Address correspondence and reprint requests to Khan Shoeb Zafar, School of Pharmacy, UCHSC, Denver, CO, 80220, USA. E-mail: email@example.com
Normal cellular metabolism produces oxidants that are neutralized within cells by antioxidant enzymes and other antioxidants. An imbalance between oxidant and antioxidant has been postulated to lead the degeneration of dopaminergic neurons in Parkinson's disease. In this study, we examined whether selenium, an antioxidant, can prevent or slowdown neuronal injury in a 6-hydroxydopamine (6-OHDA) model of Parkinsonism. Rats were pre-treated with sodium selenite (0.1, 0.2 and 0.3 mg/kg body weight) for 7 days. On day 8, 2 µL 6-OHDA (12.5 µg in 0.2% ascorbic acid in normal saline) was infused in the right striatum. Two weeks after 6-OHDA infusion, rats were tested for neurobehavioral activity, and were killed after 3 weeks of 6-OHDA infusion for the estimation of glutathione peroxidase, glutathione-S-transferase, glutathione reductase, glutathione content, lipid peroxidation, and dopamine and its metabolites. Selenium was found to be successful in upregulating the antioxidant status and lowering the dopamine loss, and functional recovery returned close to the baseline dose-dependently. This study revealed that selenium, which is an essential part of our diet, may be helpful in slowing down the progression of neurodegeneration in parkinsonism.
Parkinson's disease (PD) is the second most common progressive neurodegenerative disorder primarily affecting individuals between the age of 50 and 60, although young adults and even children can be affected by this devastating disease (Dawson 2000). PD is due to selective degeneration of dopamine-containing neurons in the midbrain. In addition to the loss of dopaminergic neurons, there are indications of increased oxidative stress such as glutathione depletion, iron deposition, increased markers of lipid peroxidation, oxidative DNA damage, protein oxidation (Perry et al. 1982; Reiderer et al. 1989; Sian et al. 1994; Jenner and Olanow 1998), and reduction in the activity of complex-1 (NADPH Co Q reductase) of the mitochondrial respiratory chain (Schapira et al. 1990). The malfunction of the basal ganglia circuits is responsible for the cardinal motor symptoms of PD such as tremors at rest, muscular rigidity, bradykinesia/akinesia, stooped posture, and instability (Sian et al. 1999).
6-Hydroxydopamine (6-OHDA) is a selective catecholaminergic neurotoxin (Ungerstedt 1968) widely used to investigate the pathogenesis of PD (Breese and Breese 1998). The specific neurotoxicity of 6-OHDA has been associated with its uptake and accumulation by a transport mechanism specific for catecholaminergic neurons (Sachs and Jonsson 1975; Ljungdahl et al. 1991). The most popular animal model of PD is produced by unilateral stereotaxic injection of 6-OHDA into the substantia nigra or medial forebrain bundle of rats. However, it has been demonstrated recently that intrastriatal injection is more useful for the study of neuroprotection or neurotrophic therapies in PD (Shults et al. 2000; Kirik et al. 2001). Under physiological conditions, 6-OHDA is rapidly and non-enzymetically oxidized by molecular oxygen to form H2O2 and corresponding quinones.
The aim of the present study was to analyze the neuroprotective role of Se in a rat model of parkinsonism.
Materials and methods
Glutathione (GSH, oxidized and reduced), glutathione reductase (GR), nicotinamide adenine dinucleotide phosphate reduced form (NADPH), 1-chloro-2,4-dinitrobenzene (CDNB), 5-5′-dithio-bis-2-nitrobenzoicacid (DTNB), DA, DOPAC, HVA, 3,4-dihydroxybenzylamine (DHBA), 6-OHDA, and heptane sulfonic acid were purchased from Sigma-Aldrich Foreign Holding Chemical Company (New Delhi, India). Other chemicals were of analytical grade.
