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Reduction of lipid peroxidation in different rat brain areas after cabergoline treatment
Pharmacological Research, Vol. 42, No. 4, 2000 doi:10.1006/phrs.2000.0690, available online at http://www.idealibrary.com on
REDUCTION OF LIPID PEROXIDATION IN DIFFERENT RAT BRAIN AREAS AFTER CABERGOLINE TREATMENT NICOLETTA FINOTTI, LAURA CASTAGNA, ANTONIO MORETTI and FULVIO MARZATICO∗ Dipartimento de Scienze Fisiologiche-Farmacologiche Sezione di Farmacologia e Biotechnologie Farmacologiche Piazza Botta 11, 27100 Pavia Italy Accepted 30 March 2000
Oxidative stress and mitochondrial damage are involved in Parkinson’s disease (PD). Several drugs used for PD treatment have demonstrated antioxidant properties. To evaluate the antioxidant efficacy of cabergoline, an ergot derivative with a long plasma halflife, male Wistar rats were treated with vehicle or with 2.5 mg kg−1 and 10 mg kg−1 of the drug three, six, or 10 times at 48-h intervals. Cabergoline decreased basal lipid peroxide levels (LPO) in the hippocampus of rats given 10 mg kg−1 10 times, and in the striatum of rats given the same dose six or 10 times. Spontaneous LPO was inhibited in the hippocampus of rats given 10 mg kg−1 10 times. Stimulated LPO was decreased in the striatum of rats given 10 mg kg−1 six times and in rats given 2.5 and 10 mg kg−1 10 times. The ability of cabergoline to reduce LPO suggests its c 2000 Academic Press anti-lipoperoxidative properties.
K EY WORDS : Parkinson’s disease, oxidative stress, free radicals, cabergoline.
INTRODUCTION Parkinson’s disease (PD) is characterized by the progressive degeneration of dopaminergic neurons in the pars compacta of the substantia nigra [1–3]. The resulting dopamine deficiency in the nigro-striatal system is the basic PD neuropathology manifesting itself in the movement disorders in symptomatic parkinsonian patients [3]. These symptoms appear when approximately 80% of the nigro-striatal neurons have been lost [4, 5]. The specific mechanism of dopaminergic degeneration has not yet been established, but there is increasing support for the hypothesis that this degenerative process is mediated by reactive oxygen species (ROS) [1–3, 5–10]. It is known that ROS are involved in the lipid peroxidation process (LPO) by reaction with polyunsaturated fatty acids (PUFAs) leading to membrane cellular damage or death. The brain is extremely vulnerable to ROS damage because: (a) it is abundantly supplied with molecular oxygen; (b) brain tissue contains high concentrations of easily polyunsaturated lipids; (c) iron is preset in a high concentration in some brain areas; (d) the brain is relatively deficient in the protective mechanisms compared with other organs such as the liver [8]. ∗ Corresponding
author: Dipartimento di Scienze FisiologicheFarmacologiche, Sezione di Farmacologia e Biotecnologie Farmacologiche, Piazza Botta 11, 27100 Pavia. E-mail: [email protected] 1043–6618/00/100287–05/$35.00/0
The brain has got several enzymatic and non-enzymatic antioxidant defence mechanisms. Both isoforms of superoxide dismutase (MnSOD and CuZnSOD) convert the superoxide radical (O•− 2 ) to H2 O2 , which in turn can be deactivated by glutathione peroxidase (GSHPx) and catalase [8]. H2 O2 is a highly oxidizing species reacting with Fe++ (or Cu++ ) to form an hydroxyl radical (• OH). Hydroxyl radicals have a devastating effect on membrane lipids because they are capable of initiating the LPO process by abstracting a hydrogen atom from a polyunsaturated fatty acid in a membrane lipid. Vitamin E (a-tocopherol) and other antioxidants interact with alcoxy and peroxyradical to break the LPO process [8]. The oxidative metabolism of the dopamine is itself involved in the degeneration of dopaminergic neurons. Monoamine oxidase deaminates dopamine through enzymatic action, with H2 O2 as one of the products of the reaction [11]. It has also been shown that dopamine and the drug L-dopa can have a cytotoxic effect, causing the death of cultured neurons in the presence of Fe++ . The substantia nigra is at risk of damage through this mechanism, because it is rich in dopamine and Fe++ [12]. The abnormal metabolism of dopamine may in part explain the paradoxical L-dopa toxicity, sometimes seen in PD treatment [11]. Several drugs used in PD treatment have been shown to have pharmacological antioxidant effect. Pergolide (a dopamine agonist) and (−)deprenil (MAOB inhibitor) increased SOD activity in rat striatum [13–16], while (−) c 2000 Academic Press
288
Pharmacological Research, Vol. 42, No. 4, 2000
CH3
Some volumes of cabergoline solution and vehicle were given orally. Twenty-four hours after the last administration of cabergoline or vehicle, the animals were sacrificed. The brains were removed and the striatum and hippocampus areas were collected. The samples were homogenized in 20 mM Tris-HCl, pH 7.4. Homogenate aliquots were used for LPO assay while the evaluation of CuZnSOD, catalase and GSHPx was performed on postmitochondrial supernatants (the cytosolic fractions) and the MnSOD was evaluated on the crude mitochondrial fractions.
