The influence and the mechanism of docosahexaenoic acid on a mouse model of Parkinson’s disease

Neurochemistry International 59 (2011) 664–670

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The influence and the mechanism of docosahexaenoic acid on a mouse model of Parkinson’s disease Ozlem Ozsoy a, Yasemin Seval-Celik b, Gulay Hacioglu a, Piraye Yargicoglu c, Ramazan Demir b, Aysel Agar a,⇑, Mutay Aslan d a

Akdeniz University, Faculty of Medicine, Department of Physiology, Antalya, Turkey Akdeniz University, Faculty of Medicine, Department of Histology and Embryology, Antalya, Turkey Akdeniz University, Faculty of Medicine, Department of Biophysics, Antalya, Turkey d Akdeniz University, Faculty of Medicine, Department of Biochemistry, Antalya, Turkey b c

a r t i c l e

i n f o

Article history: Received 26 October 2010 Received in revised form 23 May 2011 Accepted 18 June 2011 Available online 28 June 2011 Keywords: Parkinson’s disease 1-Methyl-4-phenyl-1,2,3,6tetrahydropyridine Docosahexaenoic acid Lipid peroxidation Antioxidant enzymes Mice

a b s t r a c t This study aimed to investigate the effects of docosahexaenoic acid (DHA) on the oxidative stress that occurs in an experimental mouse model of Parkinson’s disease (PD). An experimental model of PD was created by four intraperitoneal injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (4  20 mg/kg, at 12 h intervals). Docosahexaenoic acid was given daily by gavage for 4 weeks (36 mg/ kg/day). The motor activity of the mice was evaluated via the pole test, and the dopaminergic lesion was determined by immunohistochemical analysis for tyrosine hydroxylase (TH)-immunopositive cells. The activity of antioxidant enzymes in the brain were determined by spectrophotometric assays and the concentration of thiobarbituric acid-reactive substances (TBARS) were measured as an index of oxidative damage. The number of apoptotic dopaminergic cells significantly increased in MPTP-treated mice compared to controls. Although DHA significantly diminished the number of cell deaths in MPTP-treated mice, it did not improve the decreased motor activity observed in the experimental PD model. Docosahexaenoic acid significantly diminished the amount of cell death in the MPTP + DHA group as compared to the MPTP group. TBARS levels in the brain were significantly increased following MPTP treatment. Glutathione peroxidase (GPx) and catalase (CAT) activities of brain were unaltered in all groups. The activity of brain superoxide dismutase (SOD) was decreased in the MPTP-treated group compared to the control group, but DHA treatment did not have an effect on SOD activity in the MPTP + DHA group. Our current data show that DHA treatment exerts neuroprotective actions on an experimental mouse model of PD. There was a decrease tendency in brain lipid oxidation of MPTP mice but it did not significantly. Ó 2011 Elsevier B.V. All rights reserved.

1. Introduction Parkinson’s disease (PD), first described by James Parkinson in 1817 (Parkinson, 1817), is a neurodegenerative disorder that is characterized by a progressive loss of neuromelanin-containing

Abbreviations: ANOVA, analysis of variance; CAT, catalase; DA, dopamine; DAB, 3,3-diaminobenzidine tetrahydrochloride dihydrate; DHA, docosahexaenoic acid; DNA, deoxyribonucleic acid; GPx, glutathione peroxidase; GR, glutathione reductase; HCl, hydrochloric acid; MDA, malondialdehyde; MPTP, 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine; 6-OHDA, 6-hydroxydopamine; PD, Parkinson’s disease; PI3-K, phosphatidylinositol-3 kinase; PUFA, polyunsaturated fatty acid; ROS, reactive oxygen species; SN, substantia nigra; SOD, superoxide dismutase; TBA, 2thiobarbituric acid; TBARS, thiobarbituric acid-reactive substances; TH, tyrosine hydroxylase; TO, tuna oil; VE, vitamin E. ⇑ Corresponding author. Address: Department of Physiology, Faculty of Medicine, Akdeniz University, Arapsuyu, 07070 Antalya, Turkey. Tel.: +90 242 249 6958; fax: +90 242 227 4483. E-mail address: [email protected] (A. Agar). 0197-0186/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.neuint.2011.06.012

