- Email: [email protected]
6J mice
Legal Medicine 14 (2012) 229–238
Contents lists available at SciVerse ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Administration of rotenone enhanced voluntary alcohol drinking behavior in C57BL/6J mice Kanji Yoshimoto a,⇑, Shuichi Ueda b, Yoshihisa Kitamura c, Masatoshi Inden c, Hiroyuki Hattori d, Noboru Ishikawa a, Stuart McLean a, Hiroshi Ikegaya a a
Department of Forensic Medicine, Kyoto Prefectural University of Medicine, Kawaramachi, Kamigyo-ku, Kyoto 602-8566, Japan Department of Anatomy and Neurobiology, Dokkyo University School of Medicine, Mibu, Tochigi 321-02, Japan Department of Neurobiology, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan d Division of General Hospital, Tsuchiura Kyodo General Hospital, Tsuchiura, Ibaraki 300-0053, Japan b c
a r t i c l e
i n f o
Article history: Received 9 August 2011 Received in revised form 13 February 2012 Accepted 19 March 2012 Available online 29 April 2012 Keywords: Rotenone Serotonin Dopamine Alcohol drinking Neural degeneration
a b s t r a c t Rotenone, a commonly used lipophic pesticide, is a high-affinity mitochondrial complex I inhibitor. The aim of this project is to study the causal relationship between changes of brain monoamine levels and drinking behavior in rotenone-treated mice. In the first experiment, we investigated the effects of acute exposure to rotenone (20 mg/kg, p.o.) on the 8-h time limited-access alcohol drinking behavior and brain monoamine levels in C57BL/6J mice at 0, 2, 8 and 24 h. Dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5HIAA) levels in the nucleus accumbens (ACC), caudate– putamen (C/P) and lateral hypothalamus (LH) of rotenone-treated mice were decreased at 2 and/or 8 h. Rotenone-exposed mice showed a suppression of voluntary alcohol intake at 4 and 8 h, but total daily alcohol intake did not differ significantly between the two groups. The effects of chronic exposure to rotenone (1, 5, 10 and 20 mg/kg, p.o. for 30 days) on the alcohol drinking behavior and monoamine levels of rotenone-exposed mice (10 mg/kg, p.o.) were investigated in the second experiment. The mice treated with rotenone showed increases in alcohol drinking behavior. Levels of DA and 5-HT in the ACC and C/P of chronic rotenone-treated mice were decreased, while the ratios of DOPAC to DA in the ACC and C/P and of 5HIAA to 5-HT in the ACC, C/P and DRN were increased significantly. Tyrosine hydroxylase immunoreactivity of chronic rotenone-treated mice (10 mg/kg, p.o.) slightly were decreased in both the striatum and the substantia nigra. Ethanol and acetaldehyde metabolism was not significantly different between mice treated with rotenone (10 mg/kg, p.o.) and controls. It was suggested that rotenone-treated mice had increased alcohol drinking behavior associated with increases in the DA turnover ratios of ACC and striatum to compensate for the neural degeneration. Ó 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Rotenone, widely used as a pesticide in vegetable gardens and in fish population selection in lakes and reservoirs, has been considered a candidate environmental neurotoxicant contributing to Parkinson’s disease (PD), because rotenone is a high-affinity and selective complex I inhibitor [1,2]. Chronic treatment with rotenone (2.5–5 mg/kg s.c. for 30–45 days) has been reported to induce parkinsonism in mice [3]. Complex I defects may result in oxidative stress and increase the susceptibility of neurons to excitotoxic death. There is evidence that reduced activity of complex I (NADA-ubiquinone reductase) of the mitochondrial respiratory chain in dopaminergic neurons plays a critical role in the pathophysiology of PD. A reduction of complex ⇑ Corresponding author. Tel./fax: +81 075 251 5345. E-mail address: [email protected] (K. Yoshimoto). 1344-6223/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.legalmed.2012.03.005
I activity has been demonstrated in the mitochondria of PD patients [4]. Subcutaneous infusions or daily injections of 2–3 mg/kg rotenone for 28–58 days to rats were found to produce behavioral, neurochemical, and neuropathological features of PD, including nigrostriatal dopaminergic degeneration and eosinophilic cytoplasmic inclusions [5]. On the other hand, it is well known that the dopaminergic neurotoxin, 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MTPP), after its conversion to one of the most prominent mitochondrial complex I inhibitors, 1-methyl-4-phenylpyridinium (MPP+), was produced in human and other species [6]. Although the symptoms and neuropathology have been well characterized the relationship between the neural degeneration and complex I inhibition, the underlying mechanisms and causes of the development of alcohol drinking behavior are still unknown. Numerous studies have shown that environmental factors, such as exposure to pesticides, are associated with an increased risk of
230
K. Yoshimoto et al. / Legal Medicine 14 (2012) 229–238
established complex I inhibitor, generally induces many key features of Parkinson’s disease and neural degeneration [10,11]. Although epidemiological studies of rotenone have implicated exposure to agricultural pesticides as increasing the chances of contracting Parkinson disease, effects of chronic treatment of rotenone on the alcohol metabolism in mice have also not been investigated extensively.
Parkinson’s diseases, one of the most common neurodegenerative diseases with decreases of monoamine levels [7]. Furthermore, rats treated with the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) (4 lg/side) showed a higher alcohol preference score for 2 weeks following the neurotoxic treatment, and lower dopamine (DA) levels in the nucleus accumbens (ACC) and midbrain [8,9]. Chronic treatment of mice with rotenone a well-
A
B
Fig. 1. Effects of acute rotenone treatment (20 mg/kg p.o.) on voluntary alcohol drinking behavior. (A) 8 h limited time access and alcohol drinking behavior (g/kg/h). (B) Alcohol consumption (g/kg/day). Rotenone (suspended in 0.5% CMC) was orally administered to C57BL/6J mice at 20 mg/kg (each group, n = 6). 0.5% CMC was administered to the control mice as vehicle (n = 6). ⁄p < 0.05 post hoc Bofferoni’s test.
Fig. 2. Effects of acute rotenone treatments (20 mg/kg p.o.) (each group, n = 6) at 0, 2, 8, and 24 h on the levels of dopamine (DA), 3,4-dihyroxyphenyacetic acid (DOPAC) in the nucleus accumbens (ACC), caudate–putamen (C/P), lateral hypothalamus (LH), dorsal raphe nucleus (DRN) and midbrain (MID). DA and DOPAC were shown in closed column and open column, respectively. ⁄p < 0.05 compared with the 0 h-group mice (n = 6).
231
K. Yoshimoto et al. / Legal Medicine 14 (2012) 229–238
Fig. 3. Effects of acute rotenone treatments (20 mg/kg p.o.)(each group, n = 6) at 0, 2, 8, and 24 h on the ratios of 3, 4-dihyroxyphenyacetic acid (DOPAC) to dopamine (DA) in the nucleus accumbens (ACC), caudate–putamen (C/P), lateral hypothalamus (LH), dorsal raphe nucleus (DRN) and midbrain (MID). ⁄p < 0.05 compared with the 0 h-group mice (n = 6).
Table 1 Effects of chronic rotenone treatment (10 mg/kg, p.o.) on the ratios of 3,4-dihydroxyphenylacetic acid (DOPAC) to dopamine (DA) and 5-hydroxyindole acetic acid (5HIAA) to serotonin (5-HT). Area
Group
n
DOPAC:DA
5HIAA:5-HT
ACC
Control Rotenone
6 6
0.18 ± 0.05 0.30 ± 0.01*
1.10 ± 0.40 2.23 ± 0.45*
C/P
Control Rotenone
6 6
0.22 ± 0.01 0.56 ± 0.02*
1.33 ± 0.66 2.53 ± 0.05*
LH
Control Rotenone
6 6
0.77 ± 0.05 0.67 ± 0.08
1.20 ± 0.40 1.75 ± 0.62
DRN
Control Rotenone
6 6
0.69 ± 0.31 0.51 ± 0.12
1.55 ± 0.71 3.46 ± 0.68*
MID
Control Rotenone
6 6
0.78 ± 0.05 0.89 ± 0.31
1.65 ± 0.81 0.51 ± 0.07
Control: 0.5% CMC was administered orally to control C57BL/6J mice as vehicle (n = 6). Rotenone: rotenone (suspended in 0.5% CMC) was orally administered to C57BL/6J mice at 10 mg/kg for 30 days (n = 6). ACC, nucleus accumbens; C/P, caudate– putamen; LH, lateral hypothalamus; DRN, dorsal raphe nucleus; MID, midbrain; n, number of animals. DOPAC:DA and 5HIAA:5-HT ratios mean the turnover rates and the index of the releases of DA and 5-HT, respectively. * p < 0.05, unpaired 2-tail t-test.
