Cytotoxicity of 1-amino-4-phenyl-1,2,3,6-tetrahydropyridine and 1- amino-4-phenylpyridinium ion, 1-amino analogues of MPTP and MPP+, to clonal pheochromocytoma PC12 cells

Article abstract of DOI:10.1021/tx980032o

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces parkinsonism in humans after its oxidation into 1-methyl-4-phenylpyridinium ion (MPP⁺) by type B monoamine oxidase. The 1-amino analogues of MPTP and MPP⁺, 1-amino- 4-phenyl-1,2,3,6-tetrahydropyridine (APTP) and 1-amino-4-phenylpyridinium ion (APP⁺), were synthesized, and their cytotoxicity to clonal pheochromocytoma PC12 cells was examined using a tetrazolium formazan assay. After incubation for 48 and 72 h, both APP⁺ and APTP were found to be cytotoxic to PC12 cells, whereas with the N-methyl analogues, only MPP⁺, but not MPTP, was cytotoxic. The cytotoxicity of APTP increased with incubation time and equaled that of MPP⁺ after 72 h. It was found that APTP was oxidized to APP⁺ by type A monoamine oxidase in PC12 cells, suggesting that APP⁺ itself may damage the cells. In addition to APTP and APP⁺, N-amino analogues of N- methylisoquinolines and related derivatives were also synthesized and examined for their cytotoxicity to PC12 cells.

Full text of DOI:10.1021/tx980032o

Chem. Res. Toxicol. 1998, 11, 1249-1253
1249
Communications
Cytotoxicity of
1-Am in o-4-p h en yl-1,2,3,6-tetr a h yd r op yr id in e a n d
1-Am in o-4-p h en ylp yr id in iu m Ion , 1-Am in o An a logu es of
MP TP a n d MP P +, to Clon a l P h eoch r om ocytom a P C12
Cells
Kohfuku Kohda,*,† Yasuhiro Noda,† Shinsuke Aoyama,† Michi Umeda,†
Tomoe Sumino,† Toyo Kaiya,† Wakako Maruyama,‡ and Makoto Naoi§
Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabedori, Mizuho-ku,
Nagoya 467, J apan, Department of Basic Gerontology, National Institute for Longevity Sciences,
Obu 4, J apan, and Department of Biosciences, Nagoya Institute of Technology, Showa-ku,
Nagoya 466, J apan
Received February 24, 1998
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces parkinsonism in humans after
its oxidation into 1-methyl-4-phenylpyridinium ion (MPP+) by type B monoamine oxidase. The
1-amino analogues of MPTP and MPP+, 1-amino-4-phenyl-1,2,3,6-tetrahydropyridine (APTP)
and 1-amino-4-phenylpyridinium ion (APP+), were synthesized, and their cytotoxicity to clonal
pheochromocytoma PC12 cells was examined using a tetrazolium formazan assay. After
incubation for 48 and 72 h, both APP+ and APTP were found to be cytotoxic to PC12 cells,
whereas with the N-methyl analogues, only MPP+, but not MPTP, was cytotoxic. The
cytotoxicity of APTP increased with incubation time and equaled that of MPP+ after 72 h. It
was found that APTP was oxidized to APP+ by type A monoamine oxidase in PC12 cells,
suggesting that APP+ itself may damage the cells. In addition to APTP and APP+, N-amino
analogues of N-methylisoquinolines and related derivatives were also synthesized and examined
for their cytotoxicity to PC12 cells.
activity against HIV than AZT (6) and that 1-amino-5-
halo-substituted uracil derivatives have anticonflict and
anesthetic activities (7).