CH2CH2CH2N-CH3 CO-N-CONH-CH2CH3
N-CH2-CH=CH2
N H Fig. 1. Cabergoline (1[6-allylergolin-8b-gl)carbonil]-1-[(3-dimethylamine)-propyl]-3-ethylurea). Table I Groups of animals treated with different doses of vehicle or cabergoline for different administration times at 24-h intervals each Group
Dose
Treatment
A (controls) B (controls) C (controls) D E F G H I
Vehicle Vehicle Vehicle 2.5 mg kg−1 2.5 mg kg−1 2.5 mg kg−1 10 mg kg−1 10 mg kg−1 10 mg kg−1
3 times 6 times 10 times 3 times 6 times 10 times 3 times 6 times 10 times
deprenil has also been shown to increase SOD in the striatum of the dog brain [17]. Cabergoline (1[6-allylergolin-8β-γ l)carbonil]-1-[(3dimethylamine)-pr opyl]-3-ethylurea), an ergot derivative (Fig. 1), it s a dopamine agonist used in the treatment of PD. Because of its long plasma half-life, it can be given once daily. This drug can confer significant benefit to the patient with PD in terms of increasing the duration of ‘on’ phase and reducing ‘off’ period distonia [18, 19]. We investigated the antioxidant effects of cabergoline, comparing the hippocampus (poor in dopamine receptors) with the striatum (rich in dopamine receptors) of rat brain, measuring LPO as a marker of oxidative stress. We also assessed the activity of the antioxidant enzymes MnSOD, CuZnSOD, catalase and GSHPx in the same brain areas.
Lipid peroxidation assay The lipid peroxidation (LPO) assay was performed using the LPO-586 method (patented by Bioxytech SA, Bonneuil-sur-Marne, France). For basal LPO (the real amount of LPO in the tissue), the samples were performed immediately after homogenization. The physiological LPO process was assayed on homogenates incubated for 30 min at 37 ◦ C (spontaneous LPO). For stimulated LPO the samples were incubated in the presence of 100 µM FeSO4 for the same time and at the same temperature to evaluate the maximum LPO level triggered by the pro-oxidant system (FeSO4 ).
Enzyme assay The activity of GSHPx was measured following the oxidation of NADPH at 340 nm. A unit of GSHPx activity is defined as the amount of enzyme that triggers the oxidation of 1 µM of NADPH per minute [20]. Catalase activity was assayed by directly monitoring the decomposition of H2 O2 . One unit of catalase activity is defined as the amount of enzyme that decompose 1 µM of H2 O2 per minute [21]. The activity of SOD was measured following the reduction of cytocrome c by O•− 2 using xanthine/xanthine oxidase as the source for O•− 2 . One unit of SOD activity is defined as the amount of enzyme that inhibits the rate of cytocrome c reduction by 50% [22]. The SOD assay performed on the post-mitochondrial supernatant (cytosolic fraction) is referred to as CuZnSOD. The assay to determine the mitochondrial isoform was performed in the presence of 1 mM NaCN. Data were analysed by the paired two-tailed Student’s t-test with P < 0.05 considered significant.
RESULTS MATERIALS AND METHODS Male Wistar rats (250 g) were divided into nine groups of eight animals each, treated at 48-h intervals with vehicle or defferent doses of cabergoline, as shown in Table I. Cabergoline solutions were prepared daily by dissolving 4.5 mg of the free base in 0.2 ml of 0.1 M H3 PO4 and titrating to the correct concentration for the target dosage.