To investigate the effect of rotenone on the changes of drinking patterns in mice, we conducted neurochemical analyses of brain
monoamines, immunohistochemical studies and alcohol drinking tests involved with the studies of alcohol metabolism.
232
K. Yoshimoto et al. / Legal Medicine 14 (2012) 229–238
2. Materials and methods 2.1. Animals Eight-week-old male C57BL/6J mice (20–25 g) were purchased from Japan CLEA (Osaka, Japan). Mice were acclimated to and maintained at 23 °C under a 12-h light/dark cycle. They were housed in standard laboratory cages and had free access to food and water throughout the experiments. All animal experiments were carried out in accordance with the ‘National Institutes of Health: Guide for the Care and Use of Laboratory Animals’, and the protocols were approved by the ‘Committee for the Care and Use of Laboratory Animals, Kyoto Prefectural University of Medicine, Kyoto, Japan (#21-1)’. 2.2. Experiment 1 2.2.1. Acute administration of rotenone Rotenone (Sigma, St. Louis, MO, USA) was administered orally at a dose of 20 mg/kg. Rotenone was suspended in 0.5% carboxymethyl cellulose sodium salt (CMC) (Nacalai, Kyoto, Japan) with Tween-20. The suspension was administered orally at a volume of 5 mL/kg body weight. The levels of brain monoamines at 0 h were shown the results of mice sacrificed immediately after the treatment of rotenone.
2.2.2. 8-h limited access alcohol drinking behavior Water was removed overnight before performing the 8-h limited access to alcohol test. On the day of the experiment, rotenone-exposed mice (n = 6) and vehicle-exposed (control) mice (n = 6) had access to 10% (v/v) alcohol from a 20-mL graduated glass tube for an 8-h period beginning at 10:00 a.m. The amounts consumed were measured at 0.5, 1.0, 2.0, 4.0 and 8.0 h during the period of restricted access. We expressed 10% (v/v) alcohol intake as grams per kilogram per hour (g/kg/h) and grams per kilogram over 24 h (g/kg/day). 2.3. Experiment 2 2.3.1. Chronic administration of rotenone Rotenone (Sigma, St. Louis, MO, USA) was administered orally once a day at doses of 1, 5, 10 and 20 mg/kg for 30 days (each group, n = 6). Rotenone was suspended in 0.5% CMC (Nacalai, Kyoto, Japan) with Tween-20. 0.5% CMC was administered orally to control mice as vehicle (n = 6). The suspension was administered orally at a volume of 5 mL/kg body weight. 2.3.2. Alcohol preference test We investigated preferences for water or an alcohol-containing solution modified with our previous standard two-bottle methods [12]. The mice had access to 10% (v/v) alcohol and tap water in
Fig. 4. Effects of acute rotenone treatments (20 mg/kg p.o.) (each group, n = 6) at 0, 2, 8, and 24 h on the levels of serotonin (5-HT) and 5-hydroxyindolacetic acid (5HIAA) in the nucleus accumbens (ACC), caudate–putamen (C/P), lateral hypothalamus (LH), dorsal raphe nucleus (DRN) and midbrain (MID). 5-HT and 5HIAA were shown in closed column and open column, respectively. ⁄p < 0.05 compared with the 0 h-group mice (n = 6).
K. Yoshimoto et al. / Legal Medicine 14 (2012) 229–238
graduated glass bottles (20 mL) for 10 days. The locations of the bottles were changed daily on a random basis. The daily intakes of water (mL) and 10% alcohol (mL) were measured to evaluate alcohol preference by means of an alcohol consumption score (g/kg body weight/day) for each mouse. Body weights were recorded every second or third day. Alcohol preference (%) was defined as the alcohol intake expressed as a percentage of the total fluid intake of each group.
233
2.3.3. Brain monoamines and their metabolites In experiments 1 and 2, the brain was removed, cooled on ice (this process took from 1 to 1.5 min), and dissected using a Rodent Brain Matrix (Zivic Miller Inc., Allison Park, PA, USA), constructed to provide coronal brain sections. Eight brain areas, the nucleus accumbens (ACC), midbrain (MID), caudate nucleus and putamen (C/P), lateral hypothalamus (LH), dorsal raphe nucleus (DRN), frontal cortex (FC), hippocampus (HP), and amygdala (AMY), were iso-
Fig. 5. Effects of acute rotenone treatments (20 mg/kg p.o.) (each group, n = 6) at 0, 2, 8, and 24 h on the ratios of 5-hydroxyindolacetic acid (5HIAA) to serotonin (5-HT) in the nucleus accumbens (ACC), caudate–putamen (C/P), lateral hypothalamus (LH), dorsal raphe nucleus (DRN) and midbrain (MID). ⁄p < 0.05 and ⁄⁄p < 0.01, unpaired 2-tail t-test. ⁄ p < 0.05 compared with the 0 h-group mice (n = 6).
A
B
Fig. 6. Effects of chronic rotenone treatments (1, 5, 10 and 20 mg/kg p.o.) (each group, n = 6) for 30 days on alcohol drinking behavior, alcohol intake (g/kg/day) (A) and alcohol preference (%) (B). ⁄p < 0.05 and ⁄⁄⁄p < 0.005 post hoc Bonferroni’s test. 0.5% CMC was administered to the control mice as vehicle (n = 6). Rotenone (suspended in 0.5% CMC) was orally administered once a day to C57BL/6J mice.
234
K. Yoshimoto et al. / Legal Medicine 14 (2012) 229–238
lated using a tissue punch (1.0 mm o.d.), as described by Ueda et al. [13]. The isolated brain tissues were rapidly frozen in liquid nitrogen and kept at 80 °C until the biochemical analysis. Each sample was weighed and homogenized using an ultrasonic-homogenizer in a mixture of 40 lL of 0.5 M perchloric acid, 30 lL of 0.1 M 2Na–EDTA, and 30 lL of 3,4-dihydroxybenzylamine (DHBA)–HCl as an internal standard. The homogenate was centrifuged (20,000g, 20 min) and the supernatant collected and filtered using a cellulose–acetate membrane disk (Toso, Tokyo, Japan). The tissue levels of norepinephrine (NE), DA, 3,4-dihydroxyphenylacetic acid (DOPAC), 5-HT, and 5-hydroxyindoleacetic acid (5HIAA) were determined using an internal standard. Separation was performed with a reversed-phase column (TSK-Gel ODS80Ts, 5 lm particle diameter, 150 2.0 mm; Toso). The mobile phase consisted of 0.1 M sodium phosphate, containing 0.1 mM EDTA–2Na with 15% methanol, 5% acetonitrile, and 0.3 mM sodium-1-octane sulfonic acid, adjusted to pH 2.8 with phosphoric acid. All separations were performed at a flow rate of 0.8 mL/ min. The levels of DA, 5-HT, and their metabolites (DOPAC and 5HIAA) were determined by high-performance liquid chromatography (HPLC) (3201 Nanospace, Shiseido Co., Tokyo, Japan) coupled with electrochemical detection (3005 Nanospace, Shiseido Co.), using a glassy carbon electrode set at +0.6 V vs. an Ag/AgCl reference electrode. All values are expressed as pmol/mg. Additionally, DA and 5-HT turnover indexes, changes of which indicate the increased release of DA and 5-HT at terminals, are shown as ratios of the metabolites to mother monoamines.
2.3.4. Blood alcohol and acetaldehyde assay All chronic rotenone-treated mice were deprived of ethanol for 24 h before the experiment to allow ethanol and acetaldehyde to clear from their circulations. Mice were injected with 2 g/kg i.p. ethanol (10% v/v). Blood samples were collected from mice at 0, 0.5, 1.0, 2.0 and 4.0 h after the injection of ethanol. Blood samples were immediately deproteinized at 0 °C in head space chromatography vials containing 100 lL (70%) trichloroacetic acid and 1-propanol as an internal standard. Gas chromatographic analysis for ethanol and acetaldehyde in blood was patterned after the method described previously [14,15]. Gas chromatographic analysis of acetaldehyde was carried out on blood collected at 1.0 and 2.0 h after the injection of ethanol.