dopaminergic neurons in the substantia nigra (SN) and a depletion of dopamine (DA) in the striatum. Common parkinsonian symptoms are resting tremor, bradykinesia, rigidity and loss of postural reflexes (Jankovic, 2008). The clinical symptoms appear after 50– 60% of neuronal loss, cell death and degeneration of the SN (McGeer et al., 1988), which is mostly age-dependent. A large body of evidence suggests that mitochondrial dysfunction and oxidative stress-mediated mechanisms may be, at least partially, responsible for the degeneration (Schapira et al., 1990). Further evidence suggests that oxidative stress is a pathogenetic factor for PD, which emerged with the discovery of selective dopaminergic toxins, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Burns et al., 1983; Langston et al., 1983). MPTP exposure to monkeys and mice has induced many of the same biochemical and neuropathological changes in the nigrostriatal dopaminergic pathway as found in post-mortem studies of PD patients, such as a loss of dopaminergic cells in the SN that were

O. Ozsoy et al. / Neurochemistry International 59 (2011) 664–670

associated with oxidative stress (Kaul et al., 2003; Przedborski et al., 1996; Zhang et al., 2000). MPTP toxicity involves an increase in reactive oxygen species (ROS) and in damage to mitochondrial DNA (Mandavilli et al., 2000). Although MPTP produces acute intoxication, it is one of the best animal models for studying PD (Zhang et al., 2000). Studies of post-mortem brains from PD patients clearly indicate that these brains have been subjected to an oxidative challenge (Koutsilieri et al., 2002). Lipid peroxidation levels have been found to be increased in brain tissues from parkinsonian patients (Dexter et al., 1989, 1994) and MPTP-injected mice (Abdel-Wahab, 2005; Choi et al., 2005; Rajeswari and Sabesan, 2008; Rojas and Rios, 1993; Sankar et al., 2007; Xie et al., 2007). These alterations in the levels of oxidative stress were generally accompanied by changes in antioxidant defense mechanisms (Koutsilieri et al., 2002). Docosahexaenoic acid (DHA), a major polyunsaturated fatty acid (PUFA) found in the phospholipid fraction of the brain, is essential for normal neural function (Akbar et al., 2005). Docosahexaenoic acid alone constitutes >17% (by weight) of the total fatty acids in the brain of adult rats (Hamano et al., 1996; Hashimoto et al., 2002). PUFA levels were reduced in parkinsonian SN compared to other brain regions and to control tissue (Dexter et al., 1989). Docosahexaenoic acid cannot be synthesized de novo in mammals; therefore, it is most likely obtained through their diet. Studies in animals clearly show that oral intake of DHA can alter brain DHA concentrations and thereby modify brain functions (Bourre et al., 1993; Galli et al., 1971; Levant et al., 2007; Murthy et al., 2002; Pawlosky et al., 2001). This information provides us with an opportunity to use DHA as a nutraceutical or pharmaceutical tool to treat brain disorders such as PD. A neuroprotective action of DHA has been observed in a mouse model of PD (Bousquet et al., 2008). Cansev et al. (2008) has shown that DHA treatment reduced ipsilateral rotations by 47% and significantly elevated striatal DA, tyrosine hydroxylase (TH) activity and TH protein on the lesioned side in a rat model of PD. One of the first proposed mechanisms for the neuroprotective action of DHA is exerting anti-oxidative activity in vivo (Bazan, 2005; Calon et al., 2004; Hashimoto et al., 2002; Wu et al., 2004; Yavin et al., 2002). Indeed, evidence of DHA increasing glutathione reductase (GR) activity (Hashimoto et al., 2002) and decreasing the accumulation of oxidized proteins (Calon et al., 2004; Wu et al., 2004) as well as lipid peroxide and ROS levels (Hashimoto et al., 2002, 2005) in the brain have been published. We expect that DHA may have beneficial effects on the symptoms of PD; however, the relationship between DHA and lipid peroxidation/antioxidant enzyme activities in the brain of experimental Parkinson’s has not been investigated. The purpose of this present study is to establish the extent to which alterations in brain lipid peroxidation/antioxidant status occurs in PD and to evaluate whether DHA may help to overcome these alterations. 2. Materials and methods Male C57BL/6 mice (10 months old, weighing 25–30 g) were obtained from the Marmara Research Unit. The animals were housed in stainless steel cages (10 per cage) in an air-conditioned room (22 ± 2 °C with a 12:12 h, light:dark cycle). All experimental protocols conducted on mice were performed in accordance with the standards established by the Institutional Animal Care and Use Committee at Akdeniz University Medical School. 2.1. Experimental design Mice were randomly divided into four experimental groups as follows: control (n = 15); DHA-treated (DHA) (n = 15); MPTP-in-