In tr od u ction
Introducing an amino group at the ring nitrogen of
heterocyclic compounds or replacing the N-methyl group
of bioactive compounds with an N-amino group results
in retention or enhancement of the biological activities,
because the amino group can interact with surrouding
macromolecules by hydrogen bonding. We have been
working on the preparation of N-amino purine and
pyrimidine derivarives and their biological activities (1-
5). We reported previously that 3-aminothymidine in-
hibits the growth of CCRF leukemia cells more markedly
than 3-methylthymidine (1). It was also reported that
3-amino-3′-azido-3′-deoxythymidine, a 3-amino derivative
of 3′-azido-3′-deoxythymidine (AZT1), has better selective
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
and its oxidation product, 1-methyl-4-phenylpyridinium
ion (MPP+), are well-known dopaminergic neurotoxins
which cause parkinsonism in humans (8). Oxidation of
MPTP by type B monoamine oxidase [amine oxidase,
amine:oxygen oxidoreductase (deaminating), EC 1.4.3.4]
(MAO-B) into MPP+ is required for selective transport
into dopamine neurons and cytotoxicity. The mechanism
of the cytotoxicity is proposed to be the inhibition of ATP
synthesis (9), or the induction of apoptosis (10). To
examine the effect of the replacement of a methyl group
with an amino group, 1-amino-4-phenyl-1,2,3,6-tetrahy-
dropyridine (APTP) and 1-amino-4-phenylpyridinium ion
(APP+), the 1-amino analogues of MPTP and MPP+,
respectively, were synthesized and examined for their
cytotoxic activity to PC12 cells using an XTT assay. The
cytotoxicities of 2-amino analogues of other simple iso-
* To whom correspondence should be addressed. Telephone:
(81)-52-836-3796.
phar.nagoya-cu.ac.jp.
Fax:
(81)-52-834-9309.
E-mail:
kohda@
† Nagoya City University.
‡ National Institute for Longevity Sciences.
§ Nagoya Institute of Technology.
1Abbreviations: AZT, 3′-azido-3′-deoxythymidine; MPTP, 1-methyl-
4-phenyl-1,2,3,6-tetrahydropyridine; MPP+, 1-methyl-4-phenylpyri-
dinium ion; APTP, 1-amino-4-phenyl-1,2,3,6-tetrahydropyridine; APP+,
1-amino-4-phenylpyridinium ion; MAO-A, type A monoamine oxidase;
MAO-B, type B monoamine oxidase; EPTP, 1-ethyl-4-phenyl-1,2,3,6-
tetrahydropyridine; EPP+, 1-ethyl-4-phenylpyridinium ion; MAPP+,
1-(methylamino)-4-phenylpyridinium ion; MTIQ, 2-methyl-1,2,3,4-
tetrahydroisoquinoline; MIQ+, 2-methylisoquinolinium ion; ATIQ,
2-amino-1,2,3,4-tetrahydroisoquinoline; AIQ+, 2-aminoisoquinolinium
ion; DMEM, Dulbecco’s Modified Eagle’s Medium; PMS, phenazine
methosulfate; MEM, minimal essential medium; XTT, 2,3-bis(2-
methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.
10.1021/tx980032o CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/22/1998

1250 Chem. Res. Toxicol., Vol. 11, No. 11, 1998
Communications
yielding 30 mg. The product was solubilized in 10 mL of 10%
HCl and evaporated to dryness. Recrystallization from AcOEt/
MeOH gave colorless pillars. Mp: 206-207 °C [lit. (13), 192-
194 °C]. 1H NMR (CDCl3): δ 1.18 (t, 3H, J ) 7.3 Hz, CH3),
2.56 (m, 2H, 3-H), 2.57 (q, 2H, CH3CH2), 2.74 (m, 2H, 2-H), 3.19
(d, 2H, J ) 3.1 Hz, 6-H), 6.06 (br s, 1H, 5-H), 7.23 (t, 1H, J )
7.3 Hz, 4′-H), 7.31 (m, 2H, 3′- and 5′-H), 7.38 (d, 2H, J ) 7.3
Hz, 2′- and 6′-H). Anal. Calcd for C13H17N‚HCl: C, 69.78; H,
8.11; N, 6.26. Found: C, 69.62; H, 8.12; N, 6.40.
quinolines,2-methyl-1,2,3,4-tetrahydroisoquinoline(MTIQ),2-
methylisoquinolinium ion (MIQ+), and related derivatives
were also examined.
