Dopamine transporter and catechol-O-methyltransferase activities are required for the toxicity of 1-(3′,4′-dihydroxybenzyl)-1,2,3,4-tetrahydroisoquinoline

Article abstract of DOI:10.1021/tx000047y

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 ν_max, N-methylation markedly increased K_m, 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.

Full text of DOI:10.1021/tx000047y

1
294
Chem. Res. Toxicol. 2000, 13, 1294-1301
Dop a m in e Tr a n sp or ter a n d
Ca tech ol-O-m eth yltr a n sfer a se Activities Ar e Requ ir ed
for th e Toxicity of
1
-(3′,4′-Dih yd r oxyben zyl)-1,2,3,4-tetr a h yd r oisoqu in olin e
Hiroshi Kawai,†,‡ Yaichiro Kotake, and Shigeru Ohta*
§
,§
Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, J apan,
and Institute of Pharmaceutical Sciences, Hiroshima University School of Medicine,
Hiroshima 734-8551, J apan
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-methy-
lated (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
v
m
max, N-methylation markedly increased K , 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.
In tr od u ction
,2,3,4-Tetrahydroisoquinoline (TIQ) derivatives exist
TIQ) (5), 1-(3′,4′-dihydroxybenzyl)-1,2,3,4-tetrahydroiso-
quinoline [3′,4′DHBnTIQ (1)] (8), and salsolinols (10) are
all candidates for endogenous parkinsonogenic com-
pounds.
1
1
widely in nature, including in mammals (1). Since the
discovery in 1983 that 1-methyl-4-phenyl-1,2,3,6-tetrahy-
dropyridine (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 be-
cause 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-
Parkinson’s disease is a neurodegenerative disease
characterized by degeneration of nigro-striatal dopam-
inergic 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-methyl-
+
4
-phenylpyridinium cation (MPP ) by monoamine oxi-
dase and by autoxidation in astroglial cells in the brain
+
(
11, 12). MPP is specifically taken up into dopaminergic
*
To whom correspondence should be addressed: Institute of
Pharmaceutical Sciences, Hiroshima University School of Medicine,
-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, J apan. Telephone:
81-82-257-5325. Fax: E-mail: ohta@
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
1
+
+81-82-257-5329.
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, J apan.
§
1
Hiroshima University.
+
Abbreviations: Ara-C, cytosine â-D-arabinofuranoside; 1BnTIQ,
mitochondrial respiration as efficiently as MPP , conver-
1
-benzyl-1,2,3,4-tetrahydroisoquinoline; COMT, catechol-O-methyl-
+
sion to MPP is also critical. Therefore, metabolism,
transferase; 3′,4′DHBnTIQ, 1-(3′,4′-dihydroxybenzyl)-1,2,3,4-tetrahy-
droisoquinoline; DIV, days in vitro; DMEM, Dulbecco’s modified Eagle’s
medium; ECD, electrochemical detector; GBR12909, 1-{2-[bis(4-fluo-
especially oxidation, dopamine transporter selectivity,
and suppression of mitochondrial respiration may be
important factors for idiopathic Parkinson’s disease.
+
rophenyl)methoxy]ethyl}-4-(3-phenylpropyl)piperazine; MPP , 1-meth-
yl-4-phenylpyridinium; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydro-
pyridine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; PBS, phosphate-buffered saline; Ro41-0960, 2′-fluoro-3,4-
dihydroxy-5-nitrobenzophenone; SAM, S-adenosyl-L-methionine; TIQ,
Previously, we have reported that a TIQ derivative, 1,
is one of the endogenous parkinsonism-inducing sub-
stances. This compound is present in mouse brain, and
1
,2,3,4-tetrahydroisoquinoline.
1
0.1021/tx000047y CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/22/2000

Effect of Metabolism on Neurotoxicity of TIQs
Chem. Res. Toxicol., Vol. 13, No. 12, 2000 1295
Sch em e 1. P u ta tive Meta bolic P a th w a y of 3′,4′DHBn TIQ (1)
can induce parkinsonism in mice in vivo when adminis-
tered 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 neuro-
toxicity is discussed on the basis of the results.
1-(3′,4′-Dim eth oxyben zyl)-2-m eth yl-1,2,3,4-tetr ah ydr oiso-
qu in olin e. 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′-dimethoxy-
benzyl)-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.
Exp er im en ta l P r oced u r es
Ma ter ia ls. Animals were purchased from Nippon Biosupply
Center. The animals were handled in accordance with the Guide
for Animal Experimentation, Graduate School of Pharmaceuti-
1-(3′,4′-Dih ydr oxyben zyl)-2-m eth yl-1,2,3,4-tetr ah ydr oiso-
qu in olin e (3). 1-(3′,4′-Dimethoxybenzyl)-2-methyl-1,2,3,4-tet-
rahydroisoquinoline 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-tetrahydroisoquino-
2
cal Sciences, University of Tokyo. EDTA2Na2H O was pur-
chased from Dojin (Kumamoto, J apan). 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.
1
line hydrobromide (3‚HBr, 0.1 g, mp 233-235 °C). H NMR (400
(Rockville, MD). Deoxyribonuclease I, cytosine â-D-arabinofura-
MHz, DMSO-d
H1, H3 and H4), 3.79 (s, 3H, CH
6
): δ 3.15-3.34 and 4.11-4.15 (m, 3H and 2H,
), 4.52 (s, 2H, CH ), 6.48 (dd,
noside (Ara-C), and (3,4-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium 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.
3
2
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), 7.72-
7.76 (m, 1H, aromatic), 8.08 (d, 1H, aromatic, J ) 7.6 Hz). Anal.
Calcd for C17H19NO ‚HBr: C, 58.36; H, 5.76; N, 4.00. Found:
2
C, 58.53; H, 5.47; N, 4.16.
(
Tokyo, J apan). Other chemicals were purchased from Wako
Pure Chemical Industries (Osaka, J apan).
Ch em ica l Syn th eses. 1-(3′,4′-Dimethoxybenzyl)-3,4-dihy-
droisoquinoline (8), 1-(3′,4′-dimethoxybenzyl)-1,2,3,4-tetrahy-
droisoquinoline (8), 1-(3′,4′-dihydroxybenzyl)-1,2,3,4-tetrahy-
droisoquinoline (1) (8), and 1-(3′,4′-dimethoxybenzyl)isoquinoline
1-(3′,4′-Dih yd r oxyb en zyl)isoq u in olin e (2). 1-(3′,4′-Di-
methoxybenzyl)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 hydrobro-
mide (2‚HBr, 172 mg, mp 168-170 °C). 1H NMR (400 MHz,
(17) were synthesized by the cited methods.
1
-(4′-Hyd r oxy-3′-m eth oxyben zyl)-1,2,3,4-tetr a h yd r oiso-
qu in olin e (5). 1-(3′,4′-Dimethoxybenzyl)-1,2,3,4-tetrahydroiso-
quinoline 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
6 2
DMSO-d ): δ 4.72 (s, 2H, CH ), 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 C16H13NO ‚HBr: C, 57.87; H, 4.22; N, 4.22. Found: C, 58.02;
2
H, 4.06; N, 4.10.
1-(3′,4′-Dim eth oxyben zyl)-2-m eth ylisoqu in olin iu m . Meth-
yl 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
function was confirmed by the NOE signal observed between
1
OCH
H, CH
H1), 6.72 (dd, 1H, H6′, J ) 8.4, 1.6 Hz), 6.75 (d, 1H, H5′, J )
.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 C17 ‚HBr: C,
8.36; H, 5.76; N, 4.00. Found: C, 58.34; H, 5.83; N, 4.18.
3
and H2′. H NMR (400 MHz, DMSO-d
6
): δ 3.01-3.47 (m,
6
2
, H3 and H4), 3.74 (s, 3H, OCH
3
), 4.73-4.77 (m, 1H,
8
H
19NO
2
5

1
296 Chem. Res. Toxicol., Vol. 13, No. 12, 2000
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 NaH PO , 27 mM
NaHCO , 5.4 mM glucose, 0.1 mM ascorbic acid, 1.0 mM KCl,
.0 mM MgCl , and 1.0 mM CaCl and was previously saturated
with a 95% O /5% CO mixture. The reaction was started by
2
4
3
1
2
2
2
2
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
-
5
containing 10 M nomifensine, followed by centrifugation at
1500g for 10 min. The pellet was washed with ice-cold medium
1
and sonicated in 0.1 M perchloric acid containing 40 µM sodium
bisulfite and 10 µM EDTA. After centrifugation at 11500g for
1
0 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 Cu ltu r e. The ventral mesencephalon was dissected out
from a Wistar/ST rat embryo (E16) in Hanks’ medium supple-
mented with 0.4% glucose and 0.035% NaHCO . The dissected
3
areas contain the dopaminergic A8, A9 (substantia nigra), and
A10 (ventral tegmental area) neurons. The dissected mesen-
cephalic 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 superna-
tant 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
F igu r e 1. Results of HPLC/ECD. (A) Authentic compounds (10
pmol) were applied. Peaks are identified as follows: (a) 3 and
4
3
, (b) 2, (c) 1, and (d) 5. (B) An example of an in vitro assay of
′,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′-Dih yd r oxyben zyl)-2-m et h ylisoqu in olin iu m (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. Recrystal-
lization from methanol diethyl ether of the precipitate afforded
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
-
1
1
4
2
-(3′,4′-dihydroxybenzyl)-2-methylisoquinolinium bromide (4‚Br ,
1
50 mg, mp 231-233 °C). H NMR (400 MHz, DMSO-d
.38 (s, 3H, CH ), 4.96 (s, 2H, CH ), 6.35 (dd, 1H, H6′, J ) 8.0,
.0 Hz), 6.46 (d, 1H, H2′, J ) 2.0 Hz), 6.67 (d, 1H, H5′, J ) 8.0
6
): δ
5
% CO
culture medium consisted of DMEM/F-12 supplemented with
.46% glucose, 0.24% NaHCO , 5% fetal bovine serum, 5% horse
2
/95% air mixture with 100% relative humidity. The
3
2
0
3
Hz), 8.06 (m, 1H, H6 or H7), 8.24 (m, 1H, H6 or H7), 8.35 (d,
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.
1
1
1
5
H, H5 or H8, J ) 8.4 Hz), 8.49 (d, 1H, H4, J ) 6.8 Hz), 8.72 (d,
H, H3, J ) 6.8 Hz), 8.78 (d, 1H, H5 or H8, J ) 8.4 Hz), 8.88 (s,
2
H, OH), 8.93 (s, 1H, OH). Anal. Calcd for C17H16NO ‚Br: C,
8.98; H, 4.66; N, 4.05. Found: C, 58.75; H, 4.38; N, 4.19.
HP LC/ECD. The Eicom EP-300/Eicom ECD-100 system was
Eva lu a tion of in Vitr o Cytotoxicity. In vitro cytotoxicity
of 3′,4′DHBnTIQ derivatives (1-5) toward mesencephalic neu-
rons was evaluated by the MTT dye conversion assay (20). The
cells were plated on 96-well culture plates at a density of 1 ×
used under the following conditions: column, Eicompak MA-
ODS (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
5
(
5
5
2
1
0
cells per 100 µL per well (∼3 × 10 cells/cm ). Test
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 elec-
trode, 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 deriva-
tives (3 and 4) were not separated from each other, but this did
not disturb our experiments.
P r oced u r e for th e Up ta k e Assa y. Male adult Sprague-
Dawley 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 super-
natant 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.
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) solu-
tion 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.
P r oced u r e for th e Assa y of th e in Vivo Meta bolism of
3′,4′DHBn TIQ. SD rats (6-8 weeks old) were treated intrap-
eritoneally 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 oblon-
gata) if required. Tissue was homogenized in 2 M perchloric acid
containing 10 mM EDTA and 40 mM sodium bisulfite. After 30
(19).

Effect of Metabolism on Neurotoxicity of TIQs
Chem. Res. Toxicol., Vol. 13, No. 12, 2000 1297
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.
P r oced u r e for th e Assa y of th e in Vitr o Meta bolism of
3
′,4′DHBn TIQ. 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
F igu r e 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 <
1
0 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.
0
.05 (one asterisk) and p < 0.01 (two asterisks) by Scheff e´ ’s F
Eva lu a tion of O-Meth yla tion of 3′,4′DHBn TIQ in Cu l-
tu r ed Neu r on s. The cells were plated on 60 mm culture dishes
test.
5
5
2
be >500 and 50 pmol min-1 (mg of protein) , respec-
-1
at a density of 4 × 10 cells/dish (∼1.4 × 10 cells/cm ). 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
tively. These compounds exhibited higher K values than
m
the N-demethylated derivatives.
Toxicit y of 3′,4′DH Bn TIQ Der iva t ives t ow a r d
Mesen cep h a lic Neu r on s. We measured the toxicity of
(
10 µM Ro41-0960) was added 30 min before addition of 1, when
required. The cells were washed with PBS and suspended in
.2 M perchloric acid. After centrifugation at 14 000 rpm for 10
0
1
-5 to cultured neurons to examine the relationship
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.
between uptake activity and neurotoxicity of these com-
pounds. 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.
Resu lts
Sp ecific Up ta k e of 3′,4′DHBn TIQ Der iva tives. To
test the uptake activity for 3′,4′DHBnTIQ derivatives, we
used reserpinized rat striatal synaptosomes. These syn-
aptosomes were incubated with 1 µM 1-5 for 5 min at
3
7 °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
+
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 cyto-
toxicity of 1. As shown in Figure 5, these inhibitors
prevented the 1-induced reduction of cell viability.
GBR12909 and GBR12935, however, enhanced the toxic-
ity 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 reduc-
tion of cell viability, suggesting that active transport of
1 into the cells is important for cytotoxicity of 1.
Meta bolism of 3′,4′DHBn TIQ 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 intraperi-
toneally with 250 mg/kg 1, a dose at which it can induce
parkinsonism (8). They were killed 1 h after injection,
>
2 > 4. Thus, both N-methylation and oxidation of 1
diminished the uptake activity, and O-methylation abol-
ished 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
K values for 1 and 2 were 6.14 and 8.79 µM and the
m
-1
-1
v
max values 214.3 and 136.8 pmol min (mg of protein) ,
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
-
1
-1
pmol min
kinetic parameters could not be determined exactly, the
values should be >100 µM and the vmax values should
(mg of protein) , respectively. Although
K
m

1
298 Chem. Res. Toxicol., Vol. 13, No. 12, 2000
Kawai et al.
F igu r e 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.
F igu r e 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 equa-
tion. 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).
F igu r e 5. Effect of dopamine transporter inhibitors on 3′,4′DH-
BnTIQ-induced cytotoxicity. Cultured neurons were preincu-
bated 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 Scheff e´ ’s F test.
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-Meth yla tion of 3′,4′DHBn TIQ by COMT in Vit-
r o. We studied 3′,4′DHBnTIQ O-methylation in vitro by
using rat brain homogenates. Rat whole brain homoge-
nates 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
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 K
nmol h (mg of protein) for 1.
m
value of 1.37 mM and a vmax value of 3.86
-
1
-1
O-Meth yla tion of 3′,4′DHBn TIQ in Mesen cep h a lic
Cu ltu r e. We also examined if 3′,4′DHBnTIQ O-methy-
lation 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′DHBn TIQ-In d u ced Neu -
r otoxicity. To evaluate the importance of COMT for
1-induced cytotoxicity to cultured neurons, we used an
inhibitor (Ro41-0960) of COMT. In general, O-methyla-
tion of catechols inactivates the compounds, as in the
(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-me-
thylation, 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

Effect of Metabolism on Neurotoxicity of TIQs
Chem. Res. Toxicol., Vol. 13, No. 12, 2000 1299
Ta ble 1. Meta bolism of 3′,4′DHBn TIQ (1)
a
b
c
in vivo content
in vitro specific formation
in culture content
compd with the
COMT inhibitor
-
1
-1
6
compd
(nmol/mg of brain)
[nmol h (mg of protein)
]
(pmol/4 × 10 cells)
196.11 ( 48.05
e
1
2
3
4
5
0.3583
-
n
nd
nd
361.61 ( 26.96
d
nd
nd
nd
nd
nd
nd
nd
nd
nd
n
0.0991
0.59 ( 0.10
12.24 ( 1.23
a
Each value represents the mean of two experiments. b Whole brain homogenates were used. Homogenates were incubated with 1
c
mM 1 and 0.1 mM SAM at 37 °C for 1 h. Each value represents the mean ( SE of three experiments. Each value represents the mean
SE of three experiments. nd, not detected. -, not evaluated.
d
e
(
F igu r e 6. Subcellular localization of 3′,4′DHBnTIQ O-methy-
lation 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 Scheff e´ ’s F test.
F igu r e 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 K
m
and vmax values. Each
point represents the mean value of two to six experiments.
F igu r e 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 concen-
trations (B). Each point represents the result of one experiment.
F igu r e 9. Effect of a COMT inhibitor on 3′,4′DHBnTIQ-
induced 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
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 cy-
totoxicity in a concentration-dependent manner. Ro41-
(
one asterisk), p < 0.01 (two asterisks) vs nontreated control
white column), and p < 0.05 (dagger) vs 1 treated group (black
(
0
960 itself had no effect on cell viability at 1-100 µM
column) by Scheff e´ ’s F test.
(data not shown). Thus, the COMT inhibitor did not
m
and 2 to 4) greatly increased K , and O-methylation (1
enhance but prevented the 1-induced cytotoxicity.
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 observa-
tions 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
Discu ssion
In this study, we examined the structure-activity
relationship of the endogenous parkinsonism-inducing
substance, 3′,4′DHBnTIQ (1), and some related com-
pounds 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
(

1
300 Chem. Res. Toxicol., Vol. 13, No. 12, 2000
Kawai et al.
than that of 2. A comparison of Figures 3 and 4 suggests
that the neurotoxicity of 3′,4′DHBnTIQ derivatives de-
pends 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.
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.
Nomifensine can prevent 1-induced cytotoxicity at
-100 µM, but it can rescue at most 60-70% of the total
Morikawa et al. investigated the inhibitory effect of
catecholamines and their O-methylated derivatives on
mitochondrial respiration (35). They reported that cat-
echolamines such as dopamine and tetrahydropapavero-
line could inhibit complex I activity, but O-methyl
derivatives had more potent activity. O-Methylation of
catecholamines increased the activity to inhibit mito-
chondrial 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 N-
or 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′DHBn-
TIQ (1) itself, without structural modification, is taken
up into dopaminergic neurons by the dopamine trans-
porter, and then is O-methylated by COMT. If 1 is
O-methylated outside the neurons (in glia), this repre-
sents 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.
1
cells. This suggests that there are nomifensine-insensi-
tive alternative pathways for 1-induced cytotoxicity.
Catechol compounds can produce quinones and/or reac-
tive oxygen species, which cause oxidative stress and
cytotoxicity (23-25), and there is much evidence that
oxidative stress causes neuronal cell death and neuro-
degenerative 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 K
mg of protein)-1 min (18). MPP has kinetics similar
to those of dopamine (13). The K value of 1 is higher
m
value of 183 nM and a vmax value of 306 pmol
-1
+
(
m
+
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 sub-
stance, 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 parkin-
sonism-inducing neurotoxins such as 1,2,3,4-tetrahydro-
â-carbolines and salsolinols are also activated by me-
tabolism. 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, N-
methylated 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 intrac-
ellular content of 5, although that of 1 was increased
Ack n ow led gm en t. This work was supported by a
Grant-in-Aid for Scientific Research on Priority Areas,
from the Ministry of Education, Science, Sports, and
Culture, J apan. We thank the Research Center for
Molecular Medicine, Hiroshima University School of
Medicine (Hiroshima, J apan), for the use of their facili-
ties.
Refer en ces
(
1) Collins, M. A. (1983) Mammalian alkaloids. In The Alkaloids
Brossi, A., Ed.) Vol. 21, pp 329-358, Academic Press, New York.
(
(
2) Langston, J . W., Ballard, P., Tetrud, J . W., and Irwin, I. (1983)
Chronic parkinsonism in humans due to a product of meperidine-
analog synthesis. Science 219, 979-980.
(
3) Kohno, M., Ohta, S., and Hirobe, M. (1986) Tetrahydroisoquino-
line and 1-methyl-tetrahydroisoquinoline as novel endogenous
amines in rat brain. Biochem. Biophys. Res. Commun. 140, 448-
4
54.
(
5
Table 1). It appears that the COMT-methylated product,
, is the toxic compound, and the neurotoxicity depends
(4) Ohta, S., Kohno, M., Makino, Y., Tachikawa, O., and Hirobe, M.
(1987) Tetrahydroisoquinoline and 1-methyltetrahydroisoquino-
line are present in the human brain: relation to Parkinson’s
disease. Biomed. Res. 8, 453-456.
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
(
5) Kotake, Y., Tasaki, Y., Makino, Y., Ohta, S., and Hirobe, M. (1995)
-Benzyl-1,2,3,4-tetrahydroisoquinoline as a parkinsonism-induc-
ing agent: novel endogenous amine in mouse brain and
1
a
parkinsonian CSF. J . Neurochem. 65, 2633-2638.
(
6) Kotake, Y., Yoshida, M., Ogawa, M., Tasaki, Y., Hirobe, M., and
Ohta, S. (1996) Chronic administration of 1-benzyl-1,2,3,4-tet-
rahydroisoquinoline, an endogenous amine in the brain, induces
parkinsonism in a primate. Neurosci. Lett. 217, 69-71.

Effect of Metabolism on Neurotoxicity of TIQs
Chem. Res. Toxicol., Vol. 13, No. 12, 2000 1301
(
(
(
7) Nakagawa, H., Nihonmatsu, N., Ohta, S., and Hirobe, M. (1996)
Effects of new endogenous nonprotein amino acids, 1,2,3,4-
tetrahydroisoquinoline-3-carboxylic acid derivatives, on behavior
of mice. Biochem. Biophys. Res. Commun. 225, 1027-1034.
8) Kawai, H., Makino, Y., Hirobe, M., and Ohta, S. (1998) Novel
endogenous 1,2,3,4-tetrahydroisoquinoline derivatives: uptake by
dopamine transporter and activity to induce parkinsonism. J .
Neurochem. 70, 745-751.
(22) Matsubara, K., Senda, T., Uezono, T., Fukushima, S., Ohta, S.,
Igarashi, K., Naoi, M., Yamashita, Y., Ohtaki, K., Hayase, N.,
Akutsu, S., and Kimura, K. (1998) Structural significance of
azaheterocyclic amines related to Parkinson’s disease for dopam-
ine transporter. Eur. J . Pharmacol. 348, 77-84.
(23) Shen, X.-M., and Dryhurst, G. (1996) Further insights into the
influence of L-cysteine on the oxidation chemistry of dopamine:
reaction pathways of potential relevance to Parkinson’s disease.
Chem. Res. Toxicol. 9, 751-763.
9) Tasaki, Y., Makino, Y., Ohta, S., and Hirobe, M. (1991) 1-Methyl-
1
1
,2,3,4-tetrahydroisoquinoline, decreasing in 1-methyl-4-phenyl-
,2,3,6-tetrahydropyridine-treated mouse, prevents parkinsonism-
(
24) Napolitano, A., Pezzella, A., and Prota, G. (1999) New reaction
pathway of dopamine under oxidative stress conditions: nonen-
zymatic iron-assisted conversion to norepinephrine and the
neurotoxins 6-hydroxydopamine and 6,7-dihydroxytetrahydroiso-
quinoline. Chem. Res. Toxicol. 12, 1090-1097.
like behavior abnormalities. J . Neurochem. 57, 1940-1943.
(
10) Maruyama, Y., Takahashi, T., Minami, M., Takahashi, A., Dos-
tert, P., Nagatsu, T., and Naoi, M. (1993) Cytotoxicity of dopam-
ine-derived 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinolines. Adv.
Neurol. 60, 224-230.
11) Chiba, K., Trevor, A., and Castagnoli, N., J r. (1984) Metabolism
of the neurotoxic tertiary amine, MPTP, by brain monoamine
oxidase. Biochem. Biophys. Res. Commun. 120, 574-578.
12) Ransom, B. R., Kunis, D. M., Irwin, I., and Langston, J . W. (1987)
Astrocytes convert the parkinsonism inducing neurotoxin, MPTP,
(
25) Bolton, J . L., Trush, M. A., Penning, T. M., Dryhurst, G., and
Monks, T. J . (2000) Role of quinones in toxicology. Chem. Res.
Toxicol. 13, 135-160.
(
(
(
(
26) Dexter, D. T., Carter, C. J ., Wells, F. R., J avoy-Agid, F., Agid, Y.,
Lees, A., J enner, P., and Marsden, C. D. (1989) Basal lipid
peroxidation in substantia nigra is increased in Parkinson’s
disease. J . Neurochem. 52, 381-389.
+
to its active metabolite, MPP . Neurosci. Lett. 75, 323-328.
13) J avitch, J . A., D’Amato, R. J ., Strittmatter, S. M., and Snyder, S.
(27) Hirsch, E. C., Brandel, J . P., Galle, P., J avoy-Agid, F., and Agid,
Y. (1991) Iron and aluminum increase in the substantia nigra of
patients with Parkinson’s disease: an X-ray microanalysis. J .
Neurochem. 56, 446-451.
H. (1985) Parkinsonism-inducing neurotoxin, N-methyl-4-phenyl-
1
4
,2,3,6-tetrahydropyridine: uptake of the metabolite N-methyl-
-phenylpyridine by dopamine neurons explains selective toxicity.
Proc. Natl. Acad. Sci. U.S.A. 82, 2173-2177.
(28) Tipton, K. F., and Singer, T. P. (1993) Advances in our under-
standing of the mechanisms of the neurotoxicity of MPTP and
related compounds. J . Neurochem. 61, 1191-1206.
(
14) Nicklas, W. J ., Vyas, I., and Heikkila, R. E. (1985) Inhibition of
NADH-linked oxidation in brain mitochondria by 1-methyl-4-
phenylpyridine, a metabolite of the neurotoxin, 1-methyl-4-
phenyl-1,2,5,6-tetrahydropyridine. Life Sci. 36, 2503-2508.
15) Ramsay, R. R., Salach, J . I., Dadgar, J ., and Singer, T. P. (1986)
Inhibition of mitochondrial NADH dehydrogenase by pyridine
derivatives and its possible relation to experimental and idio-
pathic parkinsonism. Biochem. Biophys. Res. Commun. 135, 269-
(
29) Takahashi, T., Deng, Y., Maruyama, W., Dostert, P., Kawai, M.,
and Naoi, M. (1994) Uptake of a neurotoxin-candidate, (R)-1,2-
dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline into hu-
man dopaminergic neuroblastoma SH-SY5Y cells by dopamine
transport system. J . Neural Transm. 98, 107-118.
(
(
30) Matsubara, K., Gonda, T., Sawada, H., Uezono, T., Kobayashi,
Y., Kawamura, T., Ohtani, K., Kimura, K., and Akaike, A. (1998)
Endogenously occurring â-carboline induces parkinsonism in
nonprimate animals: a possible causative protoxin in idiopathic
Parkinson’s disease. J . Neurochem. 70, 727-735.
2
75.
(
(
(
16) Morikawa, N., Naoi, M., Maruyama, Y., Ohta, S., Kotake, Y.,
Kawai, H., Niwa, T., Dostert, P., and Mizuno, Y. (1998) Effects
of various tetrahydroisoquinoline derivatives on mitochondrial
respiration and the electron-transfer complex. J . Neural Transm.
(
31) Naoi, M., Maruyama, W., Niwa, T., and Nagatsu, T. (1994) Novel
toxins and Parkinson’s disease: N-methylation and oxidation as
metabolic bioactivation of neurotoxin. J . Neural Transm., Suppl.
1
05, 677-688.
17) Neumeyer, J . L., McCarthy, M., Battista, S. P., Rosenberg, F. J .,
and Teiger, D. G. (1973) Aporphines. 9. Synthesis and pharma-
cological evaluation of (()-9,10-dihydroxyaporphine [(()-isoapor-
phine], (+)-, (-)-, and (()-1,2-dihydroxyaporphine, and (+)-
4
1, 197-205.
(
32) Karhunen, T., Tilgmann, C., Ulmanen, I., and Panula, P. (1995)
Neuronal and non-neuronal catechol-O-methyltransferase in
primary cultures of rat brain cells. Int. J . Dev. Neurosci. 13, 825-
1
,2,9,10-tetrahydroxyaporphine. J . Med. Chem. 16, 1228-1233.
18) Amejdki-Chab, N., Benmansour, S., Costentin, J ., and Bonnet,
J .-J . (1992) Effects of several cations on the neuronal uptake of
8
34.
3
(33) Kastner, A., Anglade, P., Bouaix, C., Damier, P., J avoy-Agid, F.,
Bronet, N., Agid, Y., and Hirsch, E. C. (1994) Immunohistochemi-
cal study of catechol-O-methyltransferase in the human mesos-
triatal system. Neuroscience 62, 449-457.
dopamine and the specific binding of [ H]GBR 12783: attempts
to characterize the Na+ dependence of the neuronal transport of
dopamine. J . Neurochem. 59, 1795-1804.
(
19) Lowry, O. H., Rosebrough, N. J ., Farr, A. L., and Randall, R. J .
(
1951) Protein measurement with the Folin phenol reagent. J .
(34) Michel, P. P., Ruberg, M., and Agid, Y. (1997) Rescue of mesen-
cephalic dopamine neurons by anticancer drug cytosine arabino-
side. J . Neurochem. 69, 1499-1507.
Biol. Chem. 193, 265-275.
(
20) Hansen, M. B., Nielsen, S. E., and Berg, K. (1989) Re-examination
and further development of a precise and rapid dye method for
measuring cell growth/cell kill. J . Immunol. Methods 119, 203-
(
35) Morikawa, N., Nakagawa-Hattori, Y., and Mizuno, Y. (1996) Effect
of dopamine, dimethoxyphenylethylamine, papaverine, and re-
lated compounds on mitochondrial respiration and complex I
activity. J . Neurochem. 66, 1174-1181.
2
10.
(
21) Meiergerd, S. M., and Schenk, J . O. (1994) Striatal transporter
for dopamine: catechol structure-activity studies and suscepti-
bility to chemical modification. J . Neurochem. 62, 998-1008.
TX000047Y