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jected (MPTP) (n = 15), and DHA-treated + MPTP-injected (MPTP + DHA) (n = 15). Docosahexaenoic acid (Sigma Chemical CO, St. Louis, MO, USA) was dissolved in corn oil at a concentration of 0.046 M and was given to the treatment groups for 30 days (36 mg/kg/day) by gavage (Hacioglu et al., 2006, 2007; Kremer et al., 1990; Simopoulos, 1989; Tanriover et al., 2010). To eliminate the effects of a daily gavage and vehicle, other groups received a similar volume of corn oil alone. Food and water were provided ad libitum throughout the experiments. 2.2. Treatment of mice with MPTP On the 23rd day of gavage treatment, animals in the MPTP and MPTP + DHA groups received intraperitoneal (i.p.) injections of either freshly prepared 20 mg/kg MPTP hydrochloride (M-0896, Sigma–Aldrich, St. Louis, Mo, USA) dissolved in saline or an equivalent volume of saline (pH 7.4) every 12 h with a total of four doses (Date et al., 1990). All four animal groups continued on their normal diets for an additional week after treatment. 2.3. Motor performance test To determine the degree of bradykinesia, a typical sign of Parkinsonism, the pole test was performed according to Ogawa et al. (1987) with minor modifications (Araki et al., 2001; Kobayashi et al., 1997). One-week (7 days) post-intoxication, each mouse was placed head upward at the top of a rough-surfaced pole (8 mm in diameter and 50 cm in height) double-wrapped with gauze to prevent slipping. Mice travelled the pole freely and came down to the floor (pre-trial). After animals were habituated to the test system for two or three pre-trials, the time that each mouse took to climb down the 50 cm pole was recorded and analyzed (real trial). 2.4. Tissue collection After the motor performance test and at the end of the treatment period, mice were anesthetized with a combination of ketamine (80 mg/kg, i.p.) and xylazine (16 mg/kg, i.p.) diluted in saline. Mice were perfused transcardially with heparinized saline, and their brain tissues were removed immediately and stored at 80 °C for later biochemical analysis. For immunohistochemical studies, brain tissues containing the SN were fixed in 10% formaldehyde for 24 h and washed with tap water for approximately 6 h. 2.5. Immunohistochemical evaluation of TH-positive neurons Formalin-fixed SN samples were embedded in paraffin and cut into 5 lm sections. After the slides were deparaffinized in xylene and rehydrated in a graded series of ethanol, the slides were boiled in citrate buffer (10 mM, pH: 6.0) for 7 min followed by an additional 10 min for antigen retrieval. Sections were then left to cool for 20 min before being immersed in 3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase. Slides were then incubated in a 5% blocking solution (TP-060-HL, Labvision, Fremont, CA, USA) for 10 min, in a humidified chamber at room temperature. Afterwards, excess serum was drained, and sections were incubated with a mouse monoclonal anti-tyrosine hydroxylase (TH) antibody (#657010; PC12; 1/500; Calbiochem, Merck KGaA., Darmstadt, Germany) in a humidified chamber for 2 h at room temperature. For negative controls, mouse IgG isotypes were used at the same concentrations. The sections were washed three times in PBS for 5 min and then were incubated in a biotinylated polyvalent secondary antibody (K0675; LSAB2 System-HRP; DakoCytomation, Carpinteria, CA, USA) for 20 min at room temperature. The antigen–antibody complex was detected by using an avidin–