Exp er im en ta l P r oced u r es
Ch em ica ls. MPTP hydrochloride, iodide salt of MPP+, and
2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-car-
boxanilide (XTT) were purchased from Sigma (St. Louis, MO).
Iodide salt of EPP+ was from Aldrich (Milwaukee, WI). 2-Meth-
yl-1,2,3,4-tetrahydroisoquinoline (MTIQ) hydrochloride and io-
dide salt of MIQ+ were prepared as reported previously (11).
Syn th eses of N-Am in o Der iva tives of P yr id in es a n d
Isoqu in olin es. Gen er a l. 1H and 13C NMR spectra were
recorded on a J EOL EX 270 or GSX 400 spectrometer (Tokyo,
J apan), and chemical shifts are reported in parts per million
(ppm) using tetramethylsilane as the internal standard. Mass
spectra were obtained with a J EOL DX-300 spectrometer. pKa
values were determined with UV spectra using a Shimadzu UV-
2100 spectrophotometer (Kyoto, J apan). Melting points were
measured with a Yanagimoto micro-melting point apparatus
(Kyoto, J apan) and are uncorrected. Silica gel 60 (Merck, 70-
230 mesh, 63-200 µm) and silica gel PF254 (Merck) were used
for column chromatography and preparative thin-layer chro-
matography (PLC), respectively.
1-Am in o-4-p h en ylp yr id in iu m Iod id e (Iod id e Sa lt of
AP P +). 4-Phenylpyridine (6.21 g, 40 mmol) was added to a
solution of hydroxylamine-O-sulfonic acid (1.50 g, 13.3 mmol)
in 30 mL of water, and the mixture was heated at 90 °C for 2 h.
After the mixture cooled to room temperature, water (30 mL)
and K2CO3 (1.84 g, 13.3 mmol) were added. Precipitates were
removed by filtration, and the mother liquor was washed with
40 mL of CHCl3 three times. The aqueous phase was evapo-
rated to dryness. EtOH (24 mL) was added to the residue, and
the EtOH soluble fraction was collected by filtration. Concen-
trated HI (5.3 mL) was added to the filtrate, and the mixture
was kept at -15 °C for 1 day. The dark brown needles that
appeared were collected. The yield was 1.69 g (42.8%). Re-
crystallization from EtOH gave brown needles. Mp: 173-174
°C [lit. (12), 171 °C]. 1H NMR (Me2SO-d6): δ 7.60-7.63 (m, 3H,
Ph), 7.96-7.99 (m, 2H, Ph), 8.37 (d, 2H, J ) 7.3 Hz, 3- and 5-H),
8.38 (s, 2H, NH2), 8.80 (d, 2H, 2- and 6-H). 13C NMR (Me2SO-
d6): δ 124.7 (3- and 5-C), 127.5 (2′- and 6′-C), 129.5 (3′- and
5′-C), 131.3 (4′-C), 133.7 (1′-C), 138.5 (2- and 6-C), 149.5 (4-C).
Anal. Calcd for C11H11IN2: C, 44.31; H, 3.72; N, 9.40. Found:
C, 43.99; H, 3.94; N, 9.57.
1-Am in o-4-ph en yl-1,2,3,6-tetr ah ydr opyr idin e (AP TP ) Hy-
d r och lor id e. Iodide salt of APP+ (894 mg, 3 mmol) was
dissolved in 50 mL of MeOH. NaBH4 (227 mg, 6 mmol) was
added, and the mixture was left at room temperature for 2 h.
