current investigation, we showed that mice exposed to acetone (1% v/v) intake as the only drinking fluid for 10 days exhibited an increment in the expression of CYP 2E1 in the cerebral cortex and the cerebellum when compared to control subjects without a history of acetone ingestion. This treatment also resulted in a potentiation of CYP 2E1 activity, as can be seen in the brain homogenates of animals previously exposed to acetone. These findings are in agreement with previous reports, which show that the expression and activity of this enzyme could also be augmented after several days of acetone consumption (Koop, 1992; Yadav et al.,
nchez-Catala
n et al., 2008; Hipo
lito et al., 2009). Moreover, 2006; Sa we demonstrated that CYP 2E1 expression and activity is enhanced for 24 h, and returns to basal levels 48 h after acetone withdrawal, thereby suggesting that this treatment did not induce any permanent alteration in the functioning of this isoenzyme under our experimental conditions. CYP 2E1 is an enzyme which catalyzes the metabolism and activation of xenobiotics, such as low molecular weight solvents, procarcinogens, drugs and environmental chemicals, and endobiotics, such as fatty acids and ketones (Raucy, 1995; Lieber, 1997). Under normal conditions, this enzyme is degraded in two phases, the first having a short half-life of 7 h and the second with a longer half-life of 37 h. The induction of CYP 2E1 has been demonstrated by the administration of different substrates that are metabolized by its activity, such as EtOH, imidazole, 4-methylpyrazole and acetone, among others. The regulation of CYP 2E1 expression is complex, involving transcriptional and post-transcriptional events, depending on the inductor involved in such a procedure (Porter and Coon, 1991; Lieber, 1999). In the case of acetone, it has been shown that animals exposed to this substance for several days displayed an increased expression of this enzyme by a post-transcriptional mechanism consisting of protein stabilization through a ligand-

Table 2 Effect of chronic acetone intake on blood EtOH levels. Mean ± SEM of blood EtOH concentrations (mg/dl) after an acute IP injection of EtOH (2.4 g/kg) to mice (n ¼ 6 per group) exposed to H2O- or acetone-drinking (1% v/v, for 10 days) 24 h after withdrawing acetone. Blood EtOH levels (mg/dl)