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biotin–peroxidase kit (DakoCytomation) and DAB (3,3-diaminobenzidine tetrahydrochloride dihydrate; LabVision) was used as the chromogen. Sections were counterstained with Mayer’s hematoxylin and mounted with Permount (Fisher Chemicals, Springfield, NJ) on glass slides. Two independent researchers blinded to the source and type of tissues semi-quantitatively evaluated the intensity of immunostaining and scored the intensity as follows: 0 (no staining), 1+(weak but detectable staining), 2+(moderate or distinct staining), 3+(intense staining). On each slide, five randomly selected areas were evaluated under a light microscope (40 magnification), and the average score of two researchers was used. To assess the changes in the number of dopaminergic neurons in the SN, the total numbers of TH-stained neurons were counted independently by two observers blinded to the type and source of the tissues under a light microscope (400 magnification) in three slides from each of the groups. The average of the counts was determined. 2.6. Biochemical measurements The brain tissues were analyzed for the assay of SOD, CAT and GPx activities and TBARS. The brain tissues were sonicated (Bronson Sonifier-250) on ice in 1.5 ml of phosphate buffer (pH 7.2) that included 50 mM K2HPO4 (di-potassium hydrogen phosphate, Merck-226). After centrifugation of brain homogenates at speeds specific for the different assays [TBARS (15,000g for 10 min at 4 °C); CAT and GPx (10,000g for 15 min at 4 °C); and SOD (1500g for 5 min at 4 °C)], supernatants were collected and stored at 80 °C until biochemical analysis. 2.6.1. SOD, CAT and GPx activity assay Assay kits and spectrophotometric analyses were used to measure SOD (Cayman-706002) (Malstrom et al., 1975), CAT (Cayman707002) (Johansson and Borg, 1988) and GPx (Sigma CGP-1) (Mannervik, 1985) enzymatic activity in the brain tissues and were expressed in units per milligram of protein. 2.6.2. TBARS assay TBARS levels were measured by fluorometric analysis using 1,1,3,3-tetramethoxypropane as a standard (Wasowicz et al., 1993). Briefly, tissue samples (50 ll) were placed into a tube containing 1 ml of distilled water. One milliliter of a solution containing 29 lmol/l 2-thiobarbituric acid (TBA) in acetic acid (8.75 mol/l) was added to the samples then placed in a water bath at 95–100 °C for 1 h. After the samples cooled, 25 ll of 5 mol/l hydrochloric acid (HCl) was added to the mixture. Reaction product was extracted from the samples with 3.5 ml of n-butanol, vortexed, than centrifuged at 3000g for 10 min. The butanol phase containing the extracted reaction product was separated and fluorescence was measured by spectrophotometry (Perkin Elmer Luminescence spectrometer, LS50B) using wavelengths of 525 nm for excitation and 547 nm for emission.

3. Results 3.1. Effects of DHA on bradykinesia by pole test Changes in motor activity (bradykinesia; slow-down of voluntary movements) are shown in Fig. 1. MPTP mice showed a marked motor deficit in the pole test as compared with the control (17.6 ± 8.21 s) and DHA-treated (20.2 ± 2.03 s) animals (p < 0.05). In contrast, no significant changes were observed between MPTP and MPTP + DHA groups with measured values of 88.2 ± 19.46 and 83.8 ± 7.49 s, respectively. 3.2. Effects of DHA on TH-positive neuron numbers in SN The compact, reticular and lateral parts of mice SN were easily distinguished by TH immunohistochemistry (Fig. 2). The immunoreactivity for TH was observed in neuron bodies and processes. The staining intensity scores for TH-immunoreactivity and the number of immunopositive dopaminergic cells are presented in Table 1. As expected, the TH-immunoreactive neurons in the MPTP group (154 ± 10) were significantly lower (p < 0.05) when compared to the control (440 ± 5) and DHA (210 ± 8) groups. The number of TH-positive neurons revealed a significant difference between animals treated with DHA + MPTP (244 ± 2) compared to animals treated MPTP only (154 ± 10) (p < 0.05), but they did not exceed the control values (440 ± 5) and remained significantly lower. Additionally, DHA alone reduced the TH-positive cell number compared to that of control animals (Fig. 2) (Table 1). 3.3. Effects of DHA on brain antioxidant enzymes activities and TBARS levels Antioxidant enzyme activities and TBARS values measured in the brain of experimental groups are summarized in Table 2. There was a marked decrease in brain SOD activity of MPTP and MPTP + DHA groups compared with the control group (p < 0.05). Superoxide dismutase activity levels remained unchanged in the brains in the MPTP + DHA group compared to MPTP group. However, CAT and GPx activities remained unchanged in all groups. TBARS levels in the brain increased in DHA and MPTP groups compared with control (p < 0.05). MPTP + DHA group had a 17–18% reduction tendency (but it did not significantly) in brain TBARS levels as compared to mice that received MPTP.

2.6.3. Protein determination For all experiments, protein concentrations were measured at 550 nm by a modified Bradford assay using Coomassie Plus reagent (Pierce) with bovine serum albumin as a standard (Bradford, 1976). 2.7. Statistical analysis Differences in data were analyzed by ANOVA followed by Tukey’s Post Hoc Test for suit with normal dispersion and Kruskal Wallis followed up Mann Whitney U test for not suit with normal dispersion. Significance levels were set at p < 0.05.

Fig. 1. Determination of the degree of bradykinesia by the pole test. One week (7 days) post-intoxication, each mouse was placed head upward at the top of a rough-surfaced pole (8 mm in diameter and 50 cm in height) double-wrapped with gauze to prevent slipping. Mice travelled the pole freely and came down to the floor during pre-trials. Mice were habituated to the test system for two or three pretrials, after which, the time that each mouse needed for climbing down the pole was recorded and analyzed (real trial). N = 5 per group. Data are presented as means ± SEM. ⁄: p < 0.05 vs. control and DHA groups.

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Fig. 2. Tyrosine hydroxylase (TH) immunoreactivities in experimental groups. The TH-immunopositive dopaminergic neurons in the axial cross-sections were counted under a light microscope at 400 magnification by two observers.

Table 1 TH-immunopositive dopaminergic neuron numbers and staining intensities.

TH immunoreactivity staining intensities TH immunopositive cell number/area

Control

DHA

MPTP

MPTP + DHA

+++

+++

++

++

440 ± 5

210 ± 8*

154 ± 10

244 ± 2*,**

Values are expressed as means ± S.E.M. The TH-immunopositive dopaminergic neurons were counted under a light microscope at 400 magnification. (): No reactivity, (+): weak, (++): moderate, (+++): strong immunoreactivity. * p < 0.05, vs. control group. ** p < 0.05, vs. MPTP group.

Table 2 Antioxidant status and TBARS values of the brain in studied groups. Groups

SOD (U/mg protein)

CAT (U/mg protein)

GPx (U/mg protein)

TBARS (nmol/ mg protein)

Control DHA MPTP MPTP + DHA

0.48 ± 0.07 0.44 ± 0.03 0.33 ± 0.04* 0.34 ± 0.05*

4.67 ± 0.68 4.39 ± 0.67 5.62 ± 0.27 4.37 ± 0.16

0.07 ± 0.01 0.06 ± 0.01 0.05 ± 0.01 0.05 ± 0.01

0.08 ± 0.003 0.17 ± 0.03* 0.17 ± 0.02* 0.14 ± 0.01

Values are expressed as means ± S.E.M., n = 10 in each group. p < 0.05 vs. control group.