The product was purified by column chromatography (silica gel,
20 mm × 150 mm, eluted with CHCl3 and then with 97:3 CHCl3/
MeOH). Fractions that contained product were collected, and
the solvent was removed. To obtain the HCl salt, the residue
was dissolved in 2 mL of 1 N HCl and the solvent was removed
by evaporation. Recrystallization from MeOH/AcOEt gave
crystals in a yield of 300 mg (57.5%). Mp: 185.1-186.4 °C. pKa
) 6.51. 1H NMR (Me2SO-d6): δ 2.52 (t, 2H, J ) 5.6 Hz, 3-H),
2.75 (t, 2H, 2-H), 3.18 (d, 2H, J ) 2.9 Hz, 6-H), 6.08 (t, 1H, 5-H),
7.23 (t, 1H, J ) 7.3 Hz, 4′-H), 7.32 (t, 2H, 3′- and 5′-H), 7.41 (d,
2H, J ) 7.8 Hz, 2′- and 6′-H), 9.67 (br s, 2H, NH2). 13C NMR
(Me2SO-d6): δ 27.9 (3-C), 55.8 and 58.3 (2- and 6-C), 121.6 (5-
C), 124.5 (2′- and 6′-C), 126.9 (4′-C), 128.2 (3′- and 5′-C), 133.4
(4-C), 134.5 (1′-C). Anal. Calcd for C11H14N2‚HCl: C, 62.70;
H, 7.18; N, 13.30. Found: C, 62.92; H, 7.06; N, 13.37. MS: m/z
174 (M+ of free form).
1-(Met h yla m in o)-4-p h en ylp yr id in iu m Iod id e (Iod id e
Sa lt of MAP P +). Acetic anhydride (3 mL) was added to iodide
salt of APP+ (500 mg, 1.54 mmol) in 2 mL of pyridine, and the
mixture was left at room temperature for 15 h. After the solvent
was removed by evaporation, the 1-(acetylamino)-4-phenyl-
pyridinium salt was separated by PLC (silica gel, 9:1 CHCl3/
MeOH). The 1-(acetylamino)-4-phenylpyridinium salt obtained
was dissolved in 5 mL of MeOH, and 0.5 mL of CH3I was added.
The mixture was left at room temperature for 2 days, and
precipitates of 1-(N-acetyl-N-methylamino)-4-phenylpyridinium
iodide were collected by filtration. The precipitates were
dissolved in 5 mL of concentrated HCl, and the solution was
heated at 100 °C for 1 h. After the solvent was removed by
evaporation, the product was separated by PLC (silica gel, 4:1
CHCl3/MeOH) to give iodide salt of MAPP+ in a yield of 156 mg
(29% yield starting from iodide salt of APP+). Recrystallization
from MeOH gave pale yellow pillars. Mp: 179-181 °C. 1H
NMR (Me2SO-d6): δ 3.11 (d, 3H, J ) 4.5 Hz, CH3), 7.66 (m, 3H,
Ph), 8.01 (m, 2H, Ph), 8.42 (d, 2H, J ) 6.8 Hz, 3- and 5-H), 8.58
(br, 1H, NH), 8.98 (d, 2H, 2- and 6-H). An analytically pure
sample was not obtained after repeated recrystallization.
2-Am in oisoqu in olin iu m Iod id e (Iod id e Sa lt of AIQ+).
Isoquinoline (387 mg, 3 mmol) was dissolved in 5 mL of DMF,
and O-(2,4-dinitrophenyl)hydroxylamine (14) (900 mg, 4.5 mmol)
was added. The mixture was heated at 60 °C for 15 h. After
DMF was removed by evaporation, 5 mL of 1 N HCl was added,
and the mixture was washed three times with 5 mL of AcOEt.