During the drug administration period, control rats had a normal increase in body weight while those rats treated with cabergoline lost body weight. The cohorts treated with 2.5 mg kg−1 of the drug lost 5–10% of their body weight. The animals treated with 10 mg kg−1 cabergoline lost 10–15% of their body weight and they also demonstrated aggressive behaviour. Figure 2 shows the basal and spontaneous LPO
Pharmacological Research, Vol. 42, No. 4, 2000
289
(a)
1.5 1.0
*
0.5
2.0 mmol / mg–1 protein
mmol / mg–1 protein
2.0
0.0
1.5 *
1.0
*
0.5 0.0
6
10
4
Control 2.5 mg kg–1 10 mg kg–1 (c)
3 *
2 1
3
6
10
3.0 mmol / mg–1 protein
3
mmol / mg–1 protein
(b)
(d)
2.5 2.0 1.5 1.0 0.5 0.0
0 3
6
10
3
6
10
Fig. 2. Mean ± SE of basal LPO (mol/mg proteins) in homogenate hippocampus (a) and striatum (b), and spontaneous LPO (mmol mg−1 proteins) in homogenate hippocampus (c) and striatum (d). Statistical analysis: ∗ P < 0.05 vs the control.
Table II Means ± SE of enzymatic activities evaluated in hippocampus of rat brain at different doses of cabergoline and at different treatment times Control
CuZnSOD (U mg−1 prot)
MnSOD (U mg−1 prot)
GSHPx (mU mg−1 prot)
CAT (mU mg−1 prot)
Treated 3 times
2.5 mg kg−1 10 mg kg−1
10.1 ± 2.5 17.2 ± 5.2 16.4 ± 3.6
3.1 ± 0.9 4.1 ± 1.3 4.9 ± 1.6
50.1 ± 5.2 44.8 ± 4.7 45.1 ± 3.4
5.8 ± 1.2 4.5 ± 0.6 3.7 ± 1.2
Treated 6 times
2.5 mg kg−1 10 mg kg−1
14.7 ± 3.9 17.9 ± 6.1 14.5 ± 2.2
4.5 ± 1.2 4.3 ± 1.7 3.1 ± 1.2
50.2 ± 8.7 43.4 ± 5.9 42.8 ± 6.1
3.6 ± 1.2 4.2 ± 1.3 3.4 ± 0.8
Treated 10 times
2.5 mg kg−1 10 mg kg−1
9.6 ± 2.5 13.3 ± 3.2 10.2 ± 2.7
2.9 ± 0.9 3.2 ± 0.9 3.1 ± 1.1
44.2 ± 7.2 39.3 ± 3.4 38.7 ± 2.6
3.8 ± 0.8 3.5 ± 1.2 3.6 ± 0.5
Table III Means ± SE of enzymatic activities evaluated in striatum of rat brain at different doses of cabergoline and at different treatment times Control
CuZnSOD (U mg−1 prot)
MnSOD (U mg−1 prot)
GSHPx (mU mg−1 prot)
CAT (mU mg−1 prot)
Treated 3 times
2.5 mg kg−1 10 mg kg−1
12.5 ± 2.5 18.7 ± 5.3 12.6 ± 4.1
3.5 ± 0.9 5.2 ± 1.4 4.8 ± 1.9
40.9 ± 5.1 37.5 ± 4.4 44.9 ± 4.8
4.1 ± 0.9 2.8 ± 0.6 3.5 ± 0.8
Treated 6 times
2.5 mg kg−1 10 mg kg−1
19.8 ± 2.6 17.2 ± 2.5 15.8 ± 2.5
5.8 ± 1.1 6.1 ± 1.4 4.5 ± 0.6
42.3 ± 5.1 34.8 ± 5.8 32.1 ± 6.1
3.8 ± 0.7 3.3 ± 0.5 3.0 ± 0.4
Treated 10 times
2.5 mg kg−1 10 mg kg−1
11.7 ± 1.8 12.7 ± 2.4 10.8 ± 2.5
3.2 ± 0.5 4.0 ± 0.9 3.1 ± 1.1
45.2 ± 4.7 34.7 ± 4.5 36.9 ± 4.8
2.7 ± 0.3 2.5 ± 0.2 2.6 ± 0.3
16 14 12 10 8 6 4 2 0
(a)
3
6
10
mmol / mg–1 protein
Pharmacological Research, Vol. 42, No. 4, 2000
mmol / mg–1 protein
290
16 14 12 10 8 6 4 2 0
Control 2.5 mg kg–1 10 mg kg–1
(b) *
3
6
* *
10
Fig. 3. Mean ± SE of stimulated LPO (mmol mg−1 proteins) in homogenate hippocampus (a) and striatum (b). Statistical analysis: ∗ P < 0.05 vs the control.
