Neuroprotection by bromocriptine against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity in mice¹

^a Laboratory of Neurochemistry, Division of Pharmacology and Experimental Therapeutics, Indian Institute of Chemical Biology, Calcutta 700 032, India

	ABSTRACT

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Mice were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 30 mg/kg i.p. twice, 16 h apart). This resulted in changes in motor performance and toxic insult of nigral neurons as evidenced by dopamine depletion in nucleus caudatus putamen. In vitro and in vivo treatment of MPTP caused the generation of hydroxyl radicals (·OH) as measured by a sensitive salicylate hydroxylation procedure. A dopamine agonist, bromocriptine (10 µM and 10 mg/kg i.p.), blocked ·OH formation caused by MPTP in vitro (20 µM) and in vivo (30 mg/kg i.p.). An MPTP-induced increase in the activity of catalase and superoxide dismutase in substantia nigra on the seventh day was reduced by bromocriptine pretreatment. Bromocriptine blocked MPTP-induced behavioral dysfunction as well as glutathione and dopamine depletion, indicating its potent neuroprotective action. This study suggests that bromocriptine stimulates antioxidant mechanisms in the brain and acts as a free radical scavenger in addition to its action at dopamine receptors, thus indicating its strength as a valuable neuroprotectant.—Muralikrishnan, D., Mohanakumar, K. P. Neuroprotection by bromocriptine against 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity in mice. FASEB J. 12, 905–912 (1998)

Key Words: MPTP • hydroxyl radicals • dopamine neurotoxicity • neuroprotection • GSH • free radical scavenger • antioxidant enzymes

	INTRODUCTION

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1-METHYL-4-PHENYL-1,2,3,6-TETRAHYDROPYRIDINE (MPTP)³ is a potent neurotoxin that causes selective nigral dopaminergic lesions, resulting in Parkinson's disease in humans and primates (1). It also causes depletion of dopamine (DA) in rodents when administered in high doses (2, 3). The neurotoxic action of MPTP on DA-ergic neurons is believed to be via a number of mechanisms that include selective uptake of the active metabolite 1-methyl-4-phenyl pyridinium ion (MPP⁺) through a DA uptake mechanism (4), inhibition of mitochondrial electron transport in DA-ergic neurons by MPP⁺ (see ref 5), oxidative stress after mitochondrial energy crisis, autoxidation of DA in the neuromelanin-rich substantia nigra (SN) in the presence of iron, etc. (6). A reactive oxygen species is also implicated in DA-ergic toxicity caused by iron (7), 6-hydroxydopamine (8), and head trauma resulting from ischemia/reperfusion injury (9). Many antioxidants (especially synthesized ones) and molecules such as bromocriptine (BRC; 2-bromo--ergocriptine), lipoic acid/dihydrolipoic acid, deprenyl, and melatonin have been useful in all these cases in protecting neurons from the neurotoxicity caused (10–13).

BRC is a widely used antiparkinsonian drug with potent D₂ agonistic and mild D₁ receptor antagonistic action (14). BRC has recently been shown to possess strong free radical scavenging action in in vitro (15, 16) and in vivo (17) studies. Thus, BRC could afford protection against methamphetamine-induced DA depletion (17) and 6-hydroxydopamine-induced DA-ergic toxicity (16). Moreover, behavioral effects after the administration of MPTP have been shown to be reversed in monkeys (18, 19) and mice (20).

In the present study, we looked into the acute and subacute effects of BRC administration in mice on the behavioral and neurochemical changes caused by MPTP. Since the mechanism of action of MPTP involves generation of hydroxyl radicals (·OH) (see ref 6) and BRC is widely used as an antiparkinsonian agent, we analyzed the ·OH scavenging action of BRC after MPTP administration—its effects on MPTP-induced DA depletion as well as on antioxidant molecules such as superoxide dismutase (SOD), catalase (CAT), and GSH in the nigrostriatal system of mice.