2.3.5. Tissue preparation and immunohistochemistry Treated mice were perfused under deep anesthesia with pentobarbital (100 mg/kg, i.p.) [16]. Briefly, after perfusion, the brain was quickly removed and postfixed. The brain slices were cut 20 lm-thick using a cryostat and collected in 100 mM PBS containing 0.3% Triton X-100 (PBS-T). After several washes, slices were stored until use in free-floating state at 4 °C for immunohistochemical analysis. Brain slices were incubated with mouse monoclonal anti-tyrosine hydroxylase (TH, Sigma, St. Louis, MO, USA, diluted 1:10,000) or tryptophan hydroxylase (TPH, Chemicon Int., Temecula, CA, USA, 1:1000) antibody for three days at 4 °C, and was detected by an ABC Elite kit (Vector Laboratories, Burlin-
Fig. 7. Effects of chronic rotenone treatment (10 mg/kg p.o.) for 30 days on the levels of dopamine (DA) and 3,4-dihyroxyphenyacetic acid (DOPAC) in the nucleus accumbens (ACC), caudate–putamen (C/P), lateral hypothalamus (LH), dorsal raphe nucleus (DRN) and midbrain (MID). 0.5% CMC was administered orally to control mice as vehicle (n = 6). Rotenone: rotenone (suspended in 0.5% CMC) was orally administered to C57BL/6J mice at 10 mg/kg for 30 days (n = 6). ⁄p < 0.05 compared with the control.
K. Yoshimoto et al. / Legal Medicine 14 (2012) 229–238
game, CA, USA) with 3,30 -diaminobenzidine (DAB) with nickel ammonium. 2.4. Statistical analyses Data was analyzed with one- and two-way (monoamines time) repeated measures ANOVAs using GraphPad software. Post hoc comparisons were carried out with the Bonferroni t-test. In all cases, the accepted level of significance was set at p < 0.05 (unpaired t-test) (GraphPad Prism version 5; GraphPad Software, Inc., La Jolla, CA, USA). 3. Results
235
The levels of DA and DOPAC in the ACC, C/P, and LH were decreased at 2–8 h after the treatment with rotenone (20 mg/kg p.o.) (Fig. 2). In the same areas, the level of 5HIAA was also decreased at 8 h after the treatment (Fig. 4). Furthermore, the ratio of DOPAC to DA and of 5HIAA to 5-HT in the ACC, C/P, and LH were decreased at 2–8 h (Figs. 3 and 5). It is likely that a single rotenone treatment increases the sensitivity to dopaminergic neurons more than serotonergic neurons in the striatum and temporarily downregulates the dopaminergic functions continuously over 8 h (Figs. 2 and 3). There were no significant differences in the levels of DA, 5-HT and their metabolites between the two groups at 24 h after the treatment with rotenone (Figs. 2 and 4). The levels of NE did not show any significant changes in mice treated with rotenone acutely.
3.1. Experiment 1 3.2. Experiment 2 3.1.1. Acute rotenone treatment Fig. 1A shows the results of the 8-h time limited access to alcohol drinking behavior test. Rotenone (20 mg/kg p.o.) suppressed alcohol drinking behavior (Treatment; F(1, 45) = 21.44, p < 0.0001) with significant decreases in alcohol intake at 4 and 8 h (time; F(1, 45) = 11.88, p < 0.0001). However total alcohol consumption (g/kg/day) in the control and rotenone-treated groups did not differ significantly (Fig. 1B). The neurotoxical effect on alcohol drinking behavior was evident 8 h following single treatment with rotenone.
3.2.1. Chronic rotenone treatment The 30-day rotenone treatment p.o. enhanced voluntary alcohol drinking behavior, alcohol intake (g/kg/day) (F(4, 25) = 10.34, p < 0.0001) and alcohol preference (%) (F(4, 25) = 17.41, p < 0.0001), in a dose-dependent manner (Fig. 6A and B). In the rotenone (10 mg/kg p.o.)-treated groups, the levels of DA in the ACC, C/P, and LH, and 5-HT in the ACC, C/P, and DRN decreased significantly, as compared with the control groups (Figs. 7 and 8). On the other hand, the ratios of DOPAC to DA in
Fig. 8. Effects of chronic rotenone treatment (10 mg/kg p.o.) for 30 days on the levels of serotonine (5-HT), 5-hydroxyindolacetic acid (5HIAA) and the ratios of 5HIAA to 5-HT in the nucleus accumbens (ACC), caudate–putamen (C/P), lateral hypothalamus (LH), dorsal raphe nucleus (DRN) and midbrain (MID). 0.5% CMC was administered orally to control mice as vehicle (n = 6). Rotenone: rotenone (suspended in 0.5% CMC) was orally administered to C57BL/6J mice at 10 mg/kg for 30 days (n = 6). ⁄p < 0.05 compared with the control.
236
K. Yoshimoto et al. / Legal Medicine 14 (2012) 229–238
3.2.3. Immunohistochemistry As shown in representative photomicrographs, TH immunoreactivity in the 30 days rotenone-treated mice (10 mg/kg, p.o.) for 30 days was less in both the striatum and the substantia nigra (Fig. 10). On the other hand, there was no differences in the TPH immunoreactivity between the control and rotenone-treated mice (Fig. 11).
4. Discussion
Fig. 9. Blood alcohol concentration (mg/mL) in the chronic rotenone-treated mice (10 mg/kg, p.o.) for 30 days (d) and control mice (s) receiving 2 g/kg ethanol intraperitoneally. Each curves shows the concentration obtained from six mice in each group.
the ACC and C/P and of 5HIAA to 5-HT in the ACC, C/P, and DRN increased significantly (Table 1). These increases indicate increases in metabolic turnover or release of dopaminergic and serotonergic neurons. The levels of NE did not show any significant changes in mice treated with rotenone. It was suggested that enhanced alcohol drinking behavior compensates for the decrease of monoamine levels and the neurodegeneration in the rewarding pathway in mice administered rotenone. 3.2.2. Gas chromatography analysis Although the time course of blood alcohol concentrations showed a significant change (two-way-ANOVA; time; F(4, 50) = 61.8, p < 0.0001), blood alcohol concentrations at each of the time measured did not show a significant differences between the 30 days rotenone-treated mice (10 mg/kg, p.o.) and the control mice (Bonferroni post hoc test) (Fig. 9). Furthermore, there was not significant change of blood acetaldehyde concentration (0.4– 0.6 lg/mL) at 1.0 and 2.0 h in two groups after the injection of ethanol (2 g/kg, i.p.) (data not shown).
Alcohol abuse and alcoholism represent about one third of the total public health burden worldwide and are major medical problems as well as primary health care problems [17]. Although alcohol drinking is associated with environmental factors, industrial hygiene, pesticides and genetics, but the nature of their effects are still unknown. Acute treatment with rotenone caused decreases in the levels of DA, DOPAC and 5HIAA in the ACC, C/P and LH mainly at 2 and/or 8 h (Figs. 2 and 4). Rotenone (2 g/kg, i.p. for 40 days) affected a nigro-striatal pathway indicated by a 47% decrease in striatal DA levels [18]. On the other hand, chronic treatment with rotenone caused a decrease in DA levels in the ACC, C/P and LH and a decrease of 5-HT in the ACC and C/P without affecting the level of DOPAC and 5HIAA, respectively (Figs. 7 and 8). Rotenone has been demonstrated to cause more selective dopaminergic cell degeneration in vivo [10]. The degree of specific rotenone toxicity to dopaminergic neurons depends on not only complex I but also microtubule destabilization, vesicle accumulation and oxidative stress in dopaminergic neurons [2]. In the present study, accompanying the decrease in striatal DA and 5-HT levels of the ACC and C/P in the nigrostriatal pathway after chronic rotenone injections, DOPAC and 5HIAA levels were not significantly changed, but then the ratios of DOPAC/DA and 5HIAA/5-HT in the nigrostriatal pathway were increased (Table 1). The reason for the discrepancies of the different ratios in mice with acute and chronic treatment of rotenone remains unclear. Following the chronic rotenone treatments, volun-
Fig. 10. TH immunoreactivity in the striatum and substantia nigra. At 30 days, rotenone-treated mice (10 mg/kg, p.o.) were fixed and brain slices were prepared. Sections of the striatum and substantia nigra were immunostained by anti-TH antibody. Scale bars: 1 mm (in the striatum) and 100 lm (in the substantia nigra).
