Effects of Homocysteine on the Dopaminergic System and Behavior in Rodents

NeuroToxicology 26 (2005) 361–371

Effects of Homocysteine on the Dopaminergic System and Behavior in Rodents Eun-Sook Y. Lee 1,*, Hongtao Chen 1, Karam F.A. Soliman 1, Clivel G. Charlton 2 1 2

College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL 32307, USA Department of Pharmacology, Meharry Medical College, Nashiville, TN 37208, USA Received 27 July 2004; accepted 26 January 2005 Available online 3 March 2005

Abstract Long-term treatment of levodopa for Parkinson’s disease (PD) patients is known to elevate homocysteine level in their plasma. The present study was designed to examine the possible neurotoxic effects of the increased homocysteine level on the dopaminergic system. Homocysteine was administered into Sprague–Dawley male rats intracerebroventricularly or C57BL/6 mice intraperitoneally. Following homocysteine injection the locomotor activities, the levels of dopamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and immunohistochemistry of dopaminergic neurons were examined. The results obtained indicate that homocysteine administration (1 or 2 mmol, i.c.v.) into the rat brains for 5 days significantly decreased the locomotor activities and dopamine as well as its metabolites, DOPAC and HVA, in the rat striatal regions. Two different doses of homocysteine (50 and 100 mg/100 g, i.p. daily) were administered into mice for 36 days to evaluate the effect of systemic treatment of homocysteine on the dopaminergic neurons of the brain. The intraperitoneal injections of two doses of homocysteine significantly increased homocysteine levels in the striatal regions of mouse brains by 21.5 and 39.2%, while reducing dopamine turnover rates in the striatal regions by decreasing (DOPAC + HVA)/DA, 23.7 and 51.6%, respectively. Accordingly, homocysteine decreased locomotor activities significantly by decreasing movement time by 29 and 38%, total distance by 32 and 42%, and numbers of movement by 28 and 41%, respectively. Moreover, homocysteine decreased tyrosine hydroxylase immunoreactivity in substantia nigra of mouse brain. The data obtained indicate that the potential of homocysteine to be toxic to the dopaminergic system. Consequently, long-term levodopa therapy for PD may accelerate the progression of PD, at least in part by elevated homocysteine.

# 2005 Elsevier Inc. All rights reserved. Keywords: Homocysteine; Dopamine; Locomotor activities; Neurotransmission; Levodopa

INTRODUCTION Parkinson’s disease (PD) is a progressive neurological disorder resulted from dopaminergic cell death and dopamine depletion (Hornykiewicz, 1966). Since the etiology of PD remains to be elucidated, the strategy of current treatments of PD patients is symptomatic with efforts to slowdown the progression of the disease. L-3,4-Dihydroxyphenylalanine (levodopa) is a

* Corresponding author. Tel.: +1 850 599 8445; fax: +1 850 412 7261. E-mail address: [email protected] (E.Y. Lee).

precursor of dopamine and represents the most effective medication for PD treatment. When levodopa serves as a precursor of dopamine, one main metabolizing pathway of levodopa is O-methylation to 3-O-methyldopa (3-OMD) by catechol-O-methyltransferase (COMT) (Bartholini and Pletscher, 1968; Sharpless and McCann, 1971). During this methylation process, S-adenosylmethionine (SAM) is utilized as a methyl donor and the demethylated product from SAM-dependent methylation, S-adenosylhomocysteine (SAH), is produced as a by-product. However, SAH is unstable in the biological environment and, therefore, readily hydrolyzed to homocysteine and adenosine by SAH hydrolase

0161-813X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2005.01.008

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Fig. 1. Metabolic pathway of 3-O-methyldopa, homocysteine and adenosine. While levodopa serves as a precursor of dopamine in dopamine synthesis pathway, it is also methylated to 3-OMD by COMT and SAH is produced from methylation of levodopa. SAH is unstable and readily hydrolyzed to homocysteine and adenosine by SAH hydrolase. Enzymes: 1, methionine adenosyltransferase; 2, catechol-O-methyltransferase; 3, SAH hydrolase; 4, methionine synthase. Abbreviation: THF, tetrahydrofolic acid.