Male Wistar rats obtained from Central Animal House of Jamia Hamdard (Hamdard University), weighing 200–230 g at the start of the experiment, were used. Rats were housed in groups of four animals per cage. They were maintained on a 12-h dark–light cycle (light on from 06.00 to 18.00 h) and provided free access to rat chow and water. The experiments were in accordance with university guidelines and were approved by the Animal Ethics Committee of the University.
Experiment one was carried out to evaluate the effect of sodium selenite [0.1, 0.2 and 0.3 mg/kg body weight (b.w.), intraperitoneally (i.p.)] pre-treatment for 7 days on open field test, muscular co-ordination, and dopamine and its metabolites. The rats were divided into eight groups, each having eight animals. Group 1: vehicle-treated sham-operated control group (S), group 2: sodium selenite 0.1 mg/kg b.w. treated sham-operated group (S + Se1), group 3: sodium selenite 0.2 mg/kg b.w. treated sham-operated group (S + Se2), group 4: sodium selenite 0.3 mg/kg b.w. treated sham-operated group (S + Se3), group 5: vehicle-treated lesioned group (L), group 6: sodium selenite 0.1 mg/kg b.w. treated lesioned group (L + Se1), group 7: sodium selenite 0.2 mg/kg b.w. treated lesioned group (L + Se2), group 8: sodium selenite 0.3 mg/kg b.w. treated lesioned group (L + Se3).
This experiment was carried out to evaluate the effect of sodium selenite (0.1, 0.2 and 0.3 mg/kg b.w., i.p.) pre-treatment for 7 days on circling behavior, antioxidant enzymes, glutathione content (GSH), and lipid peroxidation (LPO). The rats were divided into eight groups, each having 16 animals. Tissues of two rats in each group were pooled for the estimation of antioxidant enzymes, GSH and LPO.
On the 8th day, 2 µL vehicle (0.2% ascorbic acid in normal saline) were infused in the striatum of groups 1–4 (S, S + Se1, S + Se2 and S + Se3) and 12.5 µg 6-OHDA in 2 µL vehicular solution to groups 5–8 (L, L + Se1, L + Se2 and L + Se3) as described below.
Intrastriatal administration of 6-OHDA
Unilateral lesions in the right striatum were made in male Wistar rats. The animals were anesthetized with 400 mg/kg i.p. chloral hydrate. The rats were placed in a stereotaxic frame and the skull exposed. Through a skull hole, a 28-gauge Hamilton syringe of 5 µL was attached to microinjector unit and the piston of the syringe was lowered manually to the right striatum. Lesion co-ordinates were used as described by Lee et al. (1996). Briefly, they were AP-0.5, L-2.5, V-5 mm relative to bragma and ventral from dura with the tooth bar set at 0 mm (Paxinos and Watson 1986). Rats were either infused 2 µL vehicle or 12.5 µg 6-OHDA in 2 µL vehicular solution over 5 min and the needle was left in place for 3 min before slowly retracting it.
After 3 weeks, animals were killed and brains were taken out quickly and kept on ice. Striatum and substantia nigra were dissected out by cutting a coronal section of 1.0 mm thickness using rat brain matrix in the light of rat brain atlas (Pelligrino et al. 1981). For enzymes and GSH assays, striatum was homogenized in phosphate buffer (10% w/v, pH 7.0) and centrifuged at 10 500 g for 20 min at 4°C to get post-mitochondrial supernatant (PMS). For catecholamine estimation, the striatum (20% w/v) was sonicated in 0.4 N perchloric acid containing 100 ng/mL of the internal standard of DHBA followed by centrifugation at 15 000 g for 10 min at 4°C. The supernatant was filtered through 0.22-µm membrane filter (Millipore, Bedford, MA, USA).
Estimation of catecholamines
The method of DeVito and Wagner (1989) was used with slight modification. The filtered supernatant was injected manually through a 20 µL loop over the ODS-C18 column (Waters, Milford, MA, USA) coupled with an HPLC/Electrochemical detector (Waters 464 detector and isocratic 515 pump) for separation and quantification. The potential was set at 750 mV. The mobile phase consisted of 0.1 m potassium phosphate (pH 4.0), 10% methanol and 1.0 mm heptane sulfonic acid. Samples were separated on C18 column using a flow rate of 1.0 mL/min. The concentrations of dopamine and its metabolites were calculated using a standard curve generated by determining the ratio between three known amounts of amine or its metabolites and a constant amount of internal standard by Millennium 32 software (Waters) and represented as ng/mg of tissue.
Glutathione peroxidase (Se-dependent)
GPx (EC 18.104.22.168) activity was measured at 37°C by a coupled assay system (Wheeler et al. 1990) in which oxidation of GSH was coupled to NADPH oxidation catalyzed by GR. The reaction mixture consisted of 0.1 mL H2O2 (0.2 mM), 0.1 mL GSH (1 mM), 1.4 units of 0.1 mL GR, 0.1 mL NADPH (1.43 mM), and 0.1 mL sodium azide (1 mM) in a 1.4-mL phosphate buffer (0.1 m, pH 7.4) and 0.1 mL PMS. The enzyme activity was quantitated by measuring the disappearance of NADPH at 340 nm with a spectrophotometer (λ-20, Perkin-Elmer, Foster City, CA, USA). GPx activity was defined as nmol NADPH oxidized min/mg protein using the molar extinction coefficient of 6.22 × 103 M/cm.
Glutathione reductase activity
GR (EC 22.214.171.124) activity was measured by the method of Carlberg and Mannerviek (1975). The assay system consisted of 1.65 mL phosphate buffer (0.1 m, pH 7.6), 0.1 mL EDTA (0.5 mm), 0.05 mL oxidized glutathione (1 mm), 0.1 mL NADPH (0.1 mm), and 0.1 mL PMS in a total volume of 2.0 mL. The enzyme activity was quantitated at 25°C by measuring the disappearance of NADPH at 340 nm with a spectrophotometer (λ-20, Perkin-Elmer) and was calculated as nmol NADPH oxidized min/mg protein using the molar extinction coefficient of 6.22 × 103m/cm.
CAT (EC 126.96.36.199) activity was measured by the method of Claiborne (1985). In brief, the assay mixture consisted of 2 mL phosphate buffer (0.1 m, pH 7.4), 0.95 mL H2O2 (0.019 m) and 0.05 mL of PMS in a final volume of 3.0 mL. The changes in absorbance were recorded at 240 nm with a spectrophotometer (λ-20, Perkin-Elmer). The CAT activity was calculated in terms of nmol H2O2 consumed min/mg protein.
Glutathione-S-transferase (GST, EC 188.8.131.52) activity was measured by the method of Habig et al. (1974). The reaction mixture consisted of 2.5 mL phosphate buffer (0.1 M, pH 6.5), 0.2 mL reduced glutathione (1 mM), 0.2 mL CDNB (1 mM), and 0.1 mL of PMS in a total volume of 3 mL. The changes in absorbance was recorded at 340 nm by using a spectrophotometer (λ-20, Perkin-Elmer) and the enzymatic activity was calculated as nmol CDNB conjugate formed min/mg protein using a molar extinction coefficient of 9.6 × 103 M/cm.
LPO was measured in substantia nigra by the method of Shimaski (1974) as described by Mohankumar et al. (1994) with slight modifications. Substantia nigra (5–7 mg) was placed in microcentrifuge tubes with 750 µL of chloroform and methanol (2 : 1; v/v), each tube was capped, agitated gently, and placed in ice. The tissue was homogenized for 2 s in an ultrasonic tissue disruptor. Then, 750 µL water were added to this and the mixture was vortexed for 1 min and left on ice for 15 min. After centrifugation, 300 µL of the methanol was added to 500 µL of the separated chloroform layer. After thorough mixing, fluorescence due to oxidized product of lipid was obtained at 256 nm excitation and 426 nm emission (Fluorescence spectrophotometer, 50B, Perkin-Elmer). The fluorescence intensity of 1 µg/mL quinine sulfate in 0.1 N H2SO4 solution is used as a standard for the relative fluorescence intensities of samples (Fletcher et al. 1973). Brain LPO is expressed as relative fluorescent unit (RFU).
Estimation of reduced glutathione
The reduced GSH in the brain was determined by the method of Jollow et al. (1974) with slight modifications. PMS 0.3 mL was mixed with 0.3 mL of sulfosalicylic acid (4%). The samples were incubated at 4°C for 30 min and then subjected to centrifugation at 1200 g for 15 min at 4°C. The assay mixture contained 0.1 mL filtered aliquot, 1.7 mL phosphate buffer (0.1 m, pH 7.4), and 0.2 mL DTNB (4 mg/1 mL) in a total volume of 2.0 mL. The yellow color that developed was read immediately at 412 nm on the spectrophotometer (λ-20, Perkin-Elmer). The GSH concentration was calculated as nmol GSH/g tissue.
The behavioral tests were started after 2 weeks of lesioning. The experiment was performed between 09.00 and 16.00 h in the laboratory at standard optimal conditions. All tests were performed and analyzed by persons blind to the experiment.
Open field test
Observations were recorded for three sessions of 5 min each. Mean of sessions totals of vehicle and treatment groups were compared for locomotion (s), distance traveled (cm), stereotype events (number), and rearing (number). Turning behavior and open fields test were recorded automatically by a Video Path Analyzer (Coulborn Instrument, Allentown, PA, USA) comprising an open field chamber (50 × 50 × 35 cm), a video camera fixed over the chamber, an activity monitor, a programmer/processor, and a printer. Behavioral testing was started 10 s after placing the rat over a black surface at the center of open field chamber.
Rotarod (muscular co-ordination)
Omni Rotor (Omnitech Electronics Inc., Columbus, OH, USA) was used to evaluate the muscular inco-ordination. It consisted of a rotating rod (75 mm diameter) which was divided into four compartments, permitting testing for four rats at a time. The apparatus automatically recorded time in 0.1 s when the rats fell off the rotating shaft. The speed was set at 10 rpm and cut-off time was 180 s. Drug-naive animals were trained on the rotarod (10 rpm), so that they could stay on the rotating rod for at least 180 s (cut-off time). After 2 weeks of lesioning, rats were again tested for endurance performance (three times).
Results are expressed as mean ± SEM. anova with post-hoc analysis was used to analyze differences among the groups. The Tukey–Kramer post-hoc test was applied to serve as significant among groups.
Effect of Se on brain dopamine metabolism
Table 1 shows the content of dopamine and its metabolites in the sham-operated control group (S) and the lesioned group (L), and in the groups protected with selenium. The levels of dopamine (78%), DOPAC (70%) and HVA (68.5%) were depleted significantly in L group as compared to S group. The ratio of DOPAC/DA in L group was increased by 41.6% as compared to S group. Pre-treatment with Se in L + Se1 to L + Se3 groups protected the level of dopamine, DOPAC, and HVA significantly and dose-dependently as compared to L group. The ratio of DOPAC/DA in L + Se1 to L + Se3 groups was depleted dose-dependently (21, 29 and 41.6%) as compared to L group.
Table 1. Effect of selenium and 6-OHDA lesioning on dopamine and its metabolites in striatum
DA (ng/mg tissue)
DOPAC (ng/mg tissue)
HVA (ng/mg tissue)
Values are expressed as mean ± SEM (n = 16). ap < 0.001 compared to S, bp < 0.05, cp < 0.01, dp < 0.001 compared to L. The values in parentheses are percentage change of Se1–Se3 compared with S and of L + Se1 to L + Se3 compared with L.
Table 2 shows the effect of sodium selenite (0.1, 0.2 and 0.3 mg/kg b.w.) on the activity of GPx, GR, GST, and CAT. The activity of antioxidant enzymes in groups S + Se1 to S + Se3 was elevated dose-dependently but the elevation was not significant when compared to S group. On the other hand, the activity of these enzymes was depleted significantly in L group as compared to S group. The selenium protected the activity of these enzymes in L + Se1 to L + Se3 groups significantly and dose-dependently as compared to L group.