values control and cabergoline-treated rats at 2.5 and 10 mg kg−1 . Cabergoline treatment significantly decreased basal LPO levels vs controls in the hippocampus of animals treated with 10 mg kg−1 10 times and in striatum at the same dose given 6 and 10 times. The spontaneous LPO production was inhibited only in the hippocampus of those rats treated with 10 mg kg−1 10 times vs the controls. Figure 3 shows the stimulated LPO values of the control rats and the cabergoline-treated rats at 2.5 and 10 mg kg−1 . Cabergoline treatment decreased stimulated LPO levels vs the controls only in the striatum of rats given 10 mg kg−1 six times and in rats given both 2.5 and 10 mg kg−1 10 times. The activities of antioxidant enzymes (CuZnSOD, MnSOD, GSHPx, and catalase) were not affected by treatment with cabergoline in both striatum and hippocampus (Table II).
CONCLUSIONS Post-mortem brains of patients with PD have shown increased LPO levels and decreased antioxidant defences in the pars compacta of the substantia nigra [23]. Experimental animal models make use of oxidative stress to induce Parkinson’s syndrome: 1-methyl4-phenyl 1, 2, 3, 6 tetrahydropyridine (MPTP) and 6-hydroxydopamine (6OHDA) promote LPO processes by interaction with transition metals [11, 24, 25]. Some experimental evidence pointed out that high doses of L -dopa are involved in increasing LPO in dopaminergicdamaged neurons [26, 27]. This hypothesis is supported by evidence that abnormal higher dopamine metabolism induces neurodegeneration by intermediate radical products [11]. Several drugs given in PD therapy have antioxidant effects. Bromocriptine is widely used in treating PD; this drug permits a delay in the use of L-dopa by increasing the duration of the ‘on’ phase. It has been shown to scavenge O•− 2 as well as lipid hydroxyl radicals [28–30]. (−)Deprenil and isatin, MAOB inhibitors, increased SOD
activity [16, 31, 32] but its in vitro enhanced dismutative activity is not yet demonstrated [33]. Pergolide is an O•− scavenger that could induce 2 SOD activity [15]. Pramipexole in vivo and in vitro inhibited ROS production and protected the membrane permeability [34, 35]. Cabergoline is a long-lasting (65 h) D2 agonist drug. Clinical study has pointed out the ability of cabergoline to smooth the motor fluctuation and decrease the akinetic episodes in parkinsonian patients [18, 19, 36]. Our study points out that repeated oral administration of cabergoline can reduce basal, spontaneous and stimulated LPO in rat brain. This effect is evident after six and 10 treatments spaced at 48-h intervals at 10 mg kg−1 dose. For LPO the effect was seen also at 2.5 mg kg−1 given 10 times. This effect cannot be ascribed to induction of antioxidant enzymes such as SOD, GSHPx and catalase. The cabergoline antioxidant property should be associated with its long-lasting activity by reducing dopamine turn-over and its toxic metabolites such as H2 O2 and aldehydes. In fact stimulated LPO levels decreased significantly in striatum after six and 10 cabergoline administrations. This evidence could be explained by dopamine turnover reduction in the striatum: in the presence of Fe++ cabergoline neutralizes the LPO process that in this brain area should be exacerbated by H2 O2 derived from dopamine metabolism. In the hippocampus, where there should be no dopamine metabolism, stimulated LPO levels did not decrease after any cabergoline administrations. Cabergoline could have some antioxidant properties associated with its molecular structure but there are no data published concerning in vitro scavenger or ‘chain breaking’ action of cabergoline per se. Our data support the hypothesis that cabergoline could have antilipoperoxidative properties shown by reduction of basal and spontaneous LPO in the hippocampus and basal LPO in striatum. On the other hand we need further studies to confirm if the pharmacological antioxidant effect of cabergoline could be translate into its clinical efficacy when treating PD.