(Fig. 1). Elevated homocysteine and 3-OMD levels have been reported in PD patients using levodopa as a therapeutic agent (Allain et al., 1995; Kuhn et al., 1998; Muller et al., 1999, 2001, 2002). Homocysteine is elevated in animals fed folic aciddeficient diet in which folic acid serves as a cofactor in the metabolic pathway of homocysyeine to methionine. Moreover, homocysteine could be derived from the demethylation of the amino acid methionine that plays a pivotal role in the generation of methyl groups required for numerous biochemical reactions (Reutens and Sachdev, 2002; Fig. 1). In human, the normal concentration of homocysteine is approximately 5 mM in plasma and 0.5 mM in cerebrospinal fluid (CSF) (Hyland and Bottiglieri, 1992), but it is elevated over 200 mM in hyperhomocysteinemia (Stabler et al., 1988). Recently, the role of homocysteine has been widely investigated in the cardiovascular area because it is believed to be associated with the pathogenesis of arteriosclerosis and thrombosis (Temple et al., 2000; Andreotti et al., 2000). It has been reported that long-term use of levodopa in PD patients might be associated with elevated homocysteine level and the increased risk for vascular disease (Rogers et al., 2003). Meanwhile, homocysteine is considered a risk factor for multiple neurological disorders including Alzheimer’s disease (AD) and PD (Gottfries et al., 1998; Reutens and Sachdev, 2002; Mattson and Shea, 2003). An increased homocysteine level is closely associated with dementia, AD, stroke and depression (Perry et al., 1995; Bots et al.,

1999; Bottiglieri et al., 2000; Seshadri et al., 2002; Kruman et al., 2002). However, the molecular mechanism of homocysteine-induced neurotoxicity has not been completely established at present. Homocysteine can convey multiple neuropathological effects, including cytosolic accumulation of calcium, induction of oxidative stress, apoptosis, and NMDA-mediated excitotoxicity (Kim and Pae, 1996; Kruman et al., 2000; Ho et al., 2002; Maler et al., 2003). On the other hand, hyperhomocysteinemia in patients with PD is likely to be secondary to the metabolism of levodopa medication, in which homocysteine is produced from the hydrolysis of SAH during SAM-dependent methylation of levodopa (Fig. 1), implying that levodopa treatment could have harmful effects via the possible neurotoxic effects of homocysteine (Reutens and Sachdev, 2002). Levodopa is generally administered with dopa decarboxylase inhibitors to prevent its peripheral degradation (Miller et al., 1997; Tolosa et al., 1998). However, the therapeutic effectiveness of levodopa is decreased and most PD patients taking levodopa experience motor fluctuations such as on–off phenomena and dyskinesia after chronic levodopa therapy (Fahn, 1974; Marsden and Parkes, 1976). The mechanism of these complications from long-term use of levodopa is not clearly understood. It has been suggested that, in addition to levodopa toxicities such as oxidative stress, the accumulation of certain metabolites of levodopa might be responsible for these side effects. There is growing evidence that elevated homocysteine level might play a

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role in the pathogenesis of PD and complications of levodopa treatment. A genetic predisposition towards hyperhomocysteinemia is associated with increased risk of PD (Yasui et al., 2000). Homocysteine may also be toxic to the dopaminergic neurons (Kim et al., 1987; Meldrum, 1993). Homocysteine was found to enhance 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic dysfunction in mouse model of PD and sensitized dopaminergic neuronal culture, implicating that elevated homocysteine levels could endanger dopaminergic neurons in susceptible individuals and could accelerate the progression of the disease in levodopa therapy for PD patients (Duan et al., 2002; Mattson and Shea, 2003). Therefore, the present study was designed to investigate the possible neurotoxic effects of homocysteine on the dopaminergic system.

MATERIALS AND METHODS Animals Male Sprague–Dawley rats weighing 180–200 g and male C57BL/6 mice 4 weeks old were purchased from Harlan (Indianapolis, IN). Animals were kept in the animal care facility until the beginning of experiments and had access to food and water ad libitum, under a 12 h light/dark cycle. Temperature was maintained at 21  1 8C. Chemicals Homocysteine, 3-O-methyldopa, adenosine, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), tri-n-butylphosphine, trichloroacetic acid, sodium borate, EDTA, methanol, acetonitrile, and dimethylaformamide were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Tyrosine hydroxylase antibodies were purchased from Chemicon International (Temecula, CA, USA). Phosphoric acid, perchloric acid, citric acid, and other materials were obtained from Fisher Scientific (Pittsburgh, PA, USA). Cannulation of Rats Rats weighing 250–300 g were anesthesized with chloral hydrate (400 mg/kg, i.p.). A 22-guage stainless steel cannula was stereotaxically implanted for injections into the lateral ventricle of the rat brain.