Tap water Acetone

15 min after EtOH injection

30 min after EtOH injection

242.1 ± 5.10 237.5 ± 23.3

190.3 ± 17.8 207.9 ± 19.3

41

mediated protection against its phosphorylation. That is, because acetone is a substrate for CYP 2E1, its chronic intake abolishes the rapid phase (7 h) of CYP 2E1 degradation and stabilizes the slower phase, which occurs at 37 h (Song et al., 1989; Song and Cederbaum, 1996). Hence, previous reports indicated that the induction of CYP 2E1 in the livers of rats previously exposed to chronic acetone treatment (5% v/v, for 10 days) persists for 37 h (Song et al., 1989). In our experiments, we showed that the rise in CYP 2E1 expression and activity was detected 24 h after withdrawing acetone but not at 48 or 72 h following its withdrawal. This is in accordance with previous works in which an induction of this enzyme in the brain was also displayed after chronic acetone treatment (Yadav et al.,
n et al., 2008; Hipo
lito et al., 2009), but 2006; S
anchez-Catala additionally we have demonstrated here, for the first time, that this cerebral induction of CYP 2E1 in mice is time-dependent, occurring in a similar time pattern as appears in other tissues (i.e., with a duration close to 37 h). Thus, it seems that the mechanism by which this enzyme is regulated in the CNS is the same as in the rest of the organism, and suggests that the brain is able to control the metabolism of different xenobiotics directly via CYP 2E1 activity. EtOH, like acetone, is also a substrate for CYP 2E1, and when it is administered chronically it could increase CYP 2E1 expression and activity in different tissues, including the CNS, in a mechanism that appears to be identical to that mediated by acetone (Eliasson et al., 1988; Roberts et al., 1995; Song and Cederbaum, 1996). Earlier investigations showed that both compounds are able to increase the expression of this enzyme in different areas of the mesolimbic
nchez-Catala
n et al., 2008; Hipo
lito et al., 2009). In the system (Sa present study, in order to evaluate whether the induction of this enzyme by acetone is similar to that produced by alcohol, we investigated whether this treatment is able to augment CYP 2E1 in the cerebral cortex and the cerebellum, because previous data has demonstrated that chronic EtOH administration increases CYP 2E1 levels in these neuroanatomical regions (Warner and Gustafsson, 1994; Howard et al., 2003; Yadav et al., 2006). Our results reveal that acetone intake enhances brain CYP 2E1 expression in both areas. Therefore, we suggest that the induction of CYP 2E1 activity by acetone could be a useful tool to study the consequences of chronic alcohol intake in some of the psychopharmacological actions of EtOH in alcohol-naïve mice. In the second set of studies, we found that acetone drinking (1% v/v, for 10 days) boosts the stimulatory effects of EtOH on locomotion in a dose- and time-dependent manner. That is, the locomotor stimulation induced by the dose of 2.4 g/kg of EtOH IP injected in mice previously exposed to acetone was significantly higher than in H2O-drinking control subjects, and this outcome was observed 24 h after acetone withdrawal but not at longer time intervals (48 or 72 h). Additionally, mice exposed to acetone and treated with a 1.6 g/kg dose of EtOH also showed an increment in their locomotor activity when compared to control animals. It is important to note that the time pattern of the enhancement of EtOH-induced locomotion observed in the behavioral experiments runs parallel to the increased CYP 2E1 expression/activity obtained in the biochemical reports, thereby indicating a possible functional link between these effects. Interestingly, acetone treatment did not affect spontaneous locomotion, as can be seen in the Acetonee EtOH 0 g/kg drug treatment group (see Experiment 3 and Fig. 3), thus ruling out an unspecific effect of this manipulation on locomotion. Moreover, the locomotor-stimulating effects promoted by amphetamine and tert-butanol were not altered by acetone in our experiments, which indicates that this treatment seems to be selective only to this effect of EtOH. Furthermore, acetone did not modulate the motor impairment triggered by EtOH (1 g/kg) in mice as evaluated in a Rota-Rod apparatus, thus suggesting that this treatment did not boost EtOH-induced locomotion by reducing the