	MATERIALS AND METHODS

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Animals and MPTP/BRC/salicylate injections
Adult Balb/c mice from the institute colony were used in this study and were housed under standard conditions of temperature (22±1°C), humidity (60±5%), and illumination (12 h light; 12 h darkness). Animals were always killed in the morning. The experimental protocol met the National Guidelines on the "Proper Care and Use of Animals in Laboratory Research" (Indian National Science Academy, New Delhi) and was approved by the Animal Ethics Committee of the institute.

MPTP HCl was purchased from Research Biochemicals International (Natick, Mass.). BRC, pyrogallol, sodium salicylate, 2,3- and 2,5-dihydroxybenzoic acid (DHBA), and standards of DA and its metabolites were purchased from Sigma Chemical Co. (St. Louis, Mo.). MPTP was dissolved in saline and the solution was injected i.p., 30 mg/kg (0.1 ml/100 g) twice, 16 h apart. BRC was dissolved in traces of methanol (final methanol concentration was approximately 8–10%) and diluted in water. BRC was injected 2.5, 5.0, and 10.0 mg/kg i.p., alone or 30 min before administration of MPTP. Control animals received 10% methanol (0.1 ml/100 g). Sodium salicylate (100 mg/kg, i.p.) was injected into a group of mice in order to measure the ·OH generated after MPTP administration. All animals treated with sodium salicylate were killed at different intervals after the treatment for measurement of the ·OH adducts of salicylate, 2,3-, and 2,5-DHBA in the striatum. This group of animals was not included in the neuroprotective study conducted to prevent MPTP neurotoxicity by BRC.

Behavioral scores
For a period of 5 h after MPTP/BRC administration, mice were observed continuously by two examiners who were trained in evaluating different animal behavior and who were blind to the treatments. Body temperature, akinesia, catalepsy, straub tail, and tremor were monitored during this period following standard scoring procedures (21). Animals were subjected to a swim test (see below) 7 days after MPTP (22). About 2–4 min after receiving MPTP, mice exhibited serotonin syndromes characterized by splayed hind limb, tremor, straub tail, etc.; by 3 h, DA-mediated behavior such as akinesia and rigidity set in (21).

Tremor and hind limb abduction were scored for intensity on a scale of 0–4 (21–23). Akinesia was measured by noting the latency of the animals to move all four limbs in a unit of time (s) and the test was terminated if latency exceeded 180 s (21, 22). Catalepsy was measured by placing the animals on a flat horizontal surface with both hind limbs on a square wooden block (3 cm); latency in seconds required to move the hind limbs from the block to the ground was measured (21, 22). Swim tests were carried out on the seventh day in water tubs (40 lengthx25 widthx12 height, in cm). The depth of water (27°C±2°C) was kept at 8 cm. The animals were acclimatized for 10 min 1 day prior to experimentation. Swim score scales were: 0, hind part sinks with head floating; 1, occasional swimming using hind limbs while floating on one side; 2, occasional floating/swimming only; and 3, continuous swimming.

Biochemical Analyses
High-performance liquid chromatography (HPLC) assay
Biochemical changes after MPTP administration in mice include an initial release of serotonin and DA (6, 21), followed in 3–4 h by a decrease in the level of DA (21). For analysis of DA and its metabolites, mice were decapitated and their brains were dissected out and frozen on dry ice. Frozen sections of 1 mm were cut, then the substantia nigra (SN) and nucleus caudatus putamen (NCP) were micropunched (3, 21). Samples were weighed and sonicated in 0.2 M ice-cold perchloric acid (10% wt/vol) containing 0.5% EDTA. They were centrifuged at 10,000 x g for 10 min, and 10 µl supernatant was injected directly into the HPLC system (Waters, Milford, Mass.) to determine DA, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA). The system was equipped with an electrochemical detector (460; Waters) and an ion-pair, ultrasphere reverse-phase chromatography column (4.6 cmx25 cm) with a 5.0 µ particle size (Beckman, Fullerton, Calif.). The mobile phase contained 8.65 mM heptane sulfonic acid, 0.27 mM EDTA, 13% acetonitrile, 0.4–0.45% triethylamine, and 0.20–0.25% phosphoric acid (vol/vol). The flow rate was 0.7 ml/min, and electrodetection was performed at 0.74 V. Results are presented as pmol/mg fresh tissue and are uncorrected values.