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The position for cannula was at 1.5 mm lateral and 0.6 mm caudal with reference to the bregma from which it extended to the lateral ventricles. After the cannulations, rats were allowed to recover for 3 days before the experiments. Drugs were injected into the lateral ventricle 5 mm from the surface of the cranium through polyethylene tube (PE 20) which was attached to a 10 ml Hamilton syringe. Measurement of Locomotor Activities Phosphate buffered saline (PBS) was used as a control and homocysteine, 3-O-methyldopa, or adenosine was dissolved in PBS. After the rats were injected (i.c.v.) with PBS, homocysteine, 3-O-methyldopa, or adenosine in 5 ml once a day, rats were placed in the locomotor activity monitor. For mice, locomotor activities were measured 24 h after 36 days of daily injection (i.p.) of homocysteine. The measurement was started 3 min after the placement of animals into the monitor in a quiet isolated place with a dim light. The changes in motor activity of the animals were measured using Activity Monitor (Degiscan Instruments, Inc., Columbus, OH, USA). Movement time (MT), total distance (TD) and the number of movements (NM) were determined. The locomotor activities were determined for 40 min post-injection. HPLC Analysis For the measurements of DA, DOPAC, HVA, SAM, and SAH, after the last injections, animals were sacrificed and cortex, hippocampus and striatal regions were dissected. The dissected tissues were homogenized using Polytron in 0.4 M perchloric acid, centrifuged, and filtered for HPLC analysis as described previously (Lowry et al., 1996). The HPLC system (LC-10 AT, Shimadzu) was connected with an electrochemical (Coulochem II, ESA) and UV (Shimadzu) detectors, using a C-18 reverse-phase column (5 mm, 250 mm  4.6 mm, Whatman EQC). The mobile phase consisted of 0.1 M sodium acetate, 60 mM citric acid, 0.6 mM octanesulfonic acid sodium salt, 0.5 mM disodium EDTA, in 15% methanol in water, pH 3.5 and pumped at a rate of 1.0 ml/min. For the measurement of homocysteine, mice were sacrificed after 36 daily injections (50 and 100 mg/ 100 g, i.p.) and mouse striatal regions were dissected. The brain tissues were homogenized in 0.4 M perchloric acid, centrifuged at 9000  g for 20 min, and filtered (0.45 mm). The supernatants were derivatized with the fluorogenic reagent ammonium-7-fluoro-

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benzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F) in 1.5 M NaOH, 4 mM EDTA, 0.1 M sodium borate buffer at 60 8C for 60 min and samples were prepared for HPLC with fluorescence detector (RF-10AL, Shimadzu) at excitation 395nm and emission 515 nm. Homocysteine was separated in C-18 reverse column (5 mm, 250 mm  4.6 mm, Whatman EQC) with the mobile phase of 50 mM NaH2PO4 and 1.5% acetonitrile at pH 3.5 and a flow rate of 0.8 ml/min. Immunohistochemistry of Tyrosine Hydroxylase (TH) After the measurement of locomotor activities, randomly selected mice from each group were anesthesized with chloral hydrate (400 mg/kg) and perfused with PBS followed by 4% paraformaldehyde in PBS. Mouse brains were dissected out and stored at 70 8C after soaked in 4% paraformaldehyde in PBS for 1 day and 30% sucrose for 2 days at 4 8C. About 25 micron frozen sections were prepared in a cryostat and mounted on gelatin chrome-alum coated slides. The method for measuring TH immunoreactivities was previously described in Charlton and Mack (1994) with a minor modification. The frozen slide mounted slices were preincubated in 0.3% Triton X-100 in PBS, pH 7.4, three times for 5 min each time. Frozen sections (25 mm thickness) of mouse brain tissues were labeled with anti-rabbit polyclonal tyrosine hydroxylase (TH) (1:1000 dilution; Chemicon International, Temecula, CA) as primary antibody and anti-goat rabbit IgG tagged with FITC (1:200 dilution; Chemicon International, Temecula, CA, USA) as secondary antibody. Tyrosine hydroxylase immunoreactivities in the mouse substantia nigra regions were visualized with Confocal microscopy (PCM 2000, Nikon) Statistical Analysis The mean and standard error of the mean (S.E.M.) were determined for each set of data and one-way analysis of variance (ANOVA) followed by Newman– Keuls post-hoc test was used for statistical analysis to

compare control and treated groups. A probability of 0.05 or less was considered significant.