42

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ataxic properties of alcohol. We used only one dose of alcohol (1 g/ kg) in the Rota-Rod experiment because we considered that if acetone did not modulate the performance of this task using the lowest dose of EtOH capable of affecting this behavior in mice (see Section 2.5.4), administration of higher doses of alcohol, which further its ataxic effects, would also be unaltered by acetone treatment. Lastly, it has also been demonstrated that blood alcohol concentrations were unaffected in animals injected with EtOH (2.4 g/kg) 24 h after acetone withdrawal when measured at similar time intervals as in the behavioral reports. Hence, it seems that acetone did not modulate EtOH absorption, at least at the times and doses tested in the present research. As pointed out in the Section 1, CYP 2E1 contributes to 20% of the total metabolism of EtOH into acetaldehyde within the CNS in alcohol-naïve rats (Zimatkin et al., 1998, 2006). Numerous studies support the notion that the local metabolism of EtOH within the brain is important for the mediation of the locomotor-activating properties of this drug. In that sense, it has been widely established that the activity of Compound I, which is the main system of central EtOH metabolism (Aragon et al., 1992a,b; Gill et al., 1992; Hamby-Mason et al., 1997), positively correlates with EtOHinduced locomotor stimulation. Hence, mice treated with different brain catalase inhibitors exhibited a reduction in EtOHinduced locomotion in a dose-dependent way (Sanchis-Segura et al., 1999a,c; Correa et al., 1999b, 2001a; Escarabajal et al., 2000; Escarabajal and Aragon, 2002b). Moreover, acatalasemic mice, which are genetically deficient (50%) in the expression and activity of this enzyme, also displayed an attenuation of the locomotor stimulation elicited by an EtOH challenge when compared with their normal control mates (Aragon et al., 1992a; Aragon and Amit, 1993). In contrast, when the activity of this enzyme is pharmacologically potentiated giving different brain catalase inducers the locomotor-stimulating effects of EtOH are increased (Correa et al., 1999a, 2000, 2001a; Sanchis-Segura et al., 1999b). For this reason, it has been postulated that there exists a clear relationship between the cerebral production of EtOH-derived acetaldehyde by the activity of catalase and the locomotor-stimulating properties of this drug in mice. In agreement with this proposal, it has also been demonstrated that systemic administration of the acetaldehyde chelating agent D-penicillamine dose-dependently reduces EtOHinduced locomotion in both mice and rats (Font et al., 2005; Martí-Prats et al., 2010). Furthermore, concurrent administration of the inhibitor of the alcohol dehydrogenase I (the primary enzyme of peripheral EtOH metabolism) diethyldithiocarbamate with the ALDH inhibitor 4-methylpyrazole, which presumably leads to central acetaldehyde accumulation after EtOH dispensation, resulted in a potentiation of the EtOH-elicited locomotor activation in mice (Escarabajal and Aragon, 2002a). Although in the present work we have not made any attempt to measure central acetaldehyde levels in animals treated with acetone, the current findings could be interpreted as pointing toward the existence of a link between the cerebral production of acetaldehyde and the locomotor-stimulating properties of EtOH. That is, taking into account the significant increment in the expression and activity of CYP 2E1 obtained in our experiments after acetone dispensation, and given that it has been proven that this enzyme participates substantially in the formation of EtOHderived acetaldehyde (Zimatkin et al., 2006), it is reasonable to suggest that the brain of mice exposed to acetone treatment would have a higher rate of acetaldehyde generation after EtOH injection than control mice. Therefore, we tentatively propose that the enhancement in locomotor stimulation observed in acetone treated-mice after EtOH injection in our experiments may be the result of a higher rate of cerebral acetaldehyde formation by the activity of CYP 2E1.

Another possibility that should also be contemplated is that the potentiation of the locomotor activation produced by EtOH observed in mice with heightened CYP 2E1 activity might be a consequence of an increment in H2O2 levels. It is well established that H2O2 concentrations are a limiting factor for the peroxidative activity of catalase (Ou and Wolf, 1994; Zimatkin et al., 2006; Deng and Deitrich, 2008). That is, catalase needs the presence of H2O2 in the CNS to form Compound I (i.e., the catalase-H2O2 system) and metabolize EtOH (Aragon et al., 1992a,b; Gill et al., 1992). Therefore, the formation of EtOH-derived acetaldehyde by catalase is H2O2 dependent. The metabolism of different substrates for CYP 2E1 such as acetone, which induces its activity, boosts the formation of reactive oxygen species in organisms, especially H2O2 (Koop, 1992; Wu and Cederbaum, 1994; Buko and Sadovnichy, 1996; Song and Cederbaum, 1996; Caro and Cederbaum, 2004; Gonzalez, 2005; Shahabi et al., 2008). Recent evidence suggests a role for H2O2 in EtOH-induced locomotion. For example, it has been demonstrated that administration of H2O2 scavengers blocks EtOH-induced locomotor stimulation in mice (Ledesma and Aragon, 2012; Ledesma et al., 2012). On the other hand, it has been shown that treatments that heighten the rate of production of H2O2 in the CNS potentiate this behavioral outcome evoked by an acute injection of EtOH (Pastor et al., 2002; Manrique et al., 2006). This has been interpreted to mean that the availability of H2O2 to brain catalase determines the formation of Compound I, thereby limiting the production of EtOH-derived acetaldehyde by this enzyme system and consequently the effects of EtOH on locomotion. Thus, it would be possible that the induction of CYP 2E1 activity by acetone exposure found in our experiments could increase H2O2 levels and therefore the formation of acetaldehyde by Compound I, resulting in a higher locomotor stimulation after EtOH injection. The locomotor-activating properties of drugs of abuse such as EtOH have been widely investigated, because they are thought to involve a common brain circuitry shared by addictive drugs that involves the dopaminergic (DAergic) system, which is called the core reinforcement circuitry and is implicated in the processes that underlie drug addiction (Wise and Bozarth, 1987; Phillips and Shen, 1996; Gardner, 2011). Specifically, the effect of EtOH-induced locomotion has been associated with the activation of mesocorticolimbic dopamine (DA), which originates in the ventral tegmental area (VTA) and innervates the prefrontal cortex and the nucleus accumbens (NAc) (Wise and Rompre, 1989; Le Moal and Koob, 2007; Meyer et al., 2009). Hence, administration of different DA antagonists blocked the locomotor activation elicited by EtOH in mice, thereby indicating that this neurotransmitter is a key mediator in this behavioral effect (Risinger et al., 1992; Shen ^ et al., 1997; Jerlhag, 2008). Interestingly, it has et al., 1995; Le been shown that acetaldehyde also stimulates DA release between these structures (Foddai et al., 2004; Deehan et al., 2013) and administration of the catalase inhibitor 3-amino-1,2,4-triazole or different acetaldehyde sequestering agents prevented this outcome in rats treated with EtOH (Diana et al., 2008; Enrico et al., 2009; Sirca et al., 2011). Thus, it is plausible to propose that the locomotor enhancement caused by EtOH could be produced by DA release from the ATV to the NAc after its conversion into acetaldehyde. In other words, it seems that the locomotor stimulation induced by EtOH, which has been suggested as being mediated by the activation of the DAergic mesocorticolimbic system, could depend on its previous transformation into acetaldehyde. Previous research has demonstrated that CYP 2E1 is present in the mesocorticolimbic system, and its expression can be induced by chronic acetone or EtOH treatment in a regional-selectivity manner in both the NAc
nchez-Catala
n et al., 2008). Therefore, it is and the VTA in rats (Sa possible that the potentiation of EtOH-induced locomotor stimulation observed in mice exposed to acetone could be due to a higher

J.C. Ledesma et al. / Neuropharmacology 85 (2014) 36e44

metabolism of EtOH into acetaldehyde by CYP 2E1 in the ATV and the NAc, which resulted in a greater DA release in these areas. Hence, considering that the motor-stimulating and the reinforcing effects of drugs of abuse are at least in part mediated by mesocorticolimbic DA (Tzschentke and Schmidt, 2000; Robinson and Berridge, 2008), we suggest that CYP 2E1 induction could modulate some of the reinforcing effects of alcohol related to its addiction and dependence. Overall, the current results represent a first approach to the use of acetone as a potential pharmacological tool to induce CYP 2E1 activity/expression and thus evaluate the involvement of this enzyme in the neurobehavioral properties of EtOH. Further studies must be performed to test the hypotheses proposed in the present work, about the possible mechanism of action by which CYP 2E1 induction selectively modulates EtOH-induced locomotion. First, it will be desirable to measure the formation of acetaldehyde in brain homogenates or directly in vivo by microdialysis in acetone treatedmice after EtOH administration, to test whether the potentiation of CYP 2E1 observed in our experiments is able to significantly increase the production of this metabolite. Second, to assess whether this enzyme could contribute to the formation of EtOH-derived acetaldehyde by the activity of Compound I, the consequence of the potentiation of CYP 2E1 activity after acetone exposure on central H2O2 levels should be determined. Acknowledgments This work was supported by grants from MICINN (PSI2011
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