Pharmacological Research, Vol. 42, No. 4, 2000
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REDUCTION OF LIPID PEROXIDATION IN DIFFERENT RAT BRAIN AREAS AFTER CABERGOLINE TREATMENT NICOLETTA FINOTTI, LAURA CASTAGNA, ANTONIO MORETTI and FULVIO MARZATICO∗ Dipartimento de Scienze Fisiologiche-Farmacologiche Sezione di Farmacologia e Biotechnologie Farmacologiche Piazza Botta 11, 27100 Pavia Italy Accepted 30 March 2000
Oxidative stress and mitochondrial damage are involved in Parkinson’s disease (PD). Several drugs used for PD treatment have demonstrated antioxidant properties. To evaluate the antioxidant efficacy of cabergoline, an ergot derivative with a long plasma halflife, male Wistar rats were treated with vehicle or with 2.5 mg kg−1 and 10 mg kg−1 of the drug three, six, or 10 times at 48-h intervals. Cabergoline decreased basal lipid peroxide levels (LPO) in the hippocampus of rats given 10 mg kg−1 10 times, and in the striatum of rats given the same dose six or 10 times. Spontaneous LPO was inhibited in the hippocampus of rats given 10 mg kg−1 10 times. Stimulated LPO was decreased in the striatum of rats given 10 mg kg−1 six times and in rats given 2.5 and 10 mg kg−1 10 times. The ability of cabergoline to reduce LPO suggests its c 2000 Academic Press anti-lipoperoxidative properties.
K EY WORDS : Parkinson’s disease, oxidative stress, free radicals, cabergoline.
INTRODUCTION Parkinson’s disease (PD) is characterized by the progressive degeneration of dopaminergic neurons in the pars compacta of the substantia nigra [1–3]. The resulting dopamine deficiency in the nigro-striatal system is the basic PD neuropathology manifesting itself in the movement disorders in symptomatic parkinsonian patients [3]. These symptoms appear when approximately 80% of the nigro-striatal neurons have been lost [4, 5]. The specific mechanism of dopaminergic degeneration has not yet been established, but there is increasing support for the hypothesis that this degenerative process is mediated by reactive oxygen species (ROS) [1–3, 5–10]. It is known that ROS are involved in the lipid peroxidation process (LPO) by reaction with polyunsaturated fatty acids (PUFAs) leading to membrane cellular damage or death. The brain is extremely vulnerable to ROS damage because: (a) it is abundantly supplied with molecular oxygen; (b) brain tissue contains high concentrations of easily polyunsaturated lipids; (c) iron is preset in a high concentration in some brain areas; (d) the brain is relatively deficient in the protective mechanisms compared with other organs such as the liver [8]. ∗ Corresponding
author: Dipartimento di Scienze FisiologicheFarmacologiche, Sezione di Farmacologia e Biotecnologie Farmacologiche, Piazza Botta 11, 27100 Pavia. E-mail: [email protected] 1043–6618/00/100287–05/$35.00/0
The brain has got several enzymatic and non-enzymatic antioxidant defence mechanisms. Both isoforms of superoxide dismutase (MnSOD and CuZnSOD) convert the superoxide radical (O•− 2 ) to H2 O2 , which in turn can be deactivated by glutathione peroxidase (GSHPx) and catalase [8]. H2 O2 is a highly oxidizing species reacting with Fe++ (or Cu++ ) to form an hydroxyl radical (• OH). Hydroxyl radicals have a devastating effect on membrane lipids because they are capable of initiating the LPO process by abstracting a hydrogen atom from a polyunsaturated fatty acid in a membrane lipid. Vitamin E (a-tocopherol) and other antioxidants interact with alcoxy and peroxyradical to break the LPO process [8]. The oxidative metabolism of the dopamine is itself involved in the degeneration of dopaminergic neurons. Monoamine oxidase deaminates dopamine through enzymatic action, with H2 O2 as one of the products of the reaction [11]. It has also been shown that dopamine and the drug L-dopa can have a cytotoxic effect, causing the death of cultured neurons in the presence of Fe++ . The substantia nigra is at risk of damage through this mechanism, because it is rich in dopamine and Fe++ [12]. The abnormal metabolism of dopamine may in part explain the paradoxical L-dopa toxicity, sometimes seen in PD treatment [11]. Several drugs used in PD treatment have been shown to have pharmacological antioxidant effect. Pergolide (a dopamine agonist) and (−)deprenil (MAOB inhibitor) increased SOD activity in rat striatum [13–16], while (−) c 2000 Academic Press
288
Pharmacological Research, Vol. 42, No. 4, 2000
CH3
Some volumes of cabergoline solution and vehicle were given orally. Twenty-four hours after the last administration of cabergoline or vehicle, the animals were sacrificed. The brains were removed and the striatum and hippocampus areas were collected. The samples were homogenized in 20 mM Tris-HCl, pH 7.4. Homogenate aliquots were used for LPO assay while the evaluation of CuZnSOD, catalase and GSHPx was performed on postmitochondrial supernatants (the cytosolic fractions) and the MnSOD was evaluated on the crude mitochondrial fractions.