RESULTS Effects of Homocysteine, Adenosine, and 3-OMD on Dopamine Turnover in Rat Brain Striatum SAM-dependent methylation of levodopa results in the production of 3-OMD as well as S-adenosylhomocysteine (SAH) which is readily hydrolyzed by SAH hydrolase to adenosine and homocysteine (Fig. 1). The present study was focused on homocysteine effects to determine the possible role of elevated homocysteine in pathological conditions such as PD with levodopa treatment. Three possible metabolites of levodopa, homocysteine, 3-OMD and adenosine, were also tested. Sprague–Dawley male rats were cannulated and each compound was injected daily for two days by i.c.v. administration. One hour after the second injection, the levels of dopamine, DOPAC, and HVA in rat brain striatal regions were determined using HPLC. The short-term treatments of all three compounds to rats significantly increased dopamine levels, while homocysteine increased dopamine greatest, followed by adenosine and 3-OMD. However, only homocysteine caused a significant decrease in the levels of dopamine metabolites, DOPAC and HVA, in the rat striatum. Accordingly, all of three compounds (homocysteine, 3-OMD and adenosine) decreased dopamine turnover significantly by 70.0, 31.6, and 49.8%, respectively, indicated as a ratio of (DOPAC + HVA)/DA (Table 1). Homocysteine decreased dopamine turnover rate most severely in rat striatal regions among three compounds tested (Fig. 2, Table 1), but did not change any locomotor activities. To determine the specificity of homocysteine effects on rat striatal regions, other regions of rat brains such as cortex and hippocampus were also tested. The results revealed that other regions (cortex and hippocampus) did not cause any significant changes on the level of dopamine and its metabolites (data not shown).

Table 1 Effects of 3-OMD, homocysteine, and adenosine on the dopamine turnover rate in rats 1 mmol (DOPAC + HVA)/DA

PBS 0.209  0.021

3-OMD 0.143  0.019

Homocysteine *

**

0.063  0.009

Adenosine 0.105  0.017*

Dopamine turnover rate was determined after 2 days of injections (once a day) of each compound. Values are expressed as means  S.E.M. (N = 8). One-way ANOVA followed by Newman–Keuls test was used for statistical analysis to compare different groups * Indicates significantly decreased compared to the control (*p < 0.05, **p < 0.01).

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and 2 mmol) were administered by i.c.v. route for 5 days. The results showed that the consecutive treatments of homocysteine for 5 days significantly decreased the levels of dopamine and its metabolites and locomotor activities (Figs. 3 and 4). Two micromoles of homocysteine decreased the levels of dopamine, DOPAC and HVA by 20.4, 26.7, and 40.6%, respectively (Fig. 3, F 2,15 = 8.746; p = 0.0030 for dopamine; F 2,15 = 6.256; p = 0.0106 for DOPAC; F 2,15 = 9.196; p = 0.0025 for HVA), indicating that the repeated administration of homocysteine for a long period time could impair the dopaminergic system. The effects of homocysteine on locomotor activities were also measured. The data showed that 2 mmol of homocysteine significantly decreased movement time (MT), total distances (TD) and the number of movements (NM) by 28.1, 21.6 and 25%, respectively (Fig. 4, F 2,15 = 8.571; p = 0.0033 for MT; F 2,15 = 5.749; p = 0.014 for TD; F 2,15 = 9.040; p = 0.0027 for NM), indicating that the higher doses of homocysteine for longer time exposure could cause behavioral changes. Other amino acids, glycine and cysteine that were randomly chosen, were also tested to determine the specificity of homocysteine effects on rats. However, neither glycine nor cysteine caused significant changes on behaviors and neurochemistry of dopaminergic nervous system (data not shown). Effects of Systemic Administration of Homocysteine on Dopamine Turnover in the Mouse Brain