The aqueous phase was evaporated to dryness, and the product
was purified by column chromatography (silica gel, 30 mm ×
200 mm, eluted with CHCl3 and then with 12.5% MeOH in
CHCl3). The product was further purified by PLC (silica gel,
20% MeOH in CHCl3). Recrystallization from MeOH/AcOEt
gave yellow columns of 2-aminoisoquinolinium chloride in a
yield of 424 mg (78.3%). Mp: 162-164 °C. 1H NMR (Me2SO-
d6): δ 8.02 and 8.13 (each t, each 1H, 6- and 7-H), 8.29 (d, 1H,
J ) 7.9 Hz, 4-H), 8.42 (d, 1H, 3-H), 8.50 and 8.57 (each d, each
1H, J ) 7.3 Hz, 5- and 8-H), 8.86 (s, 2H, NH2), 9.77 (s, 1H, NH).
Anal. Calcd for C9H9ClN2: C, 59.84; H, 5.02; N, 15.51. Found:
C, 59.65; H, 5.28; N, 15.50.
Iodide salt of AIQ+ was obtained by dissolving the chloride
salt in aqueous hydriodic acid, followed by evaporation and
recrystallization from MeOH/AcOEt. Mp: 177.5-178.5 °C [lit.
(15), 180-183 °C]. Anal. Calcd for C9H9IN2: C, 39.73; H, 3.33;
N, 10.29. Found: C, 39.85; H, 3.36; N, 10.28.
2-Am in o-1,2,3,4-tetr a h yd r oisoqu in olin e (ATIQ) Hyd r o-
ch lor id e. A procedure reported previously was followed (16).
Briefly, the reaction of o-(â-chloroethyl)benzyl chloride (1.89 g,
10 mmol), which was prepared from isochroman (17), with
benzoylhydrazine (1.63 g, 12 mmol) gave 2-(benzoylamino)-
1,2,3,4-tetrahydroisoquinoline, and its subsequent acid hydroly-
sis yielded ATIQ hydrochloride in a yield of 472 mg (25.6%).
Recrystallization from MeOH/AcOEt gave pale brown needles.
Mp: 211-213 °C [lit. (16), 206-207 °C]. 1H NMR (Me2SO-d6):
δ 2.98 (t, 2H, J ) 5.6 Hz, 4-H), 3.30 (t, 2H, 3-H), 4.15 (s, 2H,
1-H), 7.15-7.21 (m, 4H, 5-, 6-, 7-, and 8-H), 9.74 (br s, 2H, NH2).
Anal. Calcd for C9H12N2‚HCl: C, 58.53; H, 7.10; N, 15.17.
Found: C, 58.50; H, 7.06; N, 14.88.
1-Eth yl-4-p h en yl-1,2,3,6-tetr a h yd r op yr id in e (EP TP ) Hy-
d r och lor id e. Iodide salt of EPP+ (50 mg, 0.16 mmol) was
dissolved in 10 mL of MeOH. After NaBH4 (25 mg, 0.66 mmol)
was added, the mixture was left at room temperature for 10
min. The solvent was removed by evaporation, and the crude
product was separated by PLC (silica gel, 9:1 CHCl3/MeOH),
XTT Assa y for Cytotoxicity. Fifty microliters of PC12 cells
(4 × 105 cells/mL) in Dulbecco’s Modified Eagle’s Medium
(DMEM) was seeded in each well of a 96-well plastic culture
dish, and the cells were incubated for 1 day at 37 °C in 5% CO2.
The test chemical, dissolved in 50 µL of DMEM, was added to
the cells, and the mixture was incubated at 37 °C for 24, 48, or

Communications
Ta ble 1. Cytotoxicity of N-Am in o a n d N-Meth yl
Chem. Res. Toxicol., Vol. 11, No. 11, 1998 1251
Der iva tives a n d Rela ted Com p ou n d s to P C12 Cells
a
After incubation for 72 h.
F igu r e 1. Cytotoxicity of APP+, APTP, MPP+, and MPTP to
PC12 cells after treatment for 24 (0), 48 (4), and 72 h (O).