CH2CH2CH2N-CH3 CO-N-CONH-CH2CH3
N-CH2-CH=CH2
N H Fig. 1. Cabergoline (1[6-allylergolin-8b-gl)carbonil]-1-[(3-dimethylamine)-propyl]-3-ethylurea). Table I Groups of animals treated with different doses of vehicle or cabergoline for different administration times at 24-h intervals each Group
Dose
Treatment
A (controls) B (controls) C (controls) D E F G H I
Vehicle Vehicle Vehicle 2.5 mg kg−1 2.5 mg kg−1 2.5 mg kg−1 10 mg kg−1 10 mg kg−1 10 mg kg−1
3 times 6 times 10 times 3 times 6 times 10 times 3 times 6 times 10 times
deprenil has also been shown to increase SOD in the striatum of the dog brain [17]. Cabergoline (1[6-allylergolin-8β-γ l)carbonil]-1-[(3dimethylamine)-pr opyl]-3-ethylurea), an ergot derivative (Fig. 1), it s a dopamine agonist used in the treatment of PD. Because of its long plasma half-life, it can be given once daily. This drug can confer significant benefit to the patient with PD in terms of increasing the duration of ‘on’ phase and reducing ‘off’ period distonia [18, 19]. We investigated the antioxidant effects of cabergoline, comparing the hippocampus (poor in dopamine receptors) with the striatum (rich in dopamine receptors) of rat brain, measuring LPO as a marker of oxidative stress. We also assessed the activity of the antioxidant enzymes MnSOD, CuZnSOD, catalase and GSHPx in the same brain areas.
Lipid peroxidation assay The lipid peroxidation (LPO) assay was performed using the LPO-586 method (patented by Bioxytech SA, Bonneuil-sur-Marne, France). For basal LPO (the real amount of LPO in the tissue), the samples were performed immediately after homogenization. The physiological LPO process was assayed on homogenates incubated for 30 min at 37 ◦ C (spontaneous LPO). For stimulated LPO the samples were incubated in the presence of 100 µM FeSO4 for the same time and at the same temperature to evaluate the maximum LPO level triggered by the pro-oxidant system (FeSO4 ).
Enzyme assay The activity of GSHPx was measured following the oxidation of NADPH at 340 nm. A unit of GSHPx activity is defined as the amount of enzyme that triggers the oxidation of 1 µM of NADPH per minute [20]. Catalase activity was assayed by directly monitoring the decomposition of H2 O2 . One unit of catalase activity is defined as the amount of enzyme that decompose 1 µM of H2 O2 per minute [21]. The activity of SOD was measured following the reduction of cytocrome c by O•− 2 using xanthine/xanthine oxidase as the source for O•− 2 . One unit of SOD activity is defined as the amount of enzyme that inhibits the rate of cytocrome c reduction by 50% [22]. The SOD assay performed on the post-mitochondrial supernatant (cytosolic fraction) is referred to as CuZnSOD. The assay to determine the mitochondrial isoform was performed in the presence of 1 mM NaCN. Data were analysed by the paired two-tailed Student’s t-test with P < 0.05 considered significant.
RESULTS MATERIALS AND METHODS Male Wistar rats (250 g) were divided into nine groups of eight animals each, treated at 48-h intervals with vehicle or defferent doses of cabergoline, as shown in Table I. Cabergoline solutions were prepared daily by dissolving 4.5 mg of the free base in 0.2 ml of 0.1 M H3 PO4 and titrating to the correct concentration for the target dosage.