Crude homogenate of SN and ·OH assays
SN from normal mice were homogenized in ice-cold artificial cerebrospinal fluid (in mM: NaCl 124.0, KCl 5.0, NaH₂PO₄ 1.25, CaCl₂ 2.00, MgSO₄ 2.0, NaHCO₃ 26.0, and glucose 10.0; pH 7.2) in a glass-Teflon homogenizer and centrifuged at 15,000 x g for 15 min at 4°C. The supernatant fractions were incubated for 1 h with or without MPTP (20 µM) and BRC (10 µM), along with the ·OH trapping agent sodium salicylate (1 mM), at room temperature. In preliminary experiments it was observed that 2,3- and 2,5-DHBA formation in vitro due to MPTP was almost completely saturated by 60 min. For analyzing the in vivo generation of ·OH, animals treated with sodium salicylate (see injections) were killed at various intervals (every 30 min up to 3 h) to optimize the yield of ·OH adducts 2,3- and 2,5-DHBA. It was observed that 2 h after sodium salicylate, which was given 30 min after MPTP, a considerable amount of 2,3-DHBA, the most reliable indicator of ·OH adduct (24), could be detected. There was no considerable increase in 2,3-DHBA during the next 1 h. Thus, all experiments were conducted according to this protocol. HPLC electrochemistry was used to analyze 2,3- and 2,5-DHBA (6, 7). The conditions of the HPLC were as explained above. A study was conducted to determine the level of salicylate in the brain 2 h after administration of 100 mg/kg sodium salicylate in BRC- or MPTP-treated animals. Salicylate was detected at 295 nm by HPLC and a UV detector in samples prepared as described above and under similar HPLC conditions. The percentage of salicylate detectable in the brain 2 h after BRC administration was found to be 3–7% more (n=3) than in MPTP-treated animals analyzed at the same time point.

Preparations of cytosolic/particulate fractions and assays of SOD, CAT, and glutathione (GSH)
The micropunched SN were homogenized in potassium phosphate buffer (pH 7.8, 0.1 M) while using a glass-Teflon homogenizer. They were centrifuged at 100,000 x g for 60 min at 4°C. The supernatant corresponds to the cytosolic fraction containing CuZn-SOD. The pellets were resuspended in the buffer, freeze-thawed three times, and centrifuged at 100,000 x g for 60 min at 4°C. The supernatant, the particulate fraction containing Mn-SOD, was mixed with the cytosolic fraction to obtain the total enzyme fraction. SOD was analyzed after inhibition by SOD of the pyrogallol autoxidation (25) at pH 8.2 and in the presence of EDTA. Briefly, a 3 ml assay mixture contained 0.2 mM pyrogallol, 1 mM EDTA, and 50 mM Tris-HCl buffer. Pyrogallol autoxidation was monitored at 420 nm for 3 min with or without the enzyme protein (approximately 500 µg). The inhibition of pyrogallol oxidation was linear with the activity of the enzyme present. Fifty percent inhibition/(mg protein·min^-1) was taken as one unit of the enzyme activity.

CAT activity was assayed based on H₂O₂ decomposition monitored at 240 nm for 30 s (26). An assay mixture of 500 µl contained suitably diluted enzyme protein (100 µg) in 50 mM phosphate buffer, pH 7.0. The reaction was started by the addition of H₂O₂ (30 mM), which provided approximately 0.50 absorbance. The decrease in absorbance was monitored and the enzyme activity was expressed as change in absorbance/(min·mg protein^-1). GSH was measured fluorimetrically according to the method of Cohn and Lyle (27), using o-phthalaldehyde condensation reaction with GSH to yield a fluorescent product at pH 8.0. Readings were taken at activation/emission wavelengths of 340/420 nm. Protein was assayed by measuring the absorbance at 280 nm and calculating the concentration by taking ₂₈₀^1% = 6.6 or using the Bradford assay procedure (28).

Statistical analysis
Student's t test was used to find significant differences between two means. Two-way analysis of variance, followed by the Pearsons test, were performed, using a statistical package (EPISTAT) to evaluate the effects of different drugs. P values of less than 0.05 were considered significant.

	RESULTS

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The hypothermic response of BRC was absent in MPTP/BRC-treated mice. BRC did not abolish tremor as induced by MPTP but did reduce intensity. BRC reversed MPTP-induced akinesia, catalepsy, and hind limb abduction. Long-lasting motor impairment caused by MPTP, as shown by significantly lowered swim scores, was reversed by BRC ( Table 1).

A major finding was the observed generation of ·OH in response to the administration of MPTP, as seen by an increase in the content of either 2,3- and 2,5-DHBA in vitro (in SN crude homogenate; Fig. 1) or 2,3-DHBA in vivo (in NCP, see Fig. 2). ·OH generation was attenuated by the presence of BRC in the incubation medium. However, BRC failed to significantly reverse the level of 2,3-DHBA caused by MPTP in vitro ( Fig. 1). Pretreatment with BRC 30 min before MPTP administration significantly blocked ·OH generation in vivo ( Fig. 2). The amount of salicylate detected in the brain after BRC or MPTP showed no difference at any given time, which excludes the possibility that either drug may interfere with the entry of salicylate into the brain and influence the concentration of 2,3- and 2,5-DHBA detected in the in vivo studies. Figure 1shows the changes of DA content in SN after administration of MPTP and BRC in vitro. A decrease in the content of DA in the homogenate of the cell body region probably indicates a decrease in membrane permeability as a result of an inhibitory action of MPTP/MPP⁺ on voltage-activated membrane currents (our unpublished observations). There is no change in the content of DA in NCP 2 h and 30 min after the administration of MPTP ( Fig. 2), as opposed to an increase in DA that occurs after 1 h (see Fig. 6). These results explain the initial DA overflow (6) and the initiation of akinesia caused by an inhibition of DA by 3 h and 30 min after MPTP (21) administration, as reported earlier.

View larger version (27K): [in this window] [in a new window]   Figure 1. In vitro ·OH generation elicited by MPTP in crude homogenates (15,000 x g) of SN from normal mice. Fractions of 1 ml were incubated with MPTP (20 µM)/BRC (10 µM) in the presence of sodium salicylate (1 mM). An equal volume of chilled 0.2 M perchloric acid was added to an aliquot of 50 µl at 1 h and centrifuged for 10 min at 10,000 x g. The ·OH adducts of salicylate, 2,3- and 2,5-DHBA were analyzed in the supernatant with the use of HPLC electrochemistry. Note the significantly high levels of ·OH adducts formed due to MPTP and its reversal by BRC (total·OH, P0.01 or 2,5-DHBA; P0.001, as compared to MPTP group). BRC did not significantly inhibit the MPTP-induced increase in 2,3-DHBA. Results are mean ± SEM. *P 0.05 as compared to the control. n = 5.

View larger version (24K): [in this window] [in a new window]   Figure 2. In vivo generation of ·OH in NCP after MPTP (30 mg/kg, i.p.). BRC (10 mg/kg i.p.) was administered 30 min before MPTP. Sodium salicylate (100 mg/kg, i.p.) was injected 30 min after MPTP. The best time to detect ·OH adducts of salicylate was determined by analyzing the samples every 30 min after MPTP, as well as administering salicylate at various intervals before and after MPTP. All animals in this group were killed 2 h after salicylate administration, when a good amount of 2,3-DHBA was detectable. The high level of MPTP-induced 2,3-DHBA in NCP was reduced significantly (P0.001) by BRC. Results are mean ± SEM. *P 0.05 as compared to the control. n = 4.

View larger version (39K): [in this window] [in a new window]   Figure 6. Effects of MPTP and/or BRC on the content of dopamine (DA) in the striatum. DA was measured by a sensitive HPLC electrochemical procedure. An increase in dopamine 1 h after MPTP indicates the release at the nerve terminal region, NCP. The severe decrease in dopamine 7 days after neurotoxin administration indicates nigral neuronal damage. Note the significant reversal by BRC of MPTP-induced dopamine depletion (P0.05 and 0.002 as compared to the MPTP group on the first and seventh day, respectively). Results are mean ± SEM. *P 0.01 as compared to the control. n = 8–10.

MPTP did not affect the content of GSH or the activity of CAT or SOD in SN for up to 24 h. One week after MPTP administration, this nucleus, which contains DA-ergic cell bodies, showed a significant decrease in GSH ( Fig. 3) and an increase in the activity of SOD ( Fig. 4) and CAT ( Fig. 5). BRC alone caused no change in the activity of the antioxidant enzymes for up to 1 week, except that on the seventh day, CAT activity was augmented ( Fig. 5). After 1 h, BRC caused the GSH level to increase and to decrease by 1 day ( Fig. 3). BRC abolished an MPTP-induced decrease in GSH on the seventh day ( Fig. 3). Effects of BRC on the MPTP-induced increase in the activity of SOD was insignificant. There was a parallel increase in the activity of SOD after MPTP or BRC ( Fig. 4). One hour after the drug treatments, CAT activity increased; the effect disappeared by 1 day and reappeared after 7 days. However, BRC treatment failed to significantly lower MPTP-induced elevated CAT activity ( Fig. 5). MPTP-induced DA depletion in the nerve terminal region, NCP, was restored by BRC pretreatment ( Fig. 6). The effect of BRC in this action was dose dependent ( Table 2). The turnover of DA in NCP after BRC alone was decreased, but the effect was independent of the dose administered. A dose-dependent reversal by BRC of DA turnover was observed in MPTP-treated mice ( Table 2).

View larger version (37K): [in this window] [in a new window]   Figure 3. Effects of MPTP and/or BRC on the content of GSH in the cytosolic and particulate fractions of SN at various intervals. GSH was analyzed using a spectrofluorimetric procedure. The chronic depletion of GSH due to MPTP was reversed by pretreatment with BRC (P0.01 as compared to MPTP group). Results are mean ± SEM. *P 0.01 as compared to the control. n = 6–8.

View larger version (45K): [in this window] [in a new window]   Figure 4. Effects of MPTP and/or BRC on the activity of total superoxide dismutase (SOD) in SN at various intervals after treatment with the drug (or drugs). SOD was analyzed using a pyrogallol oxidation method in combined cytosolic/particulate fractions from micropunched SN. BRC caused significant induction of the enzyme by day 1 as compared to the MPTP-treated group (P0.01). Results are mean ± SEM. *P 0.05 as compared to the control. n = 6–8.

View larger version (39K): [in this window] [in a new window]   Figure 5. Effects of MPTP and/or BRC on the specific activity of catalase (CAT) in SN at 1 h, 1 day, and 7 days after administration of the drug (or drugs). CAT was analyzed by monitoring the disappearance of H2O2 at 240 nm in the presence of the enzyme in combined cytosolic/particulate fractions from micropunched SN. The MPTP-induced increase in activity was not significantly blocked by BRC. Results are mean ± SEM. *P 0.05 as compared to the control. n = 6–8.