*

4. Discussion We examined the effects of DHA on motor activity and dopaminergic cell death in mice treated with MPTP, a model of PD. To explore the mechanisms of DHA effect, we determined brain TBARS levels and antioxidant enzyme activities in experimental groups. Our data show that DHA treatment partially prevented dopaminergic neuron death in the SN and exhibited a decrease tendency (not significantly) in brain lipid oxidation of experimental PD model.

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Today, MPTP represents the most important and most frequently used parkinsonian toxin applied in animal models (Beal, 2001; Przedborski et al., 2001). MPTP is highly lipophilic and after systemic administration, rapidly crosses the blood–brain barrier (Schober, 2004). In contrast to primates, rodents are less sensitive to MPTP toxicity (Schmidt and Ferger, 2001). Nevertheless, the C57BL6 mice strain was found to be sensitive to a systemic injection of MPTP and was significantly more selective than other mice strains in terms of affecting mesencephalic dopaminergic neurons (Schober, 2004). In principle, MPTP can be given by a variety of regimens, such as gavage or stereotactic injection, but the most common and reproducible form is still systemic administration (e.g., subcutaneous, intravenous, intraperitoneal or intramuscular injection) (Przedborski et al., 2001). DHA is rapidly taken up by the body and incorporated into triglycerides in the blood. Although the active transport of DHA into the brain is not well understood, strong evidence indicates that DHA easily enters into the brain (Samadi et al., 2006). Because DHA is widely available and has an excellent safety profile, it could be used as a nutraceutical defense against brain diseases. Recent studies have tried to decipher the role of DHA in PD and to probe for a therapeutic effect of DHA. To explore the protective effects of DHA, we focused upon markers of experimental PD such as TH-immunostaining and motor activity. In the present study, the number of TH-positive neurons in the MPTP group were significantly lower when compared to all other groups (p < 0.05) (Fig. 2, Table 1). There was a 65% decline in the number of TH-immunopositive neurons in the SN by 1 week after MPTP administration, which is in agreement with previous studies (Dauer and Przedborski, 2003; Jackson-Lewis et al., 1995; Schober, 2004; Viswanath et al., 2001). Immunoblotting analyses indicated that MPTP caused a significant reduction (30%) of TH expression in the SN 1 day after injection (Reksidler et al., 2007). In the present study, the number of TH-positive neurons significantly increased in the MPTP + DHA group compared to the MPTP group (p < 0.05) (Fig. 2, Table 1). Preservation of TH-positive neurons was observed in MPTP mice treated with DHA, suggesting that nigral dopaminergic neurons were protected from MPTP neurotoxicity. Cansev et al. (2008) has shown that reductions in TH protein levels in lesioned striata were partially restored (increasing by 21%) by DHA supplementation. In the same study, the authors also concluded that DHA did not diminish the initial toxic responses to the 6-OHDA as 3 days. In the PD research field, it has been recently demonstrated that a 1-month administration of DHA (100 mg/kg) before MPTP intoxication reduced the extent of levodopa-induced dyskinesias in a non-human primate model of Parkinsonism by approximately 40% (Samadi et al., 2006). Hence, in our study, animals were sacrificed 4 weeks after starting DHA administration, a period previously shown to reliably increase membrane phosphatides, synaptic proteins (Wurtman et al., 2006) and dendritic spines (Sakamoto et al., 2007). Recent data also demonstrated a protective effect of DHA against MPTP-induced neurotoxicity in a mouse model of acute Parkinsonism (Bousquet et al., 2008). In the present study, DHA treatment alone reduced the number of dopaminergic neurons in the SN. Conversely Bousquet et al. (2008) concluded that there were no differences between the control and high n-3 PUFA diet groups. There was a high degree of correlation between dopaminergic neuron degeneration and motor impairment in the MPTP-induced experimental PD model (Haobam et al., 2005). In the current study, we demonstrated that the severity of bradykinesia (a specific sign of PD) in the MPTP group was prolonged as compared to the control group (Fig. 1). These findings are in agreement with results from previous studies (Kato et al., 2004; Mitra et al., 1992; Sedelis et al., 2001). Although there was a significant increase in the number of TH-positive dopaminergic neurons following DHA adminis-

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tration in MPTP-treated mice, DHA did not reduce the severity of bradykinesia in this group, which may indicate that the increase in the number of TH cells following DHA treatment was not sufficient for the recovery of impaired motor activity in mice. In contrast, a previous study concluded that animals that received DHA for 24 days exhibited significant reductions (47%) in the number of d-amphetamine-induced rotations, compared with those in control rats (Cansev et al., 2008), which may be related to the experimental PD model and the dose differences between the two studies. In the present study, the DHA dose was selected according to the previous studies conducted in human and rats (Hacioglu et al., 2006, 2007; Kremer et al., 1990; Tanriover et al., 2010). Low DHA concentrations were found to be effective as a therapeutic agent because high doses were cytotoxic to both normal and pathologic cells (Toit-Kohn et al., 2009). DHA is known to protect the brain against free radical damage (Hashimoto et al., 2002, 2005). Hossain et al. (1998) concluded that the DHA-treated group showed a 40% decrease in the lipid peroxide (LPO) concentration in the cerebrum compared with the normal diet group in aged rats. Previous studies have indicated that DHA treatment reduces the levels of prostaglandins and leukotrienes (Kishida et al., 1998; Shikano et al., 1994). These factors are derived from the arachidonic acid cascade of endoperoxides and have free radical characteristics (Buczynski et al., 1993). In contrast, DHA has pro-oxidant properties. Less appreciated, however, is that DHA undergoes significant peroxidation in the brain. This peroxidation suggests that DHA-derived lipid–aldehyde species may play a pathological role in the CNS. Because of lipid peroxidation, TBARS accumulation was significantly increased in the cerebrum and cerebellum tissues obtained from cockerels fed on the tuna oil (TO)-supplemented diet (Surai and Sparks, 2000). TBARS levels in the brain have been found to be higher in the fish oil-supplemented diet group than in the regular diet group (ChoiKwon et al., 2004), which is supported by in vitro studies. Docosahexaenoic acid-enriched oligodendroglia cell cultures produced more TBARS and showed a greater proportion of dying cells (Brand et al., 2000). Furthermore, it has been indicated that the membranes that were rich in n-3 fatty acids are more susceptible to oxidative stress (Salvati et al., 1993). C6 glioma cells treated with different PUFA showed a significant increase in TBARS levels soon after a 24 h incubation as compared with controls (Leonardi et al., 2005), which supports the data presented in our study that showed increased TBARS levels in mice after DHA administration (Table 2). Additionally, the reduced number of TH-positive nigral neurons in the DHA group compared to the control group may be related with this DHA-related TBARS increase (Table 1) (Fig. 2). Theoretically, the intake of n-3 fatty acids may increase the unsaturation index, leading to increased lipid peroxidation (Nenseter and Drevon, 1996), which remains a concern. This hypothesis was based on the premise that fatty acid oxidizability increases as the number of double bonds increases (Liu et al., 1997). Docosahexaenoic acid is very prone to lipid peroxidation because of its unstable chemical structure due its six double bonds. Cells with a higher degree of membrane unsaturation are more susceptible to oxidative stress (Udilova et al., 2003). Nevertheless, Leonardi et al. (2005) concluded that oxidative stress does not seem to be related to the degree of membrane unsaturation by showing that DHA-enriched cells produced a 4-fold greater ROS than arachidonic acid (ARA) and eicosapentaenoic acid (EPA)-enriched cells, although the unsaturation index was similar in the three groups. It has been suggested that the total concentration of polyunsaturates, rather than the unsaturation index, may be a more important factor effecting lipid peroxidation (Brude et al., 1997). The intake of fish oil rich in DHA enhances the sensitivity to lipid peroxidation and promotes the requirement of vitamin E (VE)