Table 2. Effect of selenium and 6-OHDA lesioning on antioxidant enzymes in striatum
GPx (nmol NADPH oxidized/min/ mg protein)
GR (nmol NADPH oxidized/min/ mg protein)
GST (CDNB conjugate formed/min/ mg protein)
CAT (nmol H2O2 consumed/min/ mg protein)
Values are expressed as mean ± SEM (n = 16). ap < 0.01, bp < 0.001 compared to S, cp < 0.05, dp < 0.01, ep < 0.001 compared to L. The values in parentheses are percentage change of Se1–Se3 compared with S and of L + Se1 to L + Se3 compared with L.
Figure 1 shows the effect of various doses of sodium selenite in S groups and its protection in L groups. The level of LPO was not altered in S + Se1 to S + Se3 groups but its level was significantly elevated in L group as compared to S group. Treating the rats with various doses of selenium in L + Se1 to L + Se3 groups depleted the level of LPO significantly and dose-dependently.
Effect of Se on GSH
Figure 2 shows the protective effect of sodium selenite on the GSH level in L group. The level of GSH was not elevated significantly in S + Se1 to S + Se3 groups but its depletion in L group was significant as compared to S group. The selenium elevated its level significantly and dose-dependently in L + Se1 to L + Se3 groups as compared to L group.
The effect of sodium selenite was evaluated on amphetamine-induced rotations in lesioned rats (Fig. 3a). Rats from the lesioned groups rotated towards the lesioned side (ipsilateral rotations) following amphetamine administration, and the rotation was decreased significantly (p < 0.001) and dose-dependently in L + Se1 to L + Se3 groups as compared to L group. No significant alteration was observed in S + Se1 to S + Se3 groups as compared to S group.
Rotarod (spontaneous motor activity)
Figure 3(b) shows a significant (p < 0.001) depletion in muscle co-ordination in L group as compared to S group. Selenium (0.1, 0.2 and 0.3 mg/kg b.w.) was found to be effective in a partial recovery of muscular inco-ordination in a dose-dependent manner in L + Se1 to L + Se3 groups as compared to L group. No significant alteration was observed in S + Se1 to S + Se3 groups as compared to S group.
The rearing activity was significantly lowered (p < 0.001) in L group in comparison to S group (Fig. 3c). The rearing was increased with all three doses of selenium in L + Se1 to L + Se3 groups as compared to L group. Groups S + Se1 to S + Se3 showed no significant alteration in rearing as compared to S group.
Distance traveled was decreased significantly in L group (p < 0.01) as compared to S group and it was reversed by selenium dose-dependently in L + Se1 to L + Se3 groups as compared to L group (Fig. 3d). Time spent in locomotion was depleted significantly (p < 0.01) in L group rats as compared to S group (Fig. 3e). There was significant and dose-dependent recovery in L + Se1 to L + Se3 groups as compared to L group (52%, 63% and 82%, respectively). No alteration was observed in distance traveled and locomotion in S + Se1 to S + Se3 groups as compared to S group.
Stereo events were decreased (41%) significantly (p < 0.01) in L group and selenium supplementation elevated its level significantly and dose-dependently in L + Se1 to L + Se3 groups as compared to L group (19%, 33% and 50%, respectively; Fig. 3f).