	DISCUSSION

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The major finding of this study is the reversal by BRC of the damaging consequences of MPTP-induced ·OH generation in SN and NCP, resulting in DA depletion in the striatum of mice. This effect is shown to be through the ·OH scavenging action of BRC in vitro and in vivo. BRC's stimulant effect on GSH may also have contributed to restoration of the deleterious effects of MPTP on the brain antioxidant system. Since both MPTP and BRC induced similar changes (an increase) in CAT and SOD, a compensatory role of these enzymes in the neuroprotective action of BRC is contraindicative.

Our findings are in line with other reported observations that BRC is an antioxidant (15) and can afford neuroprotection against DA damage in methamphetamine-induced DA-ergic neurotoxicity (17) via a free radical scavenging mechanism. 6-Hydroxydopamine-induced striatal neurotoxicity has also been shown to be blocked by BRC, and this effect has been linked to in vitro results where ·OH generated by the Fenton reaction was scavenged by BRC (16). Chronic dietary supplementation of BRC has also been reported to protect nigral neuronal integrity in aged rats by suppressing the turnover of DA through a presynaptic D₂ receptor mechanism (29).

Reports during the past decade on the deficiency of mitochondrial function (see ref 5), increased iron accumulation in the SN compacta region (30), increased lipid peroxidation (31), a decrease in GSH content (32), and changes in antioxidant enzymes activity (33, 34) in the brain of parkinsonian patients or in MPTP-induced parkinsonian monkeys (35–37) emphasize the importance of oxidative damage and the involvement of free radicals in the pathogenesis of this movement disorder. Inhibition of mitochondrial respiration in vivo (38) and the generation of ·OH due to MPTP or its metabolites employing in vivo brain microdialysis (39) in rodents have been documented in the literature. We have demonstrated iron-induced ·OH formation and lipid peroxidation resulting in nigral injury (7, 11, 40) in rats that indicates a strong role of free radicals in the pathophysiology of dopaminergic neurodegeneration. The present findings support these observations and provide concrete evidence for the involvement of ·OH in the dopaminergic neurodegeneration caused by MPTP.

MPTP is metabolized to its active toxin, MPP⁺, in the brain by the action of monoamine oxidase (MAO) (41). This enzymatic conversion of MPTP to MPP⁺ is shown to involve generation of free radicals (42). Similarly, electron paramagnetic resonance studies have indicated the formation of superoxide anions in mitochondria in the presence of MPP⁺ (43). The increased activity of total SOD in SN in the present study 7 days after MPTP may indicate induction of this enzyme to an oxidative insult by the neurotoxin on the mitochondrial electron transport system. However, our results are not consistent with an earlier report in which an inhibition of SOD activity in SN is shown after MPTP (44). Yet these authors have demonstrated an overall increase in SOD activity in NCP. The difference may be due to various reasons, including differences in the animal species used, dose regime, and time of analysis. BRC alone caused a parallel increase in this enzyme, indicating no significant role of this enzyme in the antioxidant action of BRC.

MPTP treatment resulted in increased CAT activity in SN. Our observations with NCP (data not given) show a similar trend in CAT activity. The effect of BRC on CAT activity was also similar. Our observations confirm earlier data on CAT activity after administration of MPTP (44). The present observation about CAT assumes significance in view of our findings that ·OH is generated after MPTP administration in vivo and in vitro. Moreover, a significant generation of ·OH in addition to superoxide anions has been shown in the MAO-mediated conversion of MPTP to MPP⁺ (42). The induction of CAT activity observed after MPTP administration may be result from the generation of ·OH to eliminate H₂O₂ formed in the brain in a subsequent reaction.