K. Yoshimoto et al. / Legal Medicine 14 (2012) 229–238
237
Fig. 11. TPH immunoreactivity in the striatum and raphe nucleus. At 30 days, rotenone-treated mice (10 mg/kg, p.o.) were fixed and brain slices were prepared. Sections of the striatum and raphe nucleus were immunostained by anti-TPH antiboby. Scale bars: 1 mm (in the striatum) and 100 lm (in the raphe nucleus).
tary alcohol drinking behavior actually increased in mice treated with rotenone dose-dependently (Fig. 6A and B). It was suggested that decreased DA levels in the striatum induces an increase in the release of DA to sustain neural functions and in turn enhance alcohol drinking behavior. Rotenone at 30 mg/kg p.o. for 28 days caused a significant loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra of mice [19]. It was consistent with our results of TH immunohistochemistry (Fig. 10). Rotenone also shows the actions of the reactive oxygen species in the mitochondrial compartment [2]. Consequently rotenone-treated animals with neural degeneration showed abnormal behavior, e.g. enhanced alcohol drinking behavior (Fig. 6), because rotenone causes specific dopaminergic injury with the complex I inhibitor or oxidative stress [20]. It was suggested that the causal relationship between mitochondrial dysfunction and specific dopaminergic injury increases alcohol drinking behavior. The increased alcohol drinking behaviors of the chronic rotenone-treated mice was associated with an increase in the number of surviving neurons in the nigrostriatal systems. 6-OHDA dopamine toxin-treated rats showed a significant increase in alcohol preference scores associated with the depletion of DA in the ACC and C/P [10,21]. The central DA neurons are important in the control of alcohol drinking behavior. These results are consistent with the present finding that rotenone-treated mice showed an increase in alcohol drinking behavior with the depletion of DA. Injection of 5,7-dihydroxytryptamine (5,7-DHT) into the brain has been shown to increase alcohol intake in the rat [22]. We have reported a negative correlation between brain 5-HT levels and alcohol drinking behavior in inbred strains of mice [12]. This is also consistent with the result which lower brain 5-HT levels produced an increase in alcohol drinking behavior [12,23]. It was suggested that rotenone’s influence on the occasional alcohol drinking behavior was associated with the central nigrostriatal pathway’s degeneration of DA and serotonergic neurons. We have to look more closely and neurochemically at the use of pesticides with neurotoxic actions and without the alcohol metabolism (Fig. 9). Rotenone mice model seems to mimic habitual alcohol drinking within human with progressive neurodegeneration in aging.
In conclusion, rotenone caused a decrease in DA and 5-HT levels in the nigrostriatal pathway. Rotenone-treated mice showed an increased alcohol drinking behavior associated with increases of turnover ratios of DA in the ACC and striatum. Ethical standard The authors declare that the experimental project complies with the current laws of the countries in which they were performed. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgement This study was supported in part by a Grant-in-Aid for Scientific Research (C) (21590740) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References [1] Langston JW. Parkinson’s disease: current and future challenges. Neurotoxicology 2002;23:443–50. [2] Choi WS, Kruse SE, Palmiter RD, Xia Z. Mitochondorial complex I inhibition is not required for dopaminergic neurons death induced by rotenone, MPP+, or paraquat. PNAS 2008;105:15136–41. [3] Richter F, Hamann M, Richter A. Chronic rotenone treatment induces behavioral effects but no pathological signs of Parkinsonism in mice. J Neurosci Res 2007;85:681–91. [4] Abou-Sleiman PM, Muqit MM, Wood NW. Expanding insights of mitochondrial dysfunction in Parkinson’s disease. Nat Rev Neurosci 2006;7:207–19. [5] Shere C, Kim JH, Betarbet R, Greenamyre JT. Subcutaneous rotenone exposure causes highly selective dopaminergic degeneration and alpha-synuclein aggregation. Exp Neurol 2003;179:9–16. [6] Beal MF. Experimental models of Parkinson’s disease. Nat Rev Neurosci 2001;5:325–34. [7] Thiruchelvam M, Richfield EK, Baggs RB, Tank AW, Cory-Slecha DA. The nigrostriatal dopaminergic system as a preferential target of repeated exposure to combined paraquat and maneb: implications for Parkinson’s disease. J Neurosci 2000;20:9207–14.
238
K. Yoshimoto et al. / Legal Medicine 14 (2012) 229–238
[8] Yoshimoto K, Kawamura K, Yayama K, Fujimiay T, Uemura K, Komura S. The effects of neurotoxins 6-hydroxydopamine and 5,7-dihydroxytryptamine into the rat nucleus accumbens on the alcohol drinking behavior. Jpn J Legal Med 1995;49:11–9. [9] Yoshimoto K, Kawai Y, Ueda S, Takeuchi H, Matsushita K, Yuri K, et al. Treating neonatal rat with 6-hydroxydopamine induced an increase in alcohol drinking behavior. Alcohol Clin Exp Res 1999;23:2s–6s. [10] Inden M, Kitamura Y, Takeuchi H, Yanagita T, Takata K, Kobayashi Y, et al. Neurodegeneration of mouse nigrostriatal dopaminergic system induced by repeated oral administration of rotenone is prevented by 4-phenylbutyrate, a chemical chaperone. J Neurochem 2007;101:1491–504. [11] Choi WS, Palmiter RD, Xia Z. Loss of mitochondrial complex I activity potentiates dopamine neuron death induced by microtubule dysfunction in a Parkinson’s disease model. J Cell Biol 2011;192:873–82. [12] Yoshimoto K, Komura S. Reexamination of the relationship between alcohol preference and brain monoamines in inbred strains of mice including senescence-accelerated mice. Pharmacol Biochem Behav 1987;27: 317–22. [13] Ueda S, Aikawa M, Ishizuya-Oka A, Koibuchi N, Yamaoka S, Yoshimoto K. Agerelated degeneration of the serotoninergic fibers in the zitter rat brain. Synapse 1998;30:62–70. [14] Fujimiya T, Yamaoka K, Fukui Y. Parallel first-order and Michaelis–Menten elimination kinetics of ethanol: respective role of ADH, non-ADH and firstorder pathways. J Pharmcol Exp Ther 1989;249:311–7.
[15] Okada T, Mizoi Y. Studies on the problem of blood acetaldehyde determination in man and level after alcohol intake. Jpn J Alcohol Drug Depend 1982;17: 141–59. [16] Kitamura Y, Inden M, Minamino H, Abe M, Takata K, Taniguchi T. The 6hydroxydopamine-induced nigrostriatal neurodegeneration produces microglia-like NG2 glial cells in the rat substantia nigra. Glia 2010;58: 1686–700. [17] Voas RB, Fell JC. Preventing alcohol-related problems through health policy research. Alcohol Res Health 2010;33:18–28. [18] He Y, Iman SZ, Dong Z, Jankovic J, Ali SF, Appel SH, et al. Role of nitric oxide in rotenone-induced nigro-striatal injury. J Neurochem 2003;86:1336–45. [19] Inden M, Kitamura Y, Abe M, Tamaki A, Takata K, Taniguchi T. Parkinsonian rotenone mouse model: reevaluation of long-term administration of rotenone in C57BL/6 mice. Biol Pharm Bull 2011;34:92–6. [20] Jenner P. Oxidative stress in Parkinson’s disease. Ann Neurol 2003;53:S26–38. [21] Kiianmaa K. Alcohol intake and ethanol intoxication in the rat: effect of 6OHDA-induced lesion of the ascending noradrenaline pathway. Eur J Pharmacol 1980;64:9–19. [22] Zhukov VN, Varkov AI, Burov YV. Effect of destruction of the serotonergic system on alcohol intake by rats in the early stages of experimental alcoholism. Bull Exp Biol Med 1985;99:604–6. [23] Zhou FC, Lumeng L, Li TK. Quantitative immunocytochemical evaluation of seronergic innervations in alcoholic rat brain. Neurochem Int 1995;26: 135–43.