During the drug administration period, control rats had a normal increase in body weight while those rats treated with cabergoline lost body weight. The cohorts treated with 2.5 mg kg−1 of the drug lost 5–10% of their body weight. The animals treated with 10 mg kg−1 cabergoline lost 10–15% of their body weight and they also demonstrated aggressive behaviour. Figure 2 shows the basal and spontaneous LPO
Pharmacological Research, Vol. 42, No. 4, 2000
289
(a)
1.5 1.0
*
0.5
2.0 mmol / mg–1 protein
mmol / mg–1 protein
2.0
0.0
1.5 *
1.0
*
0.5 0.0
6
10
4
Control 2.5 mg kg–1 10 mg kg–1 (c)
3 *
2 1
3
6
10
3.0 mmol / mg–1 protein
3
mmol / mg–1 protein
(b)
(d)
2.5 2.0 1.5 1.0 0.5 0.0
0 3
6
10
3
6
10
Fig. 2. Mean ± SE of basal LPO (mol/mg proteins) in homogenate hippocampus (a) and striatum (b), and spontaneous LPO (mmol mg−1 proteins) in homogenate hippocampus (c) and striatum (d). Statistical analysis: ∗ P < 0.05 vs the control.
Table II Means ± SE of enzymatic activities evaluated in hippocampus of rat brain at different doses of cabergoline and at different treatment times Control
CuZnSOD (U mg−1 prot)
MnSOD (U mg−1 prot)
GSHPx (mU mg−1 prot)
CAT (mU mg−1 prot)
Treated 3 times
2.5 mg kg−1 10 mg kg−1
10.1 ± 2.5 17.2 ± 5.2 16.4 ± 3.6
3.1 ± 0.9 4.1 ± 1.3 4.9 ± 1.6
50.1 ± 5.2 44.8 ± 4.7 45.1 ± 3.4
5.8 ± 1.2 4.5 ± 0.6 3.7 ± 1.2
Treated 6 times
2.5 mg kg−1 10 mg kg−1
14.7 ± 3.9 17.9 ± 6.1 14.5 ± 2.2
4.5 ± 1.2 4.3 ± 1.7 3.1 ± 1.2
50.2 ± 8.7 43.4 ± 5.9 42.8 ± 6.1
3.6 ± 1.2 4.2 ± 1.3 3.4 ± 0.8
Treated 10 times
2.5 mg kg−1 10 mg kg−1
9.6 ± 2.5 13.3 ± 3.2 10.2 ± 2.7
2.9 ± 0.9 3.2 ± 0.9 3.1 ± 1.1
44.2 ± 7.2 39.3 ± 3.4 38.7 ± 2.6
3.8 ± 0.8 3.5 ± 1.2 3.6 ± 0.5
Table III Means ± SE of enzymatic activities evaluated in striatum of rat brain at different doses of cabergoline and at different treatment times Control
CuZnSOD (U mg−1 prot)
MnSOD (U mg−1 prot)
GSHPx (mU mg−1 prot)
CAT (mU mg−1 prot)
Treated 3 times
2.5 mg kg−1 10 mg kg−1
12.5 ± 2.5 18.7 ± 5.3 12.6 ± 4.1
3.5 ± 0.9 5.2 ± 1.4 4.8 ± 1.9
40.9 ± 5.1 37.5 ± 4.4 44.9 ± 4.8
4.1 ± 0.9 2.8 ± 0.6 3.5 ± 0.8
Treated 6 times
2.5 mg kg−1 10 mg kg−1
19.8 ± 2.6 17.2 ± 2.5 15.8 ± 2.5
5.8 ± 1.1 6.1 ± 1.4 4.5 ± 0.6
42.3 ± 5.1 34.8 ± 5.8 32.1 ± 6.1
3.8 ± 0.7 3.3 ± 0.5 3.0 ± 0.4
Treated 10 times
2.5 mg kg−1 10 mg kg−1
11.7 ± 1.8 12.7 ± 2.4 10.8 ± 2.5
3.2 ± 0.5 4.0 ± 0.9 3.1 ± 1.1
45.2 ± 4.7 34.7 ± 4.5 36.9 ± 4.8
2.7 ± 0.3 2.5 ± 0.2 2.6 ± 0.3
16 14 12 10 8 6 4 2 0
(a)
3
6
10
mmol / mg–1 protein
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mmol / mg–1 protein
290
16 14 12 10 8 6 4 2 0
Control 2.5 mg kg–1 10 mg kg–1
(b) *
3
6
* *
10
Fig. 3. Mean ± SE of stimulated LPO (mmol mg−1 proteins) in homogenate hippocampus (a) and striatum (b). Statistical analysis: ∗ P < 0.05 vs the control.