In idiopathic parkinsonism, GSH levels are reduced in SN (32). Our findings are analogous to those in humans and are comparable to earlier reports that demonstrate a decrease in the level of GSH in rats (45), mice (46), and monkeys (47) after the administration of MPTP. In these monkeys, ferrous-induced lipid peroxidation could be blocked by -dihydroergocriptine, a BRC analog (47). This report supports our finding that BRC could reverse a MPTP-induced decrease in GSH and indicates that 1) free radicals are directly involved in the neurotoxicity caused by MPTP and 2) the reversal of MPTP neurotoxicity by BRC is via free radical scavenging mechanisms. Conversely, specific inhibition of GSH biosynthesis in animals has been shown to potentiate MPTP neurotoxicity in mice (48).

Although the foregoing discussion illustrates a direct involvement of free radicals in the neurotoxic action of MPTP and free radical scavenging ability in the neuroprotective effect of BRC, it is worthwhile to examine possible alternative mechanisms. Neurotoxic action of MPTP involves a series of events in the brain such as 1) conversion of MPTP to MPP⁺ by MAO (41), 2) selective uptake of MPP⁺ by DA-ergic neurons through a DA uptake mechanism (4), and 3) inhibition of NADH ubiquinone reductase in the mitochondrial electron transport system by MPP⁺ (5, 38). The possibility that BRC may interfere at any one or more of these events cannot be ruled out. Analyzing each possibility listed, 1) BRC did not show any effect on the activity of MAO in rat brain homogenate in vitro or in vivo (49), 2) any effect of BRC on DA uptake in the brain is at the very least controversial; the issue is reviewed in detail to conclude that it is highly unlikely that BRC has any significant effect on DA uptake (see ref 50), and 3) it has been shown that mitochondrial complex system I is unaffected by BRC treatment (51). Alternatively, it has been suggested that BRC may act at the presynaptic D₂ receptor sites and, by inhibiting DA turnover, may render neuroprotection in aged rats (29). However, based on our behavioral data and biochemical results showing a reduction in the turnover of DA in NCP after administration of BRC alone (even though there is a lack of dose dependency), we cannot exclude the possibility that the neuroprotective action of BRC is affected in part by DA₂ receptor mechanisms.

The results presented here add to the existing understanding of the free radical mechanisms in the dopaminergic neurotoxicity caused by MPTP. In addition, the study unearths neuroprotective and antioxidant actions of BRC (which so far has been known for its clinical use as an antiparkinsonian drug due to its D₂ agonistic action) against MPTP-induced neurotoxicity. The study also reveals positive effects of BRC on SOD, GSH, and CAT, the major endogenous antioxidant molecules in the brain, pointing out its added benefits in the treatment of neurodegenerative disorders in which reactive oxygen species are involved.

	ACKNOWLEDGMENTS

 
The authors thank C.S.I.R., Government of India, for a Senior Research Fellowship to D. M. The goodwill and encouragement extended by Dr. C. C. Chiueh and Dr. D. L. Murphy, NIMH, National Institutes of Health (U.S.), are gratefully acknowledged. Figures were drawn by Mr. S. P. Sahoo, and the manuscript was typed by Mr. N. K. Das, IICB.

	FOOTNOTES

 
¹ Some of the data described in this paper were presented at a poster session held at the International Symposium on Free Radicals in Medicine and Biology, Udaipur, India, September 22–24, 1997.

¹ Correspondence: Laboratory of Neurochemistry, Division of Pharmacology and Experimental Therapeutics, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Calcutta 700 032, India. E-mail: iichbio{at}giascl01.vsnl.net.in

³ Abbreviations: BRC, bromocriptine (2-bromo--ergocriptine); CAT, catalase; DA, dopamine; DHBA, dihydroxybenzoic acid; DOPAC, 3,4-dihydroxyphenylacetic acid; GSH, glutathione; HPLC, high-performance liquid chromatography; HVA, homovanillic acid; MAO, monoamine oxidase; MPP⁺, 1-methyl-4-phenyl pyridinium; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NCP, nucleus caudatus putamen; ·OH, hydroxyl radical; SN, substantia nigra; SOD, superoxide dismutase.

Received for publication October 30, 1997. Accepted for publication February 15, 1998.

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