Contents lists available at SciVerse ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Administration of rotenone enhanced voluntary alcohol drinking behavior in C57BL/6J mice Kanji Yoshimoto a,⇑, Shuichi Ueda b, Yoshihisa Kitamura c, Masatoshi Inden c, Hiroyuki Hattori d, Noboru Ishikawa a, Stuart McLean a, Hiroshi Ikegaya a a
Department of Forensic Medicine, Kyoto Prefectural University of Medicine, Kawaramachi, Kamigyo-ku, Kyoto 602-8566, Japan Department of Anatomy and Neurobiology, Dokkyo University School of Medicine, Mibu, Tochigi 321-02, Japan Department of Neurobiology, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan d Division of General Hospital, Tsuchiura Kyodo General Hospital, Tsuchiura, Ibaraki 300-0053, Japan b c
a r t i c l e
i n f o
Article history: Received 9 August 2011 Received in revised form 13 February 2012 Accepted 19 March 2012 Available online 29 April 2012 Keywords: Rotenone Serotonin Dopamine Alcohol drinking Neural degeneration
a b s t r a c t Rotenone, a commonly used lipophic pesticide, is a high-affinity mitochondrial complex I inhibitor. The aim of this project is to study the causal relationship between changes of brain monoamine levels and drinking behavior in rotenone-treated mice. In the first experiment, we investigated the effects of acute exposure to rotenone (20 mg/kg, p.o.) on the 8-h time limited-access alcohol drinking behavior and brain monoamine levels in C57BL/6J mice at 0, 2, 8 and 24 h. Dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5HIAA) levels in the nucleus accumbens (ACC), caudate– putamen (C/P) and lateral hypothalamus (LH) of rotenone-treated mice were decreased at 2 and/or 8 h. Rotenone-exposed mice showed a suppression of voluntary alcohol intake at 4 and 8 h, but total daily alcohol intake did not differ significantly between the two groups. The effects of chronic exposure to rotenone (1, 5, 10 and 20 mg/kg, p.o. for 30 days) on the alcohol drinking behavior and monoamine levels of rotenone-exposed mice (10 mg/kg, p.o.) were investigated in the second experiment. The mice treated with rotenone showed increases in alcohol drinking behavior. Levels of DA and 5-HT in the ACC and C/P of chronic rotenone-treated mice were decreased, while the ratios of DOPAC to DA in the ACC and C/P and of 5HIAA to 5-HT in the ACC, C/P and DRN were increased significantly. Tyrosine hydroxylase immunoreactivity of chronic rotenone-treated mice (10 mg/kg, p.o.) slightly were decreased in both the striatum and the substantia nigra. Ethanol and acetaldehyde metabolism was not significantly different between mice treated with rotenone (10 mg/kg, p.o.) and controls. It was suggested that rotenone-treated mice had increased alcohol drinking behavior associated with increases in the DA turnover ratios of ACC and striatum to compensate for the neural degeneration. Ó 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Rotenone, widely used as a pesticide in vegetable gardens and in fish population selection in lakes and reservoirs, has been considered a candidate environmental neurotoxicant contributing to Parkinson’s disease (PD), because rotenone is a high-affinity and selective complex I inhibitor [1,2]. Chronic treatment with rotenone (2.5–5 mg/kg s.c. for 30–45 days) has been reported to induce parkinsonism in mice [3]. Complex I defects may result in oxidative stress and increase the susceptibility of neurons to excitotoxic death. There is evidence that reduced activity of complex I (NADA-ubiquinone reductase) of the mitochondrial respiratory chain in dopaminergic neurons plays a critical role in the pathophysiology of PD. A reduction of complex ⇑ Corresponding author. Tel./fax: +81 075 251 5345. E-mail address: [email protected] (K. Yoshimoto). 1344-6223/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.legalmed.2012.03.005
I activity has been demonstrated in the mitochondria of PD patients [4]. Subcutaneous infusions or daily injections of 2–3 mg/kg rotenone for 28–58 days to rats were found to produce behavioral, neurochemical, and neuropathological features of PD, including nigrostriatal dopaminergic degeneration and eosinophilic cytoplasmic inclusions [5]. On the other hand, it is well known that the dopaminergic neurotoxin, 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MTPP), after its conversion to one of the most prominent mitochondrial complex I inhibitors, 1-methyl-4-phenylpyridinium (MPP+), was produced in human and other species [6]. Although the symptoms and neuropathology have been well characterized the relationship between the neural degeneration and complex I inhibition, the underlying mechanisms and causes of the development of alcohol drinking behavior are still unknown. Numerous studies have shown that environmental factors, such as exposure to pesticides, are associated with an increased risk of
230
K. Yoshimoto et al. / Legal Medicine 14 (2012) 229–238
established complex I inhibitor, generally induces many key features of Parkinson’s disease and neural degeneration [10,11]. Although epidemiological studies of rotenone have implicated exposure to agricultural pesticides as increasing the chances of contracting Parkinson disease, effects of chronic treatment of rotenone on the alcohol metabolism in mice have also not been investigated extensively.
Parkinson’s diseases, one of the most common neurodegenerative diseases with decreases of monoamine levels [7]. Furthermore, rats treated with the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) (4 lg/side) showed a higher alcohol preference score for 2 weeks following the neurotoxic treatment, and lower dopamine (DA) levels in the nucleus accumbens (ACC) and midbrain [8,9]. Chronic treatment of mice with rotenone a well-
A
B
Fig. 1. Effects of acute rotenone treatment (20 mg/kg p.o.) on voluntary alcohol drinking behavior. (A) 8 h limited time access and alcohol drinking behavior (g/kg/h). (B) Alcohol consumption (g/kg/day). Rotenone (suspended in 0.5% CMC) was orally administered to C57BL/6J mice at 20 mg/kg (each group, n = 6). 0.5% CMC was administered to the control mice as vehicle (n = 6). ⁄p < 0.05 post hoc Bofferoni’s test.
Fig. 2. Effects of acute rotenone treatments (20 mg/kg p.o.) (each group, n = 6) at 0, 2, 8, and 24 h on the levels of dopamine (DA), 3,4-dihyroxyphenyacetic acid (DOPAC) in the nucleus accumbens (ACC), caudate–putamen (C/P), lateral hypothalamus (LH), dorsal raphe nucleus (DRN) and midbrain (MID). DA and DOPAC were shown in closed column and open column, respectively. ⁄p < 0.05 compared with the 0 h-group mice (n = 6).
231
K. Yoshimoto et al. / Legal Medicine 14 (2012) 229–238
Fig. 3. Effects of acute rotenone treatments (20 mg/kg p.o.)(each group, n = 6) at 0, 2, 8, and 24 h on the ratios of 3, 4-dihyroxyphenyacetic acid (DOPAC) to dopamine (DA) in the nucleus accumbens (ACC), caudate–putamen (C/P), lateral hypothalamus (LH), dorsal raphe nucleus (DRN) and midbrain (MID). ⁄p < 0.05 compared with the 0 h-group mice (n = 6).
Table 1 Effects of chronic rotenone treatment (10 mg/kg, p.o.) on the ratios of 3,4-dihydroxyphenylacetic acid (DOPAC) to dopamine (DA) and 5-hydroxyindole acetic acid (5HIAA) to serotonin (5-HT). Area
Group
n
DOPAC:DA
5HIAA:5-HT
ACC
Control Rotenone
6 6
0.18 ± 0.05 0.30 ± 0.01*
1.10 ± 0.40 2.23 ± 0.45*
C/P
Control Rotenone
6 6
0.22 ± 0.01 0.56 ± 0.02*
1.33 ± 0.66 2.53 ± 0.05*
LH
Control Rotenone
6 6
0.77 ± 0.05 0.67 ± 0.08
1.20 ± 0.40 1.75 ± 0.62
DRN
Control Rotenone
6 6
0.69 ± 0.31 0.51 ± 0.12
1.55 ± 0.71 3.46 ± 0.68*
MID
Control Rotenone
6 6
0.78 ± 0.05 0.89 ± 0.31
1.65 ± 0.81 0.51 ± 0.07
Control: 0.5% CMC was administered orally to control C57BL/6J mice as vehicle (n = 6). Rotenone: rotenone (suspended in 0.5% CMC) was orally administered to C57BL/6J mice at 10 mg/kg for 30 days (n = 6). ACC, nucleus accumbens; C/P, caudate– putamen; LH, lateral hypothalamus; DRN, dorsal raphe nucleus; MID, midbrain; n, number of animals. DOPAC:DA and 5HIAA:5-HT ratios mean the turnover rates and the index of the releases of DA and 5-HT, respectively. * p < 0.05, unpaired 2-tail t-test.