in the liver (Hu et al., 1989). This peroxidation may reflect decreased VE concentration because dietary n-3 PUFA, especially DHA, can decrease VE levels in plasma and tissues samples from humans and animals (Farwer et al., 1994; Kubo et al., 1997). Therefore, increased VE supplementation may be an effective measure to prevent lipid peroxidation in tissues enriched by n-3 fatty acids (Cho and Choi, 1994). A combined treatment of VE and TO caused a decrease in tissue susceptibility to lipid peroxidation as compared to TO treatment alone (Surai and Sparks, 2000). Our results indicated a significant elevation in brain TBARS levels of the MPTP-treated group compared to controls (Table 2). Xie et al. (2007) has shown that elevated malondialdehyde (MDA) content in the brain of the rat PD model compared to controls. Results from other studies clearly show that regions of the brain from patients with advanced PD (Dexter et al., 1989) or animals treated with MPTP (Abdel-Wahab, 2005; Choi et al., 2005; Rajeswari and Sabesan, 2008; Rojas and Rios, 1993; Sankar et al., 2007) have increased levels of lipid peroxidation products compared to controls. When compared to MPTP animals, administration of DHA before MPTP intoxication represented a reduce tendency (not significantly) TBARS levels in the brains of mice (Table 2). Conversely, Kabuto et al. (2009) showed that DHA ethyl ester treatment resulted in a worsening of 6-OHDA-induced neuronal damage with lipid peroxidation. This discrepancy with our study may be related to the differences in the dose of DHA, the treatment schedule, or the animal models and tissues that were studied. Previous studies have reported low levels of endogenous antioxidants and low free radical-scavenging enzyme activity in the brains of patients with PD (Ambani et al., 1975; Kish et al., 1985; Riederer et al., 1989). However, many contradicting results have been obtained regarding antioxidant enzyme activities in MPTP-treated animals. In our study, GPx and CAT activities in the brain were unaltered in all groups (Table 2). These results were supported by previous studies that concluded unaltered GPx and CAT activity in the mesolimbic and nigrostriatal systems (Hung and Lee, 1998) and unaltered GPx mRNA expression in the basal ganglia (Kunikowska and Jenner, 2003) of MPTP mice compared to control. However, there have been studies that show decreased GPx and CAT activities in the striatum due to MPTP intoxication (Chen et al., 2007; Tsai et al., 2010). We showed a reduction in brain SOD activity in the MPTP group compared to controls (Table 2), which supports previous studies that showed decreased SOD activity in the striatum (Chen et al., 2007; Choi et al., 2005) and decreased SOD mRNA expression in the basal ganglia (Kunikowska and Jenner, 2003) of MPTP mice compared to controls. In contrast to our results, Xie et al. (2007) showed that SOD activity in the brain of MPTP mice was not altered. Furthermore, previous studies have reported an increase in SOD activity (AbdelWahab, 2005; Rajeswari and Sabesan, 2008; Sankar et al., 2007) in different brain regions of MPTP mice. SOD catalyzes the decomposition of superoxide anions into hydrogen peroxide and is a primary enzyme in free radical detoxification in brain tissue. In our study, the MPTP-induced decrease in SOD activity may predispose the brain tissue to increased free radical damage. Nevertheless, DHA administration to MPTP-treated mice did not affect SOD activity levels (Table 2). Our present study shows that chronic administration of DHA by gavage partially restores dopaminergic cell death but does not improve the behavioral consequence of systemic MPTP intoxication. However this pattern of findings do not indicate that the effect observed is the result of true neuroprotection. It should be clarify whether DHA blocks the conversion of MPTP to MPP+ or prevents the uptake of MPP+ into dopaminergic terminals. Dietary supplementation with DHA may be a potential means for delaying the onset of PD and/or the rate of progression. However, further studies are necessary to understand the neuroprotective mechanisms of DHA in experimental PD.

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Acknowledgements This study was supported by the Akdeniz University Research Projects Unit (Project number: 2004.02.0122.016).

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