The experiment presented here demonstrates the ability of selenium to partially protect the toxicity of the nigrostriatal dopaminergic neurons induced by 6-OHDA injection. Selenium reduces the ipsiversive rotations induced by amphetamine (Fig. 3a). These rotations can be considered as a reliable indicator of nigrostriatal dopaminergic depletion (Schwarting and Hudson 1996). Schwarting and Huston (1997) have shown that ipsilateral rotation may take place when nigrostriatal lesioning is more than 74%, where the partially lesioned rats do not rotate after such treatment. Thus a marked decrease in rotation may be due to a protective effect of selenium on dopaminergic neurons against 6-OHDA toxicity. Selenium reduced the increase in dopamine utilization. It is noteworthy that the greater the dopamine depletion, the higher the increase in dopamine metabolism (Zigmond et al. 1990), which is probably linked to a compensatory mechanism on the part of the remaining neurons (Agid et al. 1973; Lavieller et al. 1978; Robinson and Whishaw 1988; Robinson et al. 1990, 1994). Moreover, the turnover of dopamine is a better index of neuronal functioning than dopamine levels. It may be noted that treatment with selenium in combination with 6-OHDA allowed the remaining neurons to recover at the level of functioning closer to that of their initial activity. Such an improvement is again in good agreement with the antioxidant enzyme and behavioral data reported in the present work. The present study demonstrates that selenium supplementation prevents the striatal dopamine, also proving our earlier results (Imam et al. 1999).
The rotarod and open field experiments have indicated that vehicle injection did not cause deterioration of motor performance in the rats, while the lesioned group (L) showed depletion in locomotion, low stereotypic events, and poor co-ordination. The pre-injection of selenium protected these events significantly. The behavioral effects are closely linked to the degree of dopamine dysfunction (Schwarting et al. 1991).
The brain dopamine has long been thought to play a role in neurotoxicity which undergoes spontaneous oxidation to toxic quinone and other electrophilic species, causing PD (Bing et al. 1994; Halliwell 1992). It was also found that dopamine in solution undergoes auto-oxidation, resulting in the production of reactive quinone derivatives and H2O2 (Graham et al. 1978; Sinet et al. 1980; Slivka and Cohen 1985). The in vivo administration of antioxidant inhibits reduction in brain dopamine levels (Roghani and Behzadi 2001). Thus, the preventive effect of Se might be due to reduction in auto-oxidation of dopamine by enhancing antioxidant enzymes activity, particularly GPx which shows relatively high activity in striatum and substantia nigra compared to other brain regions. It is also well known that brain has poor CAT activity and moderate activity of GPx and SOD (Brannan et al. 1980; Halliwell 1992; Coyle and Puttfarcken 1993). H2O2 is detoxified to H2O by GPx and partially by CAT (Halliwell 1992) and GST catalyzes the detoxification of oxidized metabolites of catecholamines (o-quinone) and may serve as an antioxidant system preventing degenerative cellular process (Baez et al. 1997).
6-OHDA-generated oxidative stress was particularly blocked by selenium by inducing antioxidant defense mechanism. 6-OHDA and auto-oxidation of dopamine produced H2O2 with subsequent Fe2 + catalyzed conversion to hydroxyl radicals (Graham 1984; Gotz et al. 1994; Ebadi et al. 1996) which trigger the reaction and damage the DNA, membrane lipid, carbohydrate, and proteins. These radicals also damage the mitochondrial electron transport system, decompartmentation of intracellular calcium homeostasis, induction of proteases, increase membrane lipid peroxidation and, finally, cell damage (Halliwell 1992; Youdim et al. 1993). 6-OHDA potentiates LPO in substantia nigra and striatum, which may be the result of diminished activities of antioxidant enzymes and low levels of GSH. Reduction of brain glutathione by l-buthionine sulfoximine potentiate the dopamine depleting action of 6-OHDA in rat striatum (Garcia et al. 2000).
The impairment of Ca2 + homeostasis in the CNS may be the triggering event that leads to the development of neurodegeneration during oxidative stress (Orrenius et al. 1992). Selenium has been shown to block Ca2 + channel with the efficacy comparable to calcium antagonist verapamil (An et al. 1992). Hence, in the light of the above data, it may be suggested that the protective effect of Se against 6-OHDA neurotoxicity may be at least in part due to its calcium blocking activity. The selenium which is an essential part of our diet may be used best as a tool for the protection of neurodegeneration, especially in PD.