To investigate the effect of rotenone on the changes of drinking patterns in mice, we conducted neurochemical analyses of brain
monoamines, immunohistochemical studies and alcohol drinking tests involved with the studies of alcohol metabolism.
232
K. Yoshimoto et al. / Legal Medicine 14 (2012) 229–238
2. Materials and methods 2.1. Animals Eight-week-old male C57BL/6J mice (20–25 g) were purchased from Japan CLEA (Osaka, Japan). Mice were acclimated to and maintained at 23 °C under a 12-h light/dark cycle. They were housed in standard laboratory cages and had free access to food and water throughout the experiments. All animal experiments were carried out in accordance with the ‘National Institutes of Health: Guide for the Care and Use of Laboratory Animals’, and the protocols were approved by the ‘Committee for the Care and Use of Laboratory Animals, Kyoto Prefectural University of Medicine, Kyoto, Japan (#21-1)’. 2.2. Experiment 1 2.2.1. Acute administration of rotenone Rotenone (Sigma, St. Louis, MO, USA) was administered orally at a dose of 20 mg/kg. Rotenone was suspended in 0.5% carboxymethyl cellulose sodium salt (CMC) (Nacalai, Kyoto, Japan) with Tween-20. The suspension was administered orally at a volume of 5 mL/kg body weight. The levels of brain monoamines at 0 h were shown the results of mice sacrificed immediately after the treatment of rotenone.
2.2.2. 8-h limited access alcohol drinking behavior Water was removed overnight before performing the 8-h limited access to alcohol test. On the day of the experiment, rotenone-exposed mice (n = 6) and vehicle-exposed (control) mice (n = 6) had access to 10% (v/v) alcohol from a 20-mL graduated glass tube for an 8-h period beginning at 10:00 a.m. The amounts consumed were measured at 0.5, 1.0, 2.0, 4.0 and 8.0 h during the period of restricted access. We expressed 10% (v/v) alcohol intake as grams per kilogram per hour (g/kg/h) and grams per kilogram over 24 h (g/kg/day). 2.3. Experiment 2 2.3.1. Chronic administration of rotenone Rotenone (Sigma, St. Louis, MO, USA) was administered orally once a day at doses of 1, 5, 10 and 20 mg/kg for 30 days (each group, n = 6). Rotenone was suspended in 0.5% CMC (Nacalai, Kyoto, Japan) with Tween-20. 0.5% CMC was administered orally to control mice as vehicle (n = 6). The suspension was administered orally at a volume of 5 mL/kg body weight. 2.3.2. Alcohol preference test We investigated preferences for water or an alcohol-containing solution modified with our previous standard two-bottle methods [12]. The mice had access to 10% (v/v) alcohol and tap water in
Fig. 4. Effects of acute rotenone treatments (20 mg/kg p.o.) (each group, n = 6) at 0, 2, 8, and 24 h on the levels of serotonin (5-HT) and 5-hydroxyindolacetic acid (5HIAA) in the nucleus accumbens (ACC), caudate–putamen (C/P), lateral hypothalamus (LH), dorsal raphe nucleus (DRN) and midbrain (MID). 5-HT and 5HIAA were shown in closed column and open column, respectively. ⁄p < 0.05 compared with the 0 h-group mice (n = 6).
K. Yoshimoto et al. / Legal Medicine 14 (2012) 229–238
graduated glass bottles (20 mL) for 10 days. The locations of the bottles were changed daily on a random basis. The daily intakes of water (mL) and 10% alcohol (mL) were measured to evaluate alcohol preference by means of an alcohol consumption score (g/kg body weight/day) for each mouse. Body weights were recorded every second or third day. Alcohol preference (%) was defined as the alcohol intake expressed as a percentage of the total fluid intake of each group.
233
2.3.3. Brain monoamines and their metabolites In experiments 1 and 2, the brain was removed, cooled on ice (this process took from 1 to 1.5 min), and dissected using a Rodent Brain Matrix (Zivic Miller Inc., Allison Park, PA, USA), constructed to provide coronal brain sections. Eight brain areas, the nucleus accumbens (ACC), midbrain (MID), caudate nucleus and putamen (C/P), lateral hypothalamus (LH), dorsal raphe nucleus (DRN), frontal cortex (FC), hippocampus (HP), and amygdala (AMY), were iso-
Fig. 5. Effects of acute rotenone treatments (20 mg/kg p.o.) (each group, n = 6) at 0, 2, 8, and 24 h on the ratios of 5-hydroxyindolacetic acid (5HIAA) to serotonin (5-HT) in the nucleus accumbens (ACC), caudate–putamen (C/P), lateral hypothalamus (LH), dorsal raphe nucleus (DRN) and midbrain (MID). ⁄p < 0.05 and ⁄⁄p < 0.01, unpaired 2-tail t-test. ⁄ p < 0.05 compared with the 0 h-group mice (n = 6).
A
B
Fig. 6. Effects of chronic rotenone treatments (1, 5, 10 and 20 mg/kg p.o.) (each group, n = 6) for 30 days on alcohol drinking behavior, alcohol intake (g/kg/day) (A) and alcohol preference (%) (B). ⁄p < 0.05 and ⁄⁄⁄p < 0.005 post hoc Bonferroni’s test. 0.5% CMC was administered to the control mice as vehicle (n = 6). Rotenone (suspended in 0.5% CMC) was orally administered once a day to C57BL/6J mice.
234
K. Yoshimoto et al. / Legal Medicine 14 (2012) 229–238
lated using a tissue punch (1.0 mm o.d.), as described by Ueda et al. [13]. The isolated brain tissues were rapidly frozen in liquid nitrogen and kept at 80 °C until the biochemical analysis. Each sample was weighed and homogenized using an ultrasonic-homogenizer in a mixture of 40 lL of 0.5 M perchloric acid, 30 lL of 0.1 M 2Na–EDTA, and 30 lL of 3,4-dihydroxybenzylamine (DHBA)–HCl as an internal standard. The homogenate was centrifuged (20,000g, 20 min) and the supernatant collected and filtered using a cellulose–acetate membrane disk (Toso, Tokyo, Japan). The tissue levels of norepinephrine (NE), DA, 3,4-dihydroxyphenylacetic acid (DOPAC), 5-HT, and 5-hydroxyindoleacetic acid (5HIAA) were determined using an internal standard. Separation was performed with a reversed-phase column (TSK-Gel ODS80Ts, 5 lm particle diameter, 150 2.0 mm; Toso). The mobile phase consisted of 0.1 M sodium phosphate, containing 0.1 mM EDTA–2Na with 15% methanol, 5% acetonitrile, and 0.3 mM sodium-1-octane sulfonic acid, adjusted to pH 2.8 with phosphoric acid. All separations were performed at a flow rate of 0.8 mL/ min. The levels of DA, 5-HT, and their metabolites (DOPAC and 5HIAA) were determined by high-performance liquid chromatography (HPLC) (3201 Nanospace, Shiseido Co., Tokyo, Japan) coupled with electrochemical detection (3005 Nanospace, Shiseido Co.), using a glassy carbon electrode set at +0.6 V vs. an Ag/AgCl reference electrode. All values are expressed as pmol/mg. Additionally, DA and 5-HT turnover indexes, changes of which indicate the increased release of DA and 5-HT at terminals, are shown as ratios of the metabolites to mother monoamines.
2.3.4. Blood alcohol and acetaldehyde assay All chronic rotenone-treated mice were deprived of ethanol for 24 h before the experiment to allow ethanol and acetaldehyde to clear from their circulations. Mice were injected with 2 g/kg i.p. ethanol (10% v/v). Blood samples were collected from mice at 0, 0.5, 1.0, 2.0 and 4.0 h after the injection of ethanol. Blood samples were immediately deproteinized at 0 °C in head space chromatography vials containing 100 lL (70%) trichloroacetic acid and 1-propanol as an internal standard. Gas chromatographic analysis for ethanol and acetaldehyde in blood was patterned after the method described previously [14,15]. Gas chromatographic analysis of acetaldehyde was carried out on blood collected at 1.0 and 2.0 h after the injection of ethanol.