values control and cabergoline-treated rats at 2.5 and 10 mg kg−1 . Cabergoline treatment significantly decreased basal LPO levels vs controls in the hippocampus of animals treated with 10 mg kg−1 10 times and in striatum at the same dose given 6 and 10 times. The spontaneous LPO production was inhibited only in the hippocampus of those rats treated with 10 mg kg−1 10 times vs the controls. Figure 3 shows the stimulated LPO values of the control rats and the cabergoline-treated rats at 2.5 and 10 mg kg−1 . Cabergoline treatment decreased stimulated LPO levels vs the controls only in the striatum of rats given 10 mg kg−1 six times and in rats given both 2.5 and 10 mg kg−1 10 times. The activities of antioxidant enzymes (CuZnSOD, MnSOD, GSHPx, and catalase) were not affected by treatment with cabergoline in both striatum and hippocampus (Table II).
CONCLUSIONS Post-mortem brains of patients with PD have shown increased LPO levels and decreased antioxidant defences in the pars compacta of the substantia nigra [23]. Experimental animal models make use of oxidative stress to induce Parkinson’s syndrome: 1-methyl4-phenyl 1, 2, 3, 6 tetrahydropyridine (MPTP) and 6-hydroxydopamine (6OHDA) promote LPO processes by interaction with transition metals [11, 24, 25]. Some experimental evidence pointed out that high doses of L -dopa are involved in increasing LPO in dopaminergicdamaged neurons [26, 27]. This hypothesis is supported by evidence that abnormal higher dopamine metabolism induces neurodegeneration by intermediate radical products [11]. Several drugs given in PD therapy have antioxidant effects. Bromocriptine is widely used in treating PD; this drug permits a delay in the use of L-dopa by increasing the duration of the ‘on’ phase. It has been shown to scavenge O•− 2 as well as lipid hydroxyl radicals [28–30]. (−)Deprenil and isatin, MAOB inhibitors, increased SOD
activity [16, 31, 32] but its in vitro enhanced dismutative activity is not yet demonstrated [33]. Pergolide is an O•− scavenger that could induce 2 SOD activity [15]. Pramipexole in vivo and in vitro inhibited ROS production and protected the membrane permeability [34, 35]. Cabergoline is a long-lasting (65 h) D2 agonist drug. Clinical study has pointed out the ability of cabergoline to smooth the motor fluctuation and decrease the akinetic episodes in parkinsonian patients [18, 19, 36]. Our study points out that repeated oral administration of cabergoline can reduce basal, spontaneous and stimulated LPO in rat brain. This effect is evident after six and 10 treatments spaced at 48-h intervals at 10 mg kg−1 dose. For LPO the effect was seen also at 2.5 mg kg−1 given 10 times. This effect cannot be ascribed to induction of antioxidant enzymes such as SOD, GSHPx and catalase. The cabergoline antioxidant property should be associated with its long-lasting activity by reducing dopamine turn-over and its toxic metabolites such as H2 O2 and aldehydes. In fact stimulated LPO levels decreased significantly in striatum after six and 10 cabergoline administrations. This evidence could be explained by dopamine turnover reduction in the striatum: in the presence of Fe++ cabergoline neutralizes the LPO process that in this brain area should be exacerbated by H2 O2 derived from dopamine metabolism. In the hippocampus, where there should be no dopamine metabolism, stimulated LPO levels did not decrease after any cabergoline administrations. Cabergoline could have some antioxidant properties associated with its molecular structure but there are no data published concerning in vitro scavenger or ‘chain breaking’ action of cabergoline per se. Our data support the hypothesis that cabergoline could have antilipoperoxidative properties shown by reduction of basal and spontaneous LPO in the hippocampus and basal LPO in striatum. On the other hand we need further studies to confirm if the pharmacological antioxidant effect of cabergoline could be translate into its clinical efficacy when treating PD.
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