Fig. 2. Effect of 3-OMD, homocysteine, and adenosine on the levels of dopamine and its metabolites in the striatal regions of rat brains. Each compound was dissolved in PBS and 1 mmol of each in 5 ml of PBS was injected into the rat brain by i.c.v. route once a day for two days. One hour after the second injection rats were sacrificed and prepared for HPLC analysis. Values are expressed as means  S.E.M. (N = 8). One-way ANOVA followed by Newman–Keuls post-hoc test was used for statistical analysis to compare control and treated groups. (*) Indicates significantly decreased compared to the control (*p < 0.05, **p < 0.01, ***p < 0.001). Abbreviation: HC, homocysteine; 3-OMD, 3-O-methyldopa.

Effects of 5 Days Treatments of Homocysteine by i.c.v. Route in Rats Since homocysteine altered the dopaminergic system most significantly, two doses of homocysteine (1

In this study, the effects of chronic and systemic administration of homocysteine on the dopaminergic system were investigated using C57BL/6 mice to determine the effects of homocysteine in different routes and animals. Two doses of homocysteine (50 and 100 mg/ 100 g, i.p. daily) into mice were administered for 36 days. The dosages used for the experiments were lower than the dosage to induce epilepsy in which 11 mmol/kg or 1485 mg/kg was injected (i.p.) (Folbergrova, 1997) and no seizure activities were observed in the present experimental conditions. Moreover, it is believed relevant to use relatively high dosages of homocysteine because PD patients who take large amount of levodopa (up to 8 g/day) daily for several years might accumulate the toxic effects of homocysteine. First, it was attempted to determine whether systemic treatment of homocysteine could increase homocysteine level in the mouse brain. As shown in Fig. 5, the levels of homocysteine were significantly elevated in striatal regions of C57BL/6 mice after systemic injections of homocysteine (50 and

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Fig. 3. Effect of homocysteine on the levels of dopamine and its metabolites after 5 days of consecutive treatments (i.c.v.) in the striatal regions of rat brains. Two doses (1 and 2 mmol) of homocysteine were injected into the rat brains by i.c.v. route daily for 5 days and 2 h after the last injection rats were decapitated and the rat striatal regions were dissected. After sample preparations for HPLC, the levels of dopamine, DOPAC and HVA were measured in HPLC with electrochemical detection. Values are expressed as means  S.E.M. (N = 6). One-way ANOVA followed by Newman–Keuls test was used for statistical analysis to compare different groups. * indicates significantly decreased compared to the control (*p < 0.05, **p < 0.01).

100 mg/100 g, i.p., daily) for 36 days by 21.5 and 39.2% ( F 2,18 = 13.66; p = 0.0002), respectively. Homocysteine significantly decreased dopamine metabolites, DOPAC and HVA, resulting in a significant reduction of dopamine turnover in the mouse striatal regions

Fig. 4. Effect of homocysteine on the locomotor activities after 5 days of consecutive treatments (i.c.v.) in rats. One or two micromoles of homocysteine was injected into the rat brains by i.c.v. route daily for 5 days and 1 h after the last injection rats were placed in the locomotor activity monitor for the measurement of locomotor activities. Movement time, total distances and the number of movements were recorded for 40 min. Values are expressed as means  S.E.M. (N = 6). One-way ANOVA followed by Newman–Keuls test was used for statistical analysis to compare control and treated groups. (*) Indicates significantly decreased compared to the control (*p < 0.05, **p < 0.01).

(Table 2, F 2,18 = 5.257; p = 0.0159 for DOPAC; F 2,18 = 4.405; p = 0.0277 for HVA). Accordingly, the ratios of (DOPAC + HVA)/DA as an index of dopamine turnover decreased by 23.7 and 51.6%, respectively (Table 2, F 2,18 = 5.093; p = 0.0177). Systemic treat-

Table 2 Effects of homocysteine on the levels of DA, DOPAC, HVA, and DA turnover after 36 days (i.p.) of daily treatment in C57BL/6 mice HC (mg/100 g)

DA (nmol/g tissue)

DOPAC (nmol/g tissue)

HVA (nmol/g tissue)

(DOPAC + HVA)/DA

0 50 100

24.47  2.90 22.14  5.13 26.54  3.12

3.61  0.51 2.32  0.43 1.85  0.17*

3.32  0.53 2.45  0.29 1.79  0.19*

0.283  0.041 0.216  0.036 0.137  0.013*

After the administration of two doses (50 and 100 mg/100 g) of homocysteine, the levels of dopamine and its metabolites in the mouse striatal regions were measured by HPLC with electrochemical detection. One-way ANOVA followed by Newman–Keuls test was used for statistical analysis to compare different groups (N = 7). * Indicates significantly decreased compared to the control (*p < 0.05).

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Fig. 5. Homocysteine level in the striatal regions after 36 days systemic treatment of homocysteine in C57BL/6 mice. Two doses (50 and 100 mg/ 100 g wt.) of homocysteine were injected (i.p.) to mice for 36 days and one day after the last injections, homocysteine levels in the mouse striatal regions were measured by HPLC with fluorescence detector after derivatization. Values are expressed as means  S.E.M. (N = 7). One-way ANOVA followed by Newman–Keuls test was used for statistical analysis to compare different groups. (*) Indicates significantly decreased compared to the control (*p < 0.05, ***p < 0.001).

ment of homocysteine to mice showed similar results to those of rats treated by i.c.v. route in the dopamine turnover rates. The effects of homocysteine on the other regions of mouse brains such as cortex and hippocampus were also tested to determine whether the effects are specific on the striatal regions of mice. The results revealed that homocysteine did not change significantly the levels of dopamine and its metabolites in those regions (data not shown). Effects of the Systemic Treatment of Homocysteine on Locomotor Activities in Mice Daily injections (i.p.) of two doses (50 and 100 mg/ 100 g) of homocysteine for 36 days significantly decreased locomotor activities in mice (Fig. 6). The administration of 50 and 100 mg/100 g doses of homocysteine decreased MT, 29 and 38%, TD, 32 and 42%, and NM, 28 and 41%, respectively ( F 2,18 = 8.584; p = 0.0024 for MT; F 2,18 = 7.963; p = 0.0033 for TD; F 2,18 = 21.45; p < 0.0001 for NM).

Fig. 6. Effect of homocysteine on locomotor activities in C57BL/6 mice after 36 days treatment (i.p., daily). One day after the last injection of homocysteine, locomotor activities (movement time, total distance and number of movements) were measured in an Activity Monitor (Degiscan Instruments Inc., Columbus, OH, USA). The locomotor activities were determined for 40 min after mice were placed in the monitor. Values are expressed as means  S.E.M. (N = 7). One-way ANOVA followed by Newman–Keuls test was used for statistical analysis to compare different groups. (*) Indicates significantly decreased compared to the control (*p < 0.05, **p < 0.01, ***p < 0.001).

Effects of Systemic Treatment of Homocysteine on Methylation in the Mouse Brain Homocysteine is formed from the hydrolysis of SAH by SAH hydrolase (Fig. 1) and elevated in PD patients with levodopa therapy. Levodopa is metabolized by catechol O-methyltransferases (COMT) to 3OMD that is a methylated metabolite. 3-O-Methylation activity, therefore, is increased in levodopa treat-

ment, resulting in decreasing SAM and increasing SAH levels due to the utilization of a methyl donor for metabolism of levodopa (Liu et al., 2000). In the present study, it was examined that the accumulation of homocysteine could alter the methylation process. Homocysteine increased SAH ( F 2,18 = 4.484; p = 0.0298) and decreased SAM levels ( F 2,18 = 9.988;

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DISCUSSION

Fig. 7. Effect of homocysteine on the levels of SAM and SAH in C57BL/6 mice after 36 days treatment (i.p., daily). One day after the last injections, the animals were sacrificed and the striatal regions were dissected. The levels of SAM and SAH were measured by HPLC with UV detection at 254 nm. Values are expressed as means  S.E.M. (N = 7). One-way ANOVA followed by Newman–Keuls test was used for statistical analysis to compare different groups. (*) Indicates significantly decreased compared to the control (*p < 0.05, **p < 0.01).

p = 0.0017) resulting in decreasing the ratio of SAM/ SAH (Fig. 7). Homocysteine did not change significantly the levels of SAM and SAH in other regions of mouse brains such as cortex, midbrain and hippocampus (data not shown). Effects of Systemic Treatment of Homocysteine on Tyrosine Hydroxylase (TH) Immunoreactivity in Substantia Nigra of Mouse Brain Since systemic injections of homocysteine impaired the dopaminergic system in the mouse brain, TH immunoreactivities of dopamine neurons in the substantia nigra of mouse brain were examined to determine whether dopaminergic neurons are damaged by a long-term treatment of homocysteine in mice. As shown in Fig. 8A and B, TH immunoreactivity was decreased by the chronic administration of homocysteine. Two doses of homocysteine, 50 and 100 mg/ 100 g, significantly decreased TH immunoreactivity by 25 and 37%, respectively.

The findings of our study reveal that homocysteine significantly decreased locomotor activities in rats and mice. Homocysteine also caused severe reduction of dopamine turnover in the striatum and decreased TH immunoreactivity in substantia nigra of mouse brain after chronic exposure, implicating that elevated homocysteine could cause harmful impacts on the dopaminergic nervous system. These results indicate that elevated homocysteine could be associated with the complications of levodopa treatment in PD patients and might accelerate the progression of PD. Numerous studies have reported that homocysteine is elevated in levodopa therapy for PD patients and suggested the substantial role of homocysteine in causing various neurotoxic effects (Kuhn et al., 1998; Muller et al., 2001, 2002; Rogers et al., 2003). The results obtained from the present study show that homocysteine altered the dopaminergic neurochemistry. These effects on dopaminergic nervous system might be specific for homocysteine since other amino acids such as glycine and cysteine did not alter dopaminergic function. Elevated homocysteine in the striatal region of mice after i.p. treatment indicates that homocysteine is able to cross into the brain possibly via a specific membrane transporter (Grieve et al., 1992). Both short-term treatment to the rat brain directly and long-term treatment of homocysteine systemically to C57Black mice caused a significant reduction of dopamine turnover in the striatal region of the brain. The reduction of dopaminergic activity might be associated with the decrement of locomotor activities. These results are consistent with the report that feeding mice folate deficient diet for 37 days resulted in a significant reduction in dopaminergic activity in the mouse striatum (Gospe et al., 1995). Feeding up folate deficient diet elevated homocysteine level due to the inhibition of homocysteine metabolism (Gospe et al., 1995). In this study, they observed food spilling behavior, but they did not measure locomotor activities. Folic acid deficiency diet might have different effects from homocysteine treatment because folic acid deficiency could cause not only homocysteine elevation but also other metabolic abnormalities. Duan and co-workers (2002) have also reported similar but different results possibly due to different experimental designs. From their results, homocysteine infusion into the mouse striatum or substantia nigra did not show any significant change in dopaminergic neurochemistry or immunohistochemistry, but, co-treatment of homocysteine with MPTP exacerbated MPTP-induced neurotoxicity on

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Fig. 8. (A) Effect of homocysteine (HC) on tyrosine hydroxylase immunoreactivity (TH-IR) in substantia nigra of C57BL/6 mice after 36 days treatment of homocysteine. After perfusion of mice, 25 micron frozen sections of the mouse brain were prepared. The sections were stained with primary antibody (antirabbit TH serum) and secondary antibody (anti-goat rabbit serum) tagged with FITC. TH-IR was viewed with a confocal microscopy (PCM 2000, Nikon). Bar = 20 mm. (B) Homocysteine-induced nigral cell loss. Dopaminergic neurons in substantia nigra of mice were quantified using C-imagimg analysis program. Data represent mean  S.E.M. (n = 5). (*) Indicates significantly decreased compared to the control (*p < 0.05, **p < 0.01).

dopaminergic neurons (Duan et al., 2002). In this study, homocysteine (4.3 ng in 1 ml) was directly infused into striatum or substantia nigra and 24 h later the effects were examined, whereas, in our study, different doses of homocysteine (1 or 2 mmol in 5 ml) were injected into the lataral ventricles of rat brains daily for longer period (5 days). All these results clearly indicate that elevated homocysteine from any initiating factor such as folic acid deficiency, levodopa therapy, or genetic abnormality could cause neurotoxic effects and increase vulnerability of dopaminergic neurons. Moderately elevated homocysteine may not be toxic to normal dopaminergic neurons, but in damaged or impaired neurons such as dopaminergic neurons in PD, homocysteine may decrease the threshold of vulnerability of dopamine neurons and cause toxic effects. Homocysteine increases MPTP-induced dopaminergic neuronal toxicity in animals, likewise dopaminergic neurons in PD patients are already damaged (Hornykiewicz, 1982) and, therefore, elevated homocysteine from levodopa treatment could exacerbate dopaminergic neurotoxicity and accelerate dopaminergic neuronal degeneration. The reduction of TH immunoreactivities by the treatments of homocysteine for long-term exposure in mice without a significant change of dopamine levels in the striatum might indicate that dopaminergic cell bodies in the substantia

nigra are preferentially damaged. The possible explanation for these results could be that homocysteine might initially attack dopamine cell bodies in the substantia nigra, unlike MPTP toxicity mechanism in which MPTP (via MPP+) attacks dopaminergic nerve terminal first in the striatum and retrogradely damages dopaminergic neurons to cell bodies in the substantia nigra. The possible molecular mechanism for damaging the dopaminergic cell bodies is unknown, but it could be oxidative stress by which paraquat is known to attack dopaminergic cell bodies (McCormack et al., 2002). In their study, paraquat did not change striatal dopamine levels although it damaged dopaminergic cell bodies in the subtantia nigra. Enhanced dopamine synthesis was found by an increase of TH activity in the striatum, indicating the compensatory mechanisms by which damaged neurons by toxicants are capable of restoring neurotransmitter tissue levels (McCormack et al., 2002). It is of interest to know the effect of homocysteine on methylation activity since homocysteine is closely related to the methylation cycle. The results showed that homocysteine treatment decreased SAM and increased SAH, possibly due to the negative feedback effect of homocysteine resulting in the accumulation of SAH which in turn may act as an inhibitor of methylation. From the present study, it is unclear

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how methylation activity is altered because the levels of SAM and SAH cannot indicate the methylation activity although homocysteine increased the ratio of SAH/SAM, possibly by the accumulation of SAH. There are controversial reports for methylation activity in homo-cysteine-related studies. Homocysteine could be elevated in other conditions such as folic acid or Vitamin B12 deficiency resulting from the impeded synthesis of methionine from homocysteine which requires folic acid and Vitamin B12 as a cofactor (Stabler et al., 1988; Lindenbaum et al., 1988). Methionine is a precursor of SAM that serves as a methyl donor in various methylation reactions in the biological system (Baldessarini, 1987). Therefore, hypomethylation could be induced in folic acid deficient state and has been suggested to be associated with depression (Bottiglieri et al., 2000). In the case of PD patients with levodopa therapy, methylation activity would be different from folic acid deficiency condition because levodopa is metabolized to 3-OMD by COMT and 3-OMD as well as L-methionine S-adenosyltransferase (MAT) activity which is responsible for the synthesis of SAM was significantly increased (Cheng et al., 1997). Since SAM is rapidly utilized to metabolize levodopa to 3-OMD, SAM and methionine are decreased and homocysteine is increased in levodopa treatment (Muller et al., 2001). SAM may exert positive feedback on MAT catalytic activity resulting in upregulation of MAT activity (Benson et al., 1993; Zhao et al., 2001). Although the present study showed the inhibitory effect of homocysteine in the dopaminergic system, the present investigation did not provide clear explanation for the mechanism of action of homocysteine. It seems likely that multiple biochemical cascade events are involved in the molecular mechanism of toxicity. Oxidative stress (Wall et al., 1980; Starkebaum and Harlan, 1986), overstimulation of NMDA receptors (Kim and Pae, 1996; Lipton et al., 1997) and apoptosis (Kruman et al., 2000) have been reported to be responsible for the toxic mechanism of homocysteine. In conclusion, homocysteine appears to be a potent substance in disturbing dopamine turnover and causing dopaminergic neuronal damage in long-term exposure. The present study points out the fact that the elevation of homocysteine in PD patients with levodopa therapy might be responsible, at least in part, for motor dysfunctions and adverse effects of levodopa. In addition, our data suggest that long-term levodopa therapy for PD accelerate the progression of the disease and the elevated homocysteine might play a role on the progression.

ACKNOWLEDGEMENTS This work was supported by grants received from the National Institutes of Health (RO1 28432, RR 03020, and GM 08111).

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