2.3.5. Tissue preparation and immunohistochemistry Treated mice were perfused under deep anesthesia with pentobarbital (100 mg/kg, i.p.) [16]. Briefly, after perfusion, the brain was quickly removed and postfixed. The brain slices were cut 20 lm-thick using a cryostat and collected in 100 mM PBS containing 0.3% Triton X-100 (PBS-T). After several washes, slices were stored until use in free-floating state at 4 °C for immunohistochemical analysis. Brain slices were incubated with mouse monoclonal anti-tyrosine hydroxylase (TH, Sigma, St. Louis, MO, USA, diluted 1:10,000) or tryptophan hydroxylase (TPH, Chemicon Int., Temecula, CA, USA, 1:1000) antibody for three days at 4 °C, and was detected by an ABC Elite kit (Vector Laboratories, Burlin-
Fig. 7. Effects of chronic rotenone treatment (10 mg/kg p.o.) for 30 days on the levels of dopamine (DA) and 3,4-dihyroxyphenyacetic acid (DOPAC) in the nucleus accumbens (ACC), caudate–putamen (C/P), lateral hypothalamus (LH), dorsal raphe nucleus (DRN) and midbrain (MID). 0.5% CMC was administered orally to control mice as vehicle (n = 6). Rotenone: rotenone (suspended in 0.5% CMC) was orally administered to C57BL/6J mice at 10 mg/kg for 30 days (n = 6). ⁄p < 0.05 compared with the control.
K. Yoshimoto et al. / Legal Medicine 14 (2012) 229–238
game, CA, USA) with 3,30 -diaminobenzidine (DAB) with nickel ammonium. 2.4. Statistical analyses Data was analyzed with one- and two-way (monoamines time) repeated measures ANOVAs using GraphPad software. Post hoc comparisons were carried out with the Bonferroni t-test. In all cases, the accepted level of significance was set at p < 0.05 (unpaired t-test) (GraphPad Prism version 5; GraphPad Software, Inc., La Jolla, CA, USA). 3. Results
235
The levels of DA and DOPAC in the ACC, C/P, and LH were decreased at 2–8 h after the treatment with rotenone (20 mg/kg p.o.) (Fig. 2). In the same areas, the level of 5HIAA was also decreased at 8 h after the treatment (Fig. 4). Furthermore, the ratio of DOPAC to DA and of 5HIAA to 5-HT in the ACC, C/P, and LH were decreased at 2–8 h (Figs. 3 and 5). It is likely that a single rotenone treatment increases the sensitivity to dopaminergic neurons more than serotonergic neurons in the striatum and temporarily downregulates the dopaminergic functions continuously over 8 h (Figs. 2 and 3). There were no significant differences in the levels of DA, 5-HT and their metabolites between the two groups at 24 h after the treatment with rotenone (Figs. 2 and 4). The levels of NE did not show any significant changes in mice treated with rotenone acutely.
3.1. Experiment 1 3.2. Experiment 2 3.1.1. Acute rotenone treatment Fig. 1A shows the results of the 8-h time limited access to alcohol drinking behavior test. Rotenone (20 mg/kg p.o.) suppressed alcohol drinking behavior (Treatment; F(1, 45) = 21.44, p < 0.0001) with significant decreases in alcohol intake at 4 and 8 h (time; F(1, 45) = 11.88, p < 0.0001). However total alcohol consumption (g/kg/day) in the control and rotenone-treated groups did not differ significantly (Fig. 1B). The neurotoxical effect on alcohol drinking behavior was evident 8 h following single treatment with rotenone.
3.2.1. Chronic rotenone treatment The 30-day rotenone treatment p.o. enhanced voluntary alcohol drinking behavior, alcohol intake (g/kg/day) (F(4, 25) = 10.34, p < 0.0001) and alcohol preference (%) (F(4, 25) = 17.41, p < 0.0001), in a dose-dependent manner (Fig. 6A and B). In the rotenone (10 mg/kg p.o.)-treated groups, the levels of DA in the ACC, C/P, and LH, and 5-HT in the ACC, C/P, and DRN decreased significantly, as compared with the control groups (Figs. 7 and 8). On the other hand, the ratios of DOPAC to DA in
Fig. 8. Effects of chronic rotenone treatment (10 mg/kg p.o.) for 30 days on the levels of serotonine (5-HT), 5-hydroxyindolacetic acid (5HIAA) and the ratios of 5HIAA to 5-HT in the nucleus accumbens (ACC), caudate–putamen (C/P), lateral hypothalamus (LH), dorsal raphe nucleus (DRN) and midbrain (MID). 0.5% CMC was administered orally to control mice as vehicle (n = 6). Rotenone: rotenone (suspended in 0.5% CMC) was orally administered to C57BL/6J mice at 10 mg/kg for 30 days (n = 6). ⁄p < 0.05 compared with the control.
236
K. Yoshimoto et al. / Legal Medicine 14 (2012) 229–238
3.2.3. Immunohistochemistry As shown in representative photomicrographs, TH immunoreactivity in the 30 days rotenone-treated mice (10 mg/kg, p.o.) for 30 days was less in both the striatum and the substantia nigra (Fig. 10). On the other hand, there was no differences in the TPH immunoreactivity between the control and rotenone-treated mice (Fig. 11).
4. Discussion
Fig. 9. Blood alcohol concentration (mg/mL) in the chronic rotenone-treated mice (10 mg/kg, p.o.) for 30 days (d) and control mice (s) receiving 2 g/kg ethanol intraperitoneally. Each curves shows the concentration obtained from six mice in each group.
the ACC and C/P and of 5HIAA to 5-HT in the ACC, C/P, and DRN increased significantly (Table 1). These increases indicate increases in metabolic turnover or release of dopaminergic and serotonergic neurons. The levels of NE did not show any significant changes in mice treated with rotenone. It was suggested that enhanced alcohol drinking behavior compensates for the decrease of monoamine levels and the neurodegeneration in the rewarding pathway in mice administered rotenone. 3.2.2. Gas chromatography analysis Although the time course of blood alcohol concentrations showed a significant change (two-way-ANOVA; time; F(4, 50) = 61.8, p < 0.0001), blood alcohol concentrations at each of the time measured did not show a significant differences between the 30 days rotenone-treated mice (10 mg/kg, p.o.) and the control mice (Bonferroni post hoc test) (Fig. 9). Furthermore, there was not significant change of blood acetaldehyde concentration (0.4– 0.6 lg/mL) at 1.0 and 2.0 h in two groups after the injection of ethanol (2 g/kg, i.p.) (data not shown).
Alcohol abuse and alcoholism represent about one third of the total public health burden worldwide and are major medical problems as well as primary health care problems [17]. Although alcohol drinking is associated with environmental factors, industrial hygiene, pesticides and genetics, but the nature of their effects are still unknown. Acute treatment with rotenone caused decreases in the levels of DA, DOPAC and 5HIAA in the ACC, C/P and LH mainly at 2 and/or 8 h (Figs. 2 and 4). Rotenone (2 g/kg, i.p. for 40 days) affected a nigro-striatal pathway indicated by a 47% decrease in striatal DA levels [18]. On the other hand, chronic treatment with rotenone caused a decrease in DA levels in the ACC, C/P and LH and a decrease of 5-HT in the ACC and C/P without affecting the level of DOPAC and 5HIAA, respectively (Figs. 7 and 8). Rotenone has been demonstrated to cause more selective dopaminergic cell degeneration in vivo [10]. The degree of specific rotenone toxicity to dopaminergic neurons depends on not only complex I but also microtubule destabilization, vesicle accumulation and oxidative stress in dopaminergic neurons [2]. In the present study, accompanying the decrease in striatal DA and 5-HT levels of the ACC and C/P in the nigrostriatal pathway after chronic rotenone injections, DOPAC and 5HIAA levels were not significantly changed, but then the ratios of DOPAC/DA and 5HIAA/5-HT in the nigrostriatal pathway were increased (Table 1). The reason for the discrepancies of the different ratios in mice with acute and chronic treatment of rotenone remains unclear. Following the chronic rotenone treatments, volun-
Fig. 10. TH immunoreactivity in the striatum and substantia nigra. At 30 days, rotenone-treated mice (10 mg/kg, p.o.) were fixed and brain slices were prepared. Sections of the striatum and substantia nigra were immunostained by anti-TH antibody. Scale bars: 1 mm (in the striatum) and 100 lm (in the substantia nigra).