72 h. The medium was then changed (100 µL), and the cells
were incubated for another day. Fifty microliters of serum-free
medium containing XTT (1 mg/mL) and phenazine methosulfate
(PMS) (10 µM) was added to the cells which were then incubated
at 37 °C for 4 h. The absorbance of XTT formazan at 450 nm
was measured with a densitometer.
and fully characterized. Iodide salt of MAPP+ was
prepared by acetylation of APP+, followed by methylation
and deacetylation. We were unsuccessful in attempts to
synthesize 1-(methylamino)-4-phenyl-1,2,3,6-tetrahydro-
pyridine, the reduced form of MAPP+, by the reduction
of MAPP+ with NaBH4. Iodide salt of AIQ+ (15) was
prepared by amination of isoquinoline with O-(2,4-
dinitrophenyl)hydroxylamine (14). ATIQ hydrochloride
(16), iodide salt of MIQ+ (11), and MTIQ hydrochloride
(11) were prepared with reported procedures. Since
sufficient spectral data of iodide salt of AIQ+ and ATIQ
hydrochloride were not available, these are also pre-
sented in Experimental Procedures.
Cytotoxicity of AP TP a n d AP P + to P C12 Cells.
Cytotoxicity of APTP and APP+ to PC12 cells was
examined and compared to those of MPTP and MPP+.
The effect of the duration of treatment (24, 48, and 72 h)
on cytotoxicity was tested. As shown in Figure 1A,
treatment with APP+ for 24 h had almost no effect, while
after treatment for 48 h, there was a dose-dependent
cytotoxicity. Treatment for 72 h did not increase cyto-
toxicity, indicating that full cytotoxicity is obtained after
48 h. A similar tendency was observed with MPP+
(Figure 1B); however, MPP+ had a stronger cytotoxicity
than APP+. IC50 values of MPP+ and APP+ at 72 h were
41 and 124 µM, respectively. For APTP, treatment for
24 h showed no cytotoxicity and treatment for 48 h
showed only moderate cytotoxicity (Figure 1C). After 72
h, APTP showed strong cytotoxicity (IC50 ) 43 µM), which
was more than that of APP+ and almost the same as that
of MPP+. MPTP showed only slight cytotoxicity even
after 72 h (Figure 1D). The fact that APTP had strong
cytotoxicity (Figure 1C) whereas MPTP very weak cyto-
toxicity (Figure 1D) is an indication of a big difference
between the N-amino and N-methyl derivatives. PC12
cells primarily express the A but not the B type MAO
(19), and MPTP is not a good substrate for MAO-A
relative to MAO-B; thus, MPTP was not oxidized enough
to be cytotoxic to PC12 cells. On the other hand, APTP
was oxidized to APP+ by the MAO-A of PC12 cells and
F or m a tion of AP P + fr om AP TP . Homogenates of PC12
cells were prepared as follows. Cells (2 × 108) were collected
by centrifugation and washed with PBS. The cells were then
suspended in 4 mL of water and sonicated at 0 °C for 30 s
(Handy Sonic UR 20-P, Tomy Seiko Co., Tokyo, J apan). The
amount of protein of cell homogenates was determined by the
method of Bradford using bovine γ-globulin as a standard (18).
For the assay of APP+ formation, 200 µL of 4 mM potassium
phosphate buffer (pH 7.4) containing 0.38 or 1.25 mM APTP
and increasing amounts of PC12 cell homogenate (0.10, 0.19,
0.48, 0.71, or 0.95 mg of protein) were incubated at 37 °C for 1
h. After the addition of 25 µL of 1 M perchloric acid containing
1 mM EDTA and 1 mM sodium metabisulfate, the mixture was
centrifuged at 22000g for 10 min, and the supernatant was
passed through a Millipore filter. The amount of APP+ in the
filtrate was determined with an HPLC system composed of an
LC-9A pump and a spectrofluorometer (Shimadzu). A reversed-
phase Inertosil ODS-2 column (4.6 mm × 250 mm, GL Sciences,
Tokyo, J apan) was eluted at a flow rate of 1.0 mL/min with a
solution consisting of 90 mM sodium acetate/35 mM citric acid
buffer (pH 4.35), