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transporter, inhibits mitochondrial respiration, and induces parkinsonism in mice. We ... converted by COMT to 5, which has cytotoxic and parkinsonism...
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Chem. Res. Toxicol. 2000, 13, 1294-1301

Dopamine Transporter and Catechol-O-methyltransferase Activities Are Required for the Toxicity of 1-(3′,4′-Dihydroxybenzyl)-1,2,3,4-tetrahydroisoquinoline Hiroshi Kawai,†,‡ Yaichiro Kotake,§ and Shigeru Ohta*,§ Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan, and Institute of Pharmaceutical Sciences, Hiroshima University School of Medicine, Hiroshima 734-8551, Japan Received March 1, 2000

1-(3′,4′-Dihydroxybenzyl)-1,2,3,4-tetrahydroisoquinoline [3′,4′DHBnTIQ (1)] is an endogenous parkinsonism-inducing substance. It is taken up into dopaminergic neurons via the dopamine transporter, inhibits mitochondrial respiration, and induces parkinsonism in mice. We synthesized four derivatives [aromatized, N-methylated, N-methyl-aromatized, and O-methylated (2-5, respectively)] and studied the cellular uptake and cytotoxicity of 1-5, as well as the metabolism of 1. All except the O-methyl derivative (5) were specifically taken up by the dopamine transporter, but 1 was taken up most efficiently. Relative to 1, oxidation reduced vmax, N-methylation markedly increased Km, and O-methylation eliminated the uptake activity. The cytotoxicity of 1-5 was examined in a mesencephalic cell primary culture. Compound 1 reduced cell viability by nearly 80% at 100 µM, but the other compounds had little or no effect on cell viability. In vivo and in vitro studies revealed that 1 was O-methylated by soluble catechol-O-methyltransferase (COMT). Aromatization and N-methylation of 1 were not observed. We found that dopamine transporter inhibitors and a COMT inhibitor each blocked the cytotoxicity of 1, indicating that uptake and O-methylation are both necessary for neurotoxicity. Thus, we consider that 1 is taken up into dopaminergic neurons via the dopamine transporter and then converted by COMT to 5, which has cytotoxic and parkinsonism-inducing activities.

Introduction 1,2,3,4-Tetrahydroisoquinoline (TIQ)1 derivatives exist widely in nature, including in mammals (1). Since the discovery in 1983 that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a byproduct of meperidine analogue synthesis, could induce parkinsonism in humans (2), TIQ derivatives became of interest as candidate endogenous causative factors of idiopathic Parkinson’s disease because they structurally resemble MPTP. Several kinds of TIQ derivatives exist in the mammalian brain (3-8), and TIQ (9), 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1Bn* To whom correspondence should be addressed: Institute of Pharmaceutical Sciences, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan. Telephone: +81-82-257-5325. Fax: +81-82-257-5329. E-mail: ohta@ pharm.hiroshima-u.ac.jp. † University of Tokyo. ‡ Present address: Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. § Hiroshima University. 1 Abbreviations: Ara-C, cytosine β-D-arabinofuranoside; 1BnTIQ, 1-benzyl-1,2,3,4-tetrahydroisoquinoline; COMT, catechol-O-methyltransferase; 3′,4′DHBnTIQ, 1-(3′,4′-dihydroxybenzyl)-1,2,3,4-tetrahydroisoquinoline; DIV, days in vitro; DMEM, Dulbecco’s modified Eagle’s medium; ECD, electrochemical detector; GBR12909, 1-{2-[bis(4-fluorophenyl)methoxy]ethyl}-4-(3-phenylpropyl)piperazine; MPP+, 1-methyl-4-phenylpyridinium; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; Ro41-0960, 2′-fluoro-3,4dihydroxy-5-nitrobenzophenone; SAM, S-adenosyl-L-methionine; TIQ, 1,2,3,4-tetrahydroisoquinoline.

TIQ) (5), 1-(3′,4′-dihydroxybenzyl)-1,2,3,4-tetrahydroisoquinoline [3′,4′DHBnTIQ (1)] (8), and salsolinols (10) are all candidates for endogenous parkinsonogenic compounds. Parkinson’s disease is a neurodegenerative disease characterized by degeneration of nigro-striatal dopaminergic neurons and reduction in the level of dopamine in this area. Its pathogenesis is not clear yet. The studies on MPTP provided clues to the mechanism of cell death in Parkinson’s disease. MPTP is converted to 1-methyl4-phenylpyridinium cation (MPP+) by monoamine oxidase and by autoxidation in astroglial cells in the brain (11, 12). MPP+ is specifically taken up into dopaminergic neurons by the dopamine transporter (13) and then inhibits mitochondrial respiration (14, 15), which brings about an energy crisis followed by cell death. Selective cell death of dopaminergic neurons is thus due to the selective uptake into dopaminergic neurons via the dopamine transporter. Since MPTP is not taken up by the dopamine transporter (13) and does not inhibit mitochondrial respiration as efficiently as MPP+, conversion to MPP+ is also critical. Therefore, metabolism, especially oxidation, dopamine transporter selectivity, and suppression of mitochondrial respiration may be important factors for idiopathic Parkinson’s disease. Previously, we have reported that a TIQ derivative, 1, is one of the endogenous parkinsonism-inducing substances. This compound is present in mouse brain, and

10.1021/tx000047y CCC: $19.00 © 2000 American Chemical Society Published on Web 11/22/2000

Effect of Metabolism on Neurotoxicity of TIQs

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Scheme 1. Putative Metabolic Pathway of 3′,4′DHBnTIQ (1)

can induce parkinsonism in mice in vivo when administered chronically (8). It is taken up into dopaminergic neurons by the dopamine transporter (8) and inhibits mitochondrial respiration (16).In this study, we examined the importance of uptake and metabolism of 1 for its neurotoxicity. Four derivatives of 1 were synthesized, and their uptake and cytotoxic activities were measured. Through a structure-activity relationship study, we examined the contributions of uptake and metabolism to the neurotoxicity. The mechanism of 1-induced neurotoxicity is discussed on the basis of the results.

Experimental Procedures Materials. Animals were purchased from Nippon Biosupply Center. The animals were handled in accordance with the Guide for Animal Experimentation, Graduate School of Pharmaceutical Sciences, University of Tokyo. EDTA2Na2H2O was purchased from Dojin (Kumamoto, Japan). Hank’s balanced salt solution powder, Dulbecco’s modified Eagle’s medium (DMEM/ F12) salt solution powder, fetal bovine serum, horse serum, and amphotericin B were from Gibco BRL Life Technologies, Inc. (Rockville, MD). Deoxyribonuclease I, cytosine β-D-arabinofuranoside (Ara-C), and (3,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from Sigma (St. Louis, MO). Papain was from Worthing Biochemical Co. (Lakewood, NJ). Penicillin G and streptomycin were from Meiji Seika Co. Ltd. (Tokyo, Japan). Other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan). Chemical Syntheses. 1-(3′,4′-Dimethoxybenzyl)-3,4-dihydroisoquinoline (8), 1-(3′,4′-dimethoxybenzyl)-1,2,3,4-tetrahydroisoquinoline (8), 1-(3′,4′-dihydroxybenzyl)-1,2,3,4-tetrahydroisoquinoline (1) (8), and 1-(3′,4′-dimethoxybenzyl)isoquinoline (17) were synthesized by the cited methods. 1-(4′-Hydroxy-3′-methoxybenzyl)-1,2,3,4-tetrahydroisoquinoline (5). 1-(3′,4′-Dimethoxybenzyl)-1,2,3,4-tetrahydroisoquinoline hydrochloride (2.0 g) was refluxed in 47% hydrobromic acid for 30 min. Recrystallization from ethanol diethyl ether of the precipitate obtained after cooling afforded 1-(4′-hydroxy-3′methoxybenzyl)-1,2,3,4-tetrahydroisoquinoline hydrobromide (5‚ HBr, 1.52 g, mp 240-242 °C). The position of the methoxyl function was confirmed by the NOE signal observed between OCH3 and H2′. 1H NMR (400 MHz, DMSO-d6): δ 3.01-3.47 (m, 6H, CH2, H3 and H4), 3.74 (s, 3H, OCH3), 4.73-4.77 (m, 1H, H1), 6.72 (dd, 1H, H6′, J ) 8.4, 1.6 Hz), 6.75 (d, 1H, H5′, J ) 8.4 Hz), 6.92 (d, 1H, H2′, J ) 1.6 Hz), 7.19-7.30 (m, 4H, aromatic), 8.88 (s, 1H, OH). Anal. Calcd for C17H19NO2‚HBr: C, 58.36; H, 5.76; N, 4.00. Found: C, 58.34; H, 5.83; N, 4.18.

1-(3′,4′-Dimethoxybenzyl)-2-methyl-1,2,3,4-tetrahydroisoquinoline. 1-(3′,4′-Dimethoxybenzyl)-3,4-dihydroisoquinoline (2.86 g) was dissolved in methyl iodide, and the solution was left to stand at room temperature for 6 h. Precipitated yellow crystals were collected and dissolved in ethanol. The solution was stirred for 3 h with sodium borohydride (0.77 g). The reaction mixture was concentrated by evaporation and extracted with diethyl ether. The organic layer was extracted with 0.1 M HCl and then basified with KOH. This solution was extracted with diethyl ether. The organic phase was dried over MgSO4, filtered, and evaporated to dryness to give 1-(3′,4′-dimethoxybenzyl)-2-methyl-1,2,3,4-tetrahydroisoquinoline as an oil (1.7 g). The resultant oil was treated with HCl in dried diethyl ether. The obtained solid was washed with ethanol to afford a colorless powder, which was used for the following reaction without further purification. 1-(3′,4′-Dihydroxybenzyl)-2-methyl-1,2,3,4-tetrahydroisoquinoline (3). 1-(3′,4′-Dimethoxybenzyl)-2-methyl-1,2,3,4-tetrahydroisoquinoline hydrochloride (1.02 g) was refluxed in 47% hydrobromic acid for 2 h. Recrystallization from methanol diethyl ether of the precipitate obtained after cooling afforded 1-(3′,4′-dihydroxybenzyl)-2-methyl-1,2,3,4-tetrahydroisoquinoline hydrobromide (3‚HBr, 0.1 g, mp 233-235 °C). 1H NMR (400 MHz, DMSO-d6): δ 3.15-3.34 and 4.11-4.15 (m, 3H and 2H, H1, H3 and H4), 3.79 (s, 3H, CH3), 4.52 (s, 2H, CH2), 6.48 (dd, 1H, H6′, J ) 8.4, 2.0 Hz), 6.63 (d, 1H, H2′, J ) 2.0 Hz), 6.68 (d, 1H, H5′, J ) 8.4 Hz), 7.49-7.52 (m, 2H, aromatic H), 7.727.76 (m, 1H, aromatic), 8.08 (d, 1H, aromatic, J ) 7.6 Hz). Anal. Calcd for C17H19NO2‚HBr: C, 58.36; H, 5.76; N, 4.00. Found: C, 58.53; H, 5.47; N, 4.16. 1-(3′,4′-Dihydroxybenzyl)isoquinoline (2). 1-(3′,4′-Dimethoxybenzyl)isoquinoline was treated with HCl in dried diethyl ether, and the obtained solid was recrystallized from ethanol diethyl ether (mp 139-141 °C). This product (205 mg) was refluxed in 47% hydrobromic acid for 2 h. Recrystallization from ethanol diethyl ether of the precipitate obtained after cooling afforded 1-(3′,4′-dihydroxybenzyl)isoquinoline hydrobromide (2‚HBr, 172 mg, mp 168-170 °C). 1H NMR (400 MHz, DMSO-d6): δ 4.72 (s, 2H, CH2), 6.59 (dd, 1H, H6′, J ) 8.1, 1.9 Hz), 6.66 (d, 1H, H5′, J ) 8.1 Hz), 6.67 (d, 1H, H2′, J ) 1.9 Hz), 7.96 (m, 1H, H6 or H7), 8.10 (m, 1H, H6 or H7), 8.27 (d, 1H, H5 or H8, J ) 8.4 Hz), 8.31 (d, 1H, H4, J ) 6.4 Hz), 8.59 (d, 1H, H3, J ) 6.4 Hz), 8.63 (d, 1H, H5 or H8, J ) 8.3 Hz). Anal. Calcd for C16H13NO2‚HBr: C, 57.87; H, 4.22; N, 4.22. Found: C, 58.02; H, 4.06; N, 4.10. 1-(3′,4′-Dimethoxybenzyl)-2-methylisoquinolinium. Methyl iodide was added to 1-(3′,4′-dimethoxybenzyl)isoquinoline (458 mg), and the mixture was refluxed for 4 h. Precipitated yellow crystals were collected and washed with diethyl ether, affording

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Figure 1. Results of HPLC/ECD. (A) Authentic compounds (10 pmol) were applied. Peaks are identified as follows: (a) 3 and 4, (b) 2, (c) 1, and (d) 5. (B) An example of an in vitro assay of 3′,4′DHBnTIQ (1) metabolism. The arrow indicates 5. The large peak after 10 min is the substrate (1). 1-(3′,4′-dimethoxybenzyl)-2-methylisoquinolinium iodide (503 mg). 1-(3′,4′-Dihydroxybenzyl)-2-methylisoquinolinium (4). 1-(3′,4′-Dimethoxybenzyl)-2-methylisoquinolinium iodide (312 mg) was refluxed in 47% hydrobromic acid for 2 h. Ethanol was added, and a precipitate was obtained after cooling. Recrystallization from methanol diethyl ether of the precipitate afforded 1-(3′,4′-dihydroxybenzyl)-2-methylisoquinolinium bromide (4‚Br-, 150 mg, mp 231-233 °C). 1H NMR (400 MHz, DMSO-d6): δ 4.38 (s, 3H, CH3), 4.96 (s, 2H, CH2), 6.35 (dd, 1H, H6′, J ) 8.0, 2.0 Hz), 6.46 (d, 1H, H2′, J ) 2.0 Hz), 6.67 (d, 1H, H5′, J ) 8.0 Hz), 8.06 (m, 1H, H6 or H7), 8.24 (m, 1H, H6 or H7), 8.35 (d, 1H, H5 or H8, J ) 8.4 Hz), 8.49 (d, 1H, H4, J ) 6.8 Hz), 8.72 (d, 1H, H3, J ) 6.8 Hz), 8.78 (d, 1H, H5 or H8, J ) 8.4 Hz), 8.88 (s, 1H, OH), 8.93 (s, 1H, OH). Anal. Calcd for C17H16NO2‚Br: C, 58.98; H, 4.66; N, 4.05. Found: C, 58.75; H, 4.38; N, 4.19. HPLC/ECD. The Eicom EP-300/Eicom ECD-100 system was used under the following conditions: column, Eicompak MA5ODS (4.6 or 2.1 mm i.d. × 150 mm) with precolumn; mobile phase, 0.1 M sodium acetate and 0.1 M citric acid buffer (70%) (pH 3.5) and methanol (30%), containing 5 mg/L EDTA; flow rate, 1 or 0.23 mL/min (for column of 4.6 or 2.1 mm i.d., respectively); electrode, Eicom WE-3G graphite; reference electrode, Eicom RE-100 Ag-AgCl; applied voltage, 700 mV versus the Ag/AgCl electrode. Figure 1A shows a chromatogram of authentic 1-5 (10 pmol of each compound was injected). The two 2-methylated derivatives (3 and 4) were not separated from each other, but this did not disturb our experiments. Procedure for the Uptake Assay. Male adult SpragueDawley rats (250-350 g) were treated intraperitoneally with 5 mg/kg reserpine at 20 h and 2.5 mg/kg reserpine at 3 h before being killed, to deplete the level of striatal dopamine (18). They were killed by decapitation, and the striata were dissected out and homogenized in 10 volumes (w/v) of ice-cold 0.32 M sucrose with a Teflon-glass homogenizer. The nuclear fraction was removed by centrifugation at 900g for 10 min, and the supernatant was again centrifuged at 11500g for 20 min. The pellet from the second centrifugation was resuspended in 0.32 M sucrose with a Teflon-glass homogenizer, and this suspension was used for the uptake assay as reserpinized rat striatal synaptosomes. A portion of this suspension was used for protein determination by a modification of the method of Lowry et al. (19).

Kawai et al. The synaptosomal suspension (400-800 µg of protein) in modified Krebs-Ringer medium was preincubated at 37 °C for 5 min. The modified Krebs-Ringer medium was prepared to give final concentrations of 108 mM NaCl, 1.1 mM NaH2PO4, 27 mM NaHCO3, 5.4 mM glucose, 0.1 mM ascorbic acid, 1.0 mM KCl, 1.0 mM MgCl2, and 1.0 mM CaCl2 and was previously saturated with a 95% O2/5% CO2 mixture. The reaction was started by the addition of a test compound at various concentrations (final volume of 1 mL) and was terminated after incubation at 37 °C for an appropriate time by adding 4 mL of ice-cold medium containing 10-5 M nomifensine, followed by centrifugation at 11500g for 10 min. The pellet was washed with ice-cold medium and sonicated in 0.1 M perchloric acid containing 40 µM sodium bisulfite and 10 µM EDTA. After centrifugation at 11500g for 10 min, the supernatant was adjusted to pH ∼3 and then filtered through a 0.45 µm filter, and a portion of the filtrate was subjected to HPLC/ECD analysis. The nonspecific level of uptake determined at 0 °C was subtracted. Cell Culture. The ventral mesencephalon was dissected out from a Wistar/ST rat embryo (E16) in Hanks’ medium supplemented with 0.4% glucose and 0.035% NaHCO3. The dissected areas contain the dopaminergic A8, A9 (substantia nigra), and A10 (ventral tegmental area) neurons. The dissected mesencephalic blocks were dissociated with papain; 2 µL of papain was suspended in 10 mL of phosphate-buffered saline (PBS, pH 7.4) containing 2 mg of DL-cysteine, 2 mg of bovine albumin, 50 mg of glucose, and 100 µL of 2% deoxyribonuclease I, and tissues were added. This solution was incubated at 37 °C for 30 min with vigorous shaking. After incubation, 5 mL of the supernatant was discarded, and 5 mL of culture medium was added. Cells were mechanically dissociated by pipetting. After filtration, the cells were collected by centrifugation at 1000 rpm for 5 min and resuspended in 10 mL of culture medium. After pipetting, cells were counted, collected by centrifugation at 1000 rpm for 5 min, and resuspended in culture medium. The cells suspended in culture medium were plated on poly-L-lysine-coated plastic culture plates or dishes. Cultures were incubated at 37 °C in a 5% CO2/95% air mixture with 100% relative humidity. The culture medium consisted of DMEM/F-12 supplemented with 0.46% glucose, 0.24% NaHCO3, 5% fetal bovine serum, 5% horse serum, 32 mg/L penicillin G, 80 mg/L streptomycin, and 2.5 mg/L amphotericin B. Ara-C (1 µM final concentration) was added at 4-7 days in vitro (DIV) to obtain a pure neuronal culture, and assays were performed at 7-12 DIV, 4 days after the Ara-C treatment. Evaluation of in Vitro Cytotoxicity. In vitro cytotoxicity of 3′,4′DHBnTIQ derivatives (1-5) toward mesencephalic neurons was evaluated by the MTT dye conversion assay (20). The cells were plated on 96-well culture plates at a density of 1 × 105 cells per 100 µL per well (∼3 × 105 cells/cm2). Test compounds dissolved in DMEM/F-12 were added to the cultures, and cultivation was continued at 37 °C for 24 h. To investigate the possible mechanism of 3′,4′DHBnTIQ toxicity, dopamine transporter inhibitors and a COMT inhibitor were added during a 30 min preincubation, followed by addition of 1 for 24 h. MTT dissolved in PBS (pH 7.4) (0.4 mM final concentration) was added to cultures and left at 37 °C for 4 h. Cells were washed with PBS and lysed with lysis buffer, which consisted of a 20% sodium dodecyl sulfate/dimethylformamide (1:1) solution at pH 4.7. Cell lysates were held at 37 °C overnight to allow cell lysis and dye solubilization. The absorbance at 570 nm was measured with a microplate reader. Cell viability was evaluated in terms of A570 and represented as a percentage of the untreated control. Procedure for the Assay of the in Vivo Metabolism of 3′,4′DHBnTIQ. SD rats (6-8 weeks old) were treated intraperitoneally with 250 mg/kg 1. One hour after injection, they were killed by decapitation and the brains were removed. The brain was divided into seven parts (cerebral cortex, midbrain, thalamus/ hypothalamus, striatum, hippocampus, cerebellum, and oblongata) if required. Tissue was homogenized in 2 M perchloric acid containing 10 mM EDTA and 40 mM sodium bisulfite. After 30

Effect of Metabolism on Neurotoxicity of TIQs min on ice, the homogenate was centrifuged at 11500g for 10 min at 4 °C. The supernatant was adjusted to pH ∼3 with 1 M sodium acetate and then filtered through a 0.45 µm filter, and a portion of the filtrate was subjected to HPLC/ECD analysis. Procedure for the Assay of the in Vitro Metabolism of 3′,4′DHBnTIQ. Brains from male SD rats killed by decapitation were homogenized in 10 mM sodium phosphate buffer (pH 7.4) with a Teflon-glass homogenizer. Portions of the obtained homogenates were used for the assay. Homogenates were centrifuged at 700g for 10 min, and the supernatants were centrifuged at 11500g for 20 min. The supernatants were centrifuged at 100000g for 60 min. The pellets from the first, second, and third centrifugations were used as nuclear (P1), mitochondrial-synaptosomal (P2), and microsomal (P3) fractions, respectively, and the supernatant of the third centrifugation was used as the cytosolic (S3) fraction. The protein concentration of homogenates and fractions was determined by a modification of the method of Lowry et al. (19). The homogenate or a subcellular fraction (∼1 mg of protein) in 0.4 mL of 10 mM sodium phosphate buffer (pH 7.4) was preincubated with 1 at 37 °C for 5 min in the presence or absence of a COMT inhibitor. The reaction was started by the addition of 0.1 mL of S-adenosyl-L-methionine (SAM) and was terminated after incubation at 37 °C by adding 1 M perchloric acid and cooling on ice. After centrifugation at 14 000 rpm for 10 min, the supernatant was adjusted to pH ∼3 with 1 M sodium acetate and then filtered through a 0.45 µm filter, and a portion of the filtrate was subjected to HPLC/ECD analysis. The level of nonspecific metabolism determined at 0 °C was subtracted. Evaluation of O-Methylation of 3′,4′DHBnTIQ in Cultured Neurons. The cells were plated on 60 mm culture dishes at a density of 4 × 105 cells/dish (∼1.4 × 105 cells/cm2). 1 (10 µM) dissolved in DMEM/F-12 was added to the cultures, and cultivation was continued at 37 °C for 6 h. The COMT inhibitor (10 µM Ro41-0960) was added 30 min before addition of 1, when required. The cells were washed with PBS and suspended in 0.2 M perchloric acid. After centrifugation at 14 000 rpm for 10 min, the supernatant was adjusted to pH ∼3 with 1 M sodium acetate and then filtered through a 0.45 µm filter, and a portion of the filtrate was subjected to HPLC/ECD analysis.

Results Specific Uptake of 3′,4′DHBnTIQ Derivatives. To test the uptake activity for 3′,4′DHBnTIQ derivatives, we used reserpinized rat striatal synaptosomes. These synaptosomes were incubated with 1 µM 1-5 for 5 min at 37 °C, and the amount of specific uptake was measured by the method described in Experimental Procedures. Figure 2 shows the specific uptake of each compound. Compounds 1-4 were specifically taken up, but 5 was not. The order of uptake activity was as follows: 1 > 3 > 2 > 4. Thus, both N-methylation and oxidation of 1 diminished the uptake activity, and O-methylation abolished the activity. As shown in Figure 3A, specific uptakes of 1 and 2 were saturatable, and kinetic parameters were calculated by fitting the data to the Michaelis-Menten equation. The Km values for 1 and 2 were 6.14 and 8.79 µM and the vmax values 214.3 and 136.8 pmol min-1 (mg of protein)-1, respectively. Compound 1 is a better substrate of the dopamine transporter than 2. Figure 3B shows the specific uptakes of the 2-methyl derivatives, 3 and 4. Their uptakes were not saturated even at substrate concentrations of e300 µM. The uptake amounts at 300 µM substrate were about 513 and 56 pmol min-1 (mg of protein)-1, respectively. Although kinetic parameters could not be determined exactly, the Km values should be >100 µM and the vmax values should

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Figure 2. Specific uptake of 3′,4′DHBnTIQ derivatives 1-5 into rat striatal synaptosomes. Synaptosomes were incubated with 1 µM test compound at 37 °C for 5 min. Uptake amounts were determined as described in Experimental Procedures. Each bar represents the mean ( SE of three to six experiments. p < 0.05 (one asterisk) and p < 0.01 (two asterisks) by Scheffe´’s F test.

be >500 and 50 pmol min-1 (mg of protein)-1, respectively. These compounds exhibited higher Km values than the N-demethylated derivatives. Toxicity of 3′,4′DHBnTIQ Derivatives toward Mesencephalic Neurons. We measured the toxicity of 1-5 to cultured neurons to examine the relationship between uptake activity and neurotoxicity of these compounds. Primary-cultured mesencephalic neurons were exposed to test compounds for 24 h, and the relative cell viability was determined by the MTT assay as described in Experimental Procedures. Compound 1 reduced the number of viable cells in a concentration-dependent manner, and nearly 80% of the cells were killed by 100 µM (Figure 4). Among the other derivatives, 2 was most toxic and killed about 80% of the cells at 500 µM, but 3-5 exhibited no toxicity at 500 µM (Figure 4). The parkinsonism-inducing substance, 1, was more toxic to mesencephalic neurons than its derivatives. If 1 acts in the same way as MPTP (MPP+), uptake of the compound should be essential for cytotoxicity. We examined the effect of dopamine transporter inhibitors (GBR12909, GBR12935, and nomifensine) on the cytotoxicity of 1. As shown in Figure 5, these inhibitors prevented the 1-induced reduction of cell viability. GBR12909 and GBR12935, however, enhanced the toxicity at high concentrations. This is presumably because these compounds themselves are toxic to mesencephalic cells at 10-100 µM (data not shown). In conclusion, dopamine transporter inhibitors can prevent the reduction of cell viability, suggesting that active transport of 1 into the cells is important for cytotoxicity of 1. Metabolism of 3′,4′DHBnTIQ in Vivo. Next, we examined the relationship between the metabolism in the brain and neurotoxicity of 1. First, we examined whether 1 was metabolized to 2-5. Rats were injected intraperitoneally with 250 mg/kg 1, a dose at which it can induce parkinsonism (8). They were killed 1 h after injection,

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Figure 4. Cytotoxicity of 3′,4′DHBnTIQ derivatives 1-5 to mesencephalic neurons. Cultured neurons were exposed to the test compound for 24 h at 37 °C. Cell viability was determined by the MTT assay as described in Experimental Procedures: (9) 1, (O) 2, (4) 3, (3) 4, and (0) 5. Each point represents the mean ( SE of 4-14 experiments.

Figure 3. Concentration-activity relationship of specific uptake of 3′,4′DHBnTIQ derivatives 1-4. Synaptosomes were incubated with 0.5-300 µM test compound at 37 °C for 1 min. Uptake amounts were determined as described in Experimental Procedures. Curves were fitted to the Michaelis-Menten equation. Each point represents the mean value of three to six experiments: (A) 1 (b, data from ref 7) and 2 (0) and (B) 3 (2) and 4 (3).

and the contents of 1 and its derivatives in the brain were measured by HPLC/ECD. As shown in Table 1, 1 and 5 were detected, but the other three compounds were not. The results suggest that 1 is O-methylated to 5 in vivo. O-Methylation of 3′,4′DHBnTIQ by COMT in Vitro. We studied 3′,4′DHBnTIQ O-methylation in vitro by using rat brain homogenates. Rat whole brain homogenates were incubated with 1 mM 1 in the presence of 0.1 mM SAM. After incubation for 1 h, only 5 was detected as a metabolite of 1 (Figure 1B and Table 1). The other three compounds were not detected even at a substrate (1) concentration of 10 mM. We divided the brain into seven regions and assessed the metabolism of 1 in each region. The O-methyl derivative 5 was detected, but the other derivatives were not detected in any region (data not shown). To characterize the activity of 3′,4′DHBnTIQ O-methylation, we studied the subcellular localization and the effect of a COMT inhibitor. The activity predominantly existed in the S3 fraction, and O-methylation was almost completely inhibited by the COMT inhibitor Ro41-0960 (Figure 6). Neither 2, 3, nor 4 was detected, whether formation of 5 was inhibited. It is concluded that 1 is

Figure 5. Effect of dopamine transporter inhibitors on 3′,4′DHBnTIQ-induced cytotoxicity. Cultured neurons were preincubated in the presence or absence of each inhibitor for 30 min and exposed to 100 µM 1 for 24 h at 37 °C. Cell viability was determined by the MTT assay as described in Experimental Procedures. Each bar represents the mean ( SE of three experiments. p < 0.05 (one asterisk) and p < 0.01 (two asterisks) vs nontreated control (white column) by Scheffe´’s F test.

O-methylated by soluble COMT in the brain. 3′,4′DHBnTIQ O-methylation by COMT was linear at least during 0-90 min and at 0.5-1.2 mg of protein (Figure 7). To determine the kinetic parameters of 3′,4′DHBnTIQ O-methylation, we assessed the formation of 5 at various concentrations of 1 for 1 h (Figure 8) and obtained a Km value of 1.37 mM and a vmax value of 3.86 nmol h-1 (mg of protein)-1 for 1. O-Methylation of 3′,4′DHBnTIQ in Mesencephalic Culture. We also examined if 3′,4′DHBnTIQ O-methylation occurred in mesencephalic neurons. Cultured neurons were exposed to 10 µM 1 for 6 h, and the amounts of 1 and 5 were measured. Both 1 and 5 were detected (Table 1). In the presence of the COMT inhibitor, however, the amount of 1 was increased and that of 5 was greatly reduced (Table 1). This indicates that 1 is O-methylated by COMT in cultured cells. Effect of COMT on 3′,4′DHBnTIQ-Induced Neurotoxicity. To evaluate the importance of COMT for 1-induced cytotoxicity to cultured neurons, we used an inhibitor (Ro41-0960) of COMT. In general, O-methylation of catechols inactivates the compounds, as in the

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Table 1. Metabolism of 3′,4′DHBnTIQ (1) compd

in vivoa content (nmol/mg of brain)

in vitrob specific formation [nmol h-1 (mg of protein)-1]

in culturec content (pmol/4 × 106 cells)

compd with the COMT inhibitor

1 2 3 4 5

0.3583 ndd nd n 0.0991

-e n nd nd 0.59 ( 0.10

196.11 ( 48.05 nd nd nd 12.24 ( 1.23

361.61 ( 26.96 nd nd nd nd

a Each value represents the mean of two experiments. b Whole brain homogenates were used. Homogenates were incubated with 1 mM 1 and 0.1 mM SAM at 37 °C for 1 h. Each value represents the mean ( SE of three experiments. c Each value represents the mean ( SE of three experiments. d nd, not detected. e -, not evaluated.

Figure 6. Subcellular localization of 3′,4′DHBnTIQ O-methylation activity. P1-P3 and S3 fractions of rat brain homogenates were incubated as described in Table 1. Each bar represents the mean ( SE of three to six experiments. p < 0.01 (two asterisks) by Scheffe´’s F test.

Figure 7. Time course (A) and protein dependency (B) of 3′,4′DHBnTIQ O-methylation activity by the cytosolic fraction. Assays were performed as described in Table 1 for various incubation times (A) or at various protein homogenate concentrations (B). Each point represents the result of one experiment.

cases of dopamine and epinephrine. Thus, we expected that COMT would also inactivate 1 and that a COMT inhibitor would enhance the cytotoxicity of 1. But, as shown in Figure 9, Ro41-0960 decreased 1-induced cytotoxicity in a concentration-dependent manner. Ro410960 itself had no effect on cell viability at 1-100 µM (data not shown). Thus, the COMT inhibitor did not enhance but prevented the 1-induced cytotoxicity.

Discussion In this study, we examined the structure-activity relationship of the endogenous parkinsonism-inducing substance, 3′,4′DHBnTIQ (1), and some related compounds to elucidate the mechanism of the neurotoxicity of this compound. First, we measured the specific uptake of 1-5 into rat striatal synaptosomes. Figure 3 revealed that oxidation (1 to 2 and 3 to 4) decreased vmax, N-methylation (1 to 3

Figure 8. Kinetics of 3′,4′DHBnTIQ O-methylation activity by the cytosolic fraction. Assays were performed as described in Table 1 at various 1 concentrations. Curve fitting to the Michaelis-Menten equation gave Km and vmax values. Each point represents the mean value of two to six experiments.

Figure 9. Effect of a COMT inhibitor on 3′,4′DHBnTIQinduced cytotoxicity. Cultured neurons were preincubated in the presence or absence of Ro41-0960 for 30 min and exposed to 100 µM 1 for 24 h at 37 °C. Cell viability was determined by the MTT assay as described in Experimental Procedures. Each bar represents the mean ( SE of three experiments. p < 0.05 (one asterisk), p < 0.01 (two asterisks) vs nontreated control (white column), and p < 0.05 (dagger) vs 1 treated group (black column) by Scheffe´’s F test.

and 2 to 4) greatly increased Km, and O-methylation (1 to 5) abolished specific uptake. All the modifications reduced the uptake efficiency. The catechol structure, especially 3′-OH, is essential for interaction with the dopamine transporter, and an appropriate charge on the N atom is required for maximum transport. Our observations are consistent with previous reports on dopamine derivatives or TIQ derivatives (21, 22). The cytotoxicity of the 3′,4′DHBnTIQ derivatives was correlated with the uptake activity. As shown in Figure 4, 1 and 2 are cytotoxic to cultured neurons, although 3-5 are not. The strength of the toxicity of 1 is greater

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than that of 2. A comparison of Figures 3 and 4 suggests that the neurotoxicity of 3′,4′DHBnTIQ derivatives depends on the efficiency of their uptake. Compound 1 is accumulated in the cells efficiently by the dopamine transporter, and induces cell death. On the other hand, derivatives 3-5 cannot induce cell death because they do not accumulate well in the cells. This conclusion is supported by the finding that inhibitors of the dopamine transporter can prevent 1-induced cytotoxicity (Figure 5). Thus, uptake by the dopamine transporter is essential for the neurotoxicity of 1. Nomifensine can prevent 1-induced cytotoxicity at 1-100 µM, but it can rescue at most 60-70% of the total cells. This suggests that there are nomifensine-insensitive alternative pathways for 1-induced cytotoxicity. Catechol compounds can produce quinones and/or reactive oxygen species, which cause oxidative stress and cytotoxicity (23-25), and there is much evidence that oxidative stress causes neuronal cell death and neurodegenerative diseases (26-28). Since 1 is catecholamine, it is considered that quinones are produced and oxidative stress contributes to the 1-induced cytotoxicity. Dopamine is taken up by the dopamine transporter with a Km value of 183 nM and a vmax value of 306 pmol (mg of protein)-1 min-1 (18). MPP+ has kinetics similar to those of dopamine (13). The Km value of 1 is higher than that of dopamine or MPP+. This means that 1 is less readily taken up by dopaminergic neurons, and this may be one of the reasons why its toxicity is weaker than that of MPP+ (8, 9). MPTP, which is a strong parkinsonism-inducing substance, is converted to 1-methyl-4-phenylpyridinium (MPP+) in astroglial cells (11, 12). MPP+ is taken up into dopaminergic neurons by the dopamine transporter and then inhibits mitochondrial respiration, which leads to cell death and parkinsonism (13-15). MPTP itself is not a neurotoxin until it is converted to MPP+, so metabolism (oxidation) is critical for its neurotoxicity. Other parkinsonism-inducing neurotoxins such as 1,2,3,4-tetrahydroβ-carbolines and salsolinols are also activated by metabolism. They are oxidized and N-methylated in the brain, and the metabolites show higher uptake activity and exert higher toxicity (22, 29-31). Oxidation and N-methylation seem to be general activating pathways of parkinsonism-inducing substances. However, in the case of 1, neither N-methylation nor oxidation was observed in our experiments (Table 1). Further, Nmethylated and/or oxidized derivatives (2-4) had weak or no toxicity (Figure 4). O-Methylation of 1 by COMT occurs in preference to oxidation and N-methylation in the brain (Table 1). Our results indicate that this metabolism by COMT is crucial to the neurotoxicity of 1, because a COMT inhibitor prevented the 1-induced cytotoxicity (Figure 9). Further, the presence of the COMT inhibitor reduced the intracellular content of 5, although that of 1 was increased (Table 1). It appears that the COMT-methylated product, 5, is the toxic compound, and the neurotoxicity depends on the intracellular content of 5. However, 5 is toxic only when it is formed in the neurons, and extracellular 5 is not toxic, as shown in Figure 4, because it is not accumulated in the neurons via the dopamine transporter (Figure 2). On the other hand, extracellular 1 is toxic, because it is accumulated in the neurons via the dopamine transporter (Figure 2) and then is converted to the toxic compound 5 in the

Kawai et al.

neurons (Table 1). The neuroprotective effect of the COMT inhibitor correlates well with the reduction of the intracellular concentration of 5 (Table 1). COMT exists both in neurons and in glia (32, 33), and we consider that O-methylation of 1 results in activation in neurons, but detoxification in glia, since O-methylated 1 formed in glia cannot be taken up by neurons. In our experiments, glial cells were removed by addition of 1 µM Ara-C, leaving a nearly pure neuronal culture (data not shown; 34), so COMT acted only as an activator of 1 in neurons, and the COMT inhibitor was neuroprotective. Morikawa et al. investigated the inhibitory effect of catecholamines and their O-methylated derivatives on mitochondrial respiration (35). They reported that catecholamines such as dopamine and tetrahydropapaveroline could inhibit complex I activity, but O-methyl derivatives had more potent activity. O-Methylation of catecholamines increased the activity to inhibit mitochondrial respiration. Previously, we reported that 1 can inhibit mitochondrial respiration (complex I activity) (16). Although it has not yet been established whether 5 can inhibit mitochondrial respiration, it may well be a more potent inhibitor of complex I than 1. In conclusion, we have shown that oxidation and Nor O-methylation of 1 reduce the level of cellular uptake and that 1 is O-methylated by COMT in neuronal cells, and this may be an activating pathway of 1. 3′,4′DHBnTIQ (1) itself, without structural modification, is taken up into dopaminergic neurons by the dopamine transporter, and then is O-methylated by COMT. If 1 is O-methylated outside the neurons (in glia), this represents detoxification. Both uptake into neurons via the dopamine transporter and metabolism by COMT are necessary for 1 to exert neurotoxicity. This is not likely to be the only pathway leading to Parkinson’s disease; however, the dopamine transporter and COMT may be at least partly involved in the pathogenesis of the disease, and they may represent potential therapeutic targets.

Acknowledgment. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas, from the Ministry of Education, Science, Sports, and Culture, Japan. We thank the Research Center for Molecular Medicine, Hiroshima University School of Medicine (Hiroshima, Japan), for the use of their facilities.

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