K. Yoshimoto et al. / Legal Medicine 14 (2012) 229–238
237
Fig. 11. TPH immunoreactivity in the striatum and raphe nucleus. At 30 days, rotenone-treated mice (10 mg/kg, p.o.) were fixed and brain slices were prepared. Sections of the striatum and raphe nucleus were immunostained by anti-TPH antiboby. Scale bars: 1 mm (in the striatum) and 100 lm (in the raphe nucleus).
tary alcohol drinking behavior actually increased in mice treated with rotenone dose-dependently (Fig. 6A and B). It was suggested that decreased DA levels in the striatum induces an increase in the release of DA to sustain neural functions and in turn enhance alcohol drinking behavior. Rotenone at 30 mg/kg p.o. for 28 days caused a significant loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra of mice [19]. It was consistent with our results of TH immunohistochemistry (Fig. 10). Rotenone also shows the actions of the reactive oxygen species in the mitochondrial compartment [2]. Consequently rotenone-treated animals with neural degeneration showed abnormal behavior, e.g. enhanced alcohol drinking behavior (Fig. 6), because rotenone causes specific dopaminergic injury with the complex I inhibitor or oxidative stress [20]. It was suggested that the causal relationship between mitochondrial dysfunction and specific dopaminergic injury increases alcohol drinking behavior. The increased alcohol drinking behaviors of the chronic rotenone-treated mice was associated with an increase in the number of surviving neurons in the nigrostriatal systems. 6-OHDA dopamine toxin-treated rats showed a significant increase in alcohol preference scores associated with the depletion of DA in the ACC and C/P [10,21]. The central DA neurons are important in the control of alcohol drinking behavior. These results are consistent with the present finding that rotenone-treated mice showed an increase in alcohol drinking behavior with the depletion of DA. Injection of 5,7-dihydroxytryptamine (5,7-DHT) into the brain has been shown to increase alcohol intake in the rat [22]. We have reported a negative correlation between brain 5-HT levels and alcohol drinking behavior in inbred strains of mice [12]. This is also consistent with the result which lower brain 5-HT levels produced an increase in alcohol drinking behavior [12,23]. It was suggested that rotenone’s influence on the occasional alcohol drinking behavior was associated with the central nigrostriatal pathway’s degeneration of DA and serotonergic neurons. We have to look more closely and neurochemically at the use of pesticides with neurotoxic actions and without the alcohol metabolism (Fig. 9). Rotenone mice model seems to mimic habitual alcohol drinking within human with progressive neurodegeneration in aging.
In conclusion, rotenone caused a decrease in DA and 5-HT levels in the nigrostriatal pathway. Rotenone-treated mice showed an increased alcohol drinking behavior associated with increases of turnover ratios of DA in the ACC and striatum. Ethical standard The authors declare that the experimental project complies with the current laws of the countries in which they were performed. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgement This study was supported in part by a Grant-in-Aid for Scientific Research (C) (21590740) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References [1] Langston JW. Parkinson’s disease: current and future challenges. Neurotoxicology 2002;23:443–50. [2] Choi WS, Kruse SE, Palmiter RD, Xia Z. Mitochondorial complex I inhibition is not required for dopaminergic neurons death induced by rotenone, MPP+, or paraquat. PNAS 2008;105:15136–41. [3] Richter F, Hamann M, Richter A. Chronic rotenone treatment induces behavioral effects but no pathological signs of Parkinsonism in mice. J Neurosci Res 2007;85:681–91. [4] Abou-Sleiman PM, Muqit MM, Wood NW. Expanding insights of mitochondrial dysfunction in Parkinson’s disease. Nat Rev Neurosci 2006;7:207–19. [5] Shere C, Kim JH, Betarbet R, Greenamyre JT. Subcutaneous rotenone exposure causes highly selective dopaminergic degeneration and alpha-synuclein aggregation. Exp Neurol 2003;179:9–16. [6] Beal MF. Experimental models of Parkinson’s disease. Nat Rev Neurosci 2001;5:325–34. [7] Thiruchelvam M, Richfield EK, Baggs RB, Tank AW, Cory-Slecha DA. The nigrostriatal dopaminergic system as a preferential target of repeated exposure to combined paraquat and maneb: implications for Parkinson’s disease. J Neurosci 2000;20:9207–14.
238
K. Yoshimoto et al. / Legal Medicine 14 (2012) 229–238
[8] Yoshimoto K, Kawamura K, Yayama K, Fujimiay T, Uemura K, Komura S. The effects of neurotoxins 6-hydroxydopamine and 5,7-dihydroxytryptamine into the rat nucleus accumbens on the alcohol drinking behavior. Jpn J Legal Med 1995;49:11–9. [9] Yoshimoto K, Kawai Y, Ueda S, Takeuchi H, Matsushita K, Yuri K, et al. Treating neonatal rat with 6-hydroxydopamine induced an increase in alcohol drinking behavior. Alcohol Clin Exp Res 1999;23:2s–6s. [10] Inden M, Kitamura Y, Takeuchi H, Yanagita T, Takata K, Kobayashi Y, et al. Neurodegeneration of mouse nigrostriatal dopaminergic system induced by repeated oral administration of rotenone is prevented by 4-phenylbutyrate, a chemical chaperone. J Neurochem 2007;101:1491–504. [11] Choi WS, Palmiter RD, Xia Z. Loss of mitochondrial complex I activity potentiates dopamine neuron death induced by microtubule dysfunction in a Parkinson’s disease model. J Cell Biol 2011;192:873–82. [12] Yoshimoto K, Komura S. Reexamination of the relationship between alcohol preference and brain monoamines in inbred strains of mice including senescence-accelerated mice. Pharmacol Biochem Behav 1987;27: 317–22. [13] Ueda S, Aikawa M, Ishizuya-Oka A, Koibuchi N, Yamaoka S, Yoshimoto K. Agerelated degeneration of the serotoninergic fibers in the zitter rat brain. Synapse 1998;30:62–70. [14] Fujimiya T, Yamaoka K, Fukui Y. Parallel first-order and Michaelis–Menten elimination kinetics of ethanol: respective role of ADH, non-ADH and firstorder pathways. J Pharmcol Exp Ther 1989;249:311–7.
[15] Okada T, Mizoi Y. Studies on the problem of blood acetaldehyde determination in man and level after alcohol intake. Jpn J Alcohol Drug Depend 1982;17: 141–59. [16] Kitamura Y, Inden M, Minamino H, Abe M, Takata K, Taniguchi T. The 6hydroxydopamine-induced nigrostriatal neurodegeneration produces microglia-like NG2 glial cells in the rat substantia nigra. Glia 2010;58: 1686–700. [17] Voas RB, Fell JC. Preventing alcohol-related problems through health policy research. Alcohol Res Health 2010;33:18–28. [18] He Y, Iman SZ, Dong Z, Jankovic J, Ali SF, Appel SH, et al. Role of nitric oxide in rotenone-induced nigro-striatal injury. J Neurochem 2003;86:1336–45. [19] Inden M, Kitamura Y, Abe M, Tamaki A, Takata K, Taniguchi T. Parkinsonian rotenone mouse model: reevaluation of long-term administration of rotenone in C57BL/6 mice. Biol Pharm Bull 2011;34:92–6. [20] Jenner P. Oxidative stress in Parkinson’s disease. Ann Neurol 2003;53:S26–38. [21] Kiianmaa K. Alcohol intake and ethanol intoxication in the rat: effect of 6OHDA-induced lesion of the ascending noradrenaline pathway. Eur J Pharmacol 1980;64:9–19. [22] Zhukov VN, Varkov AI, Burov YV. Effect of destruction of the serotonergic system on alcohol intake by rats in the early stages of experimental alcoholism. Bull Exp Biol Med 1985;99:604–6. [23] Zhou FC, Lumeng L, Li TK. Quantitative immunocytochemical evaluation of seronergic innervations in alcoholic rat brain. Neurochem Int 1995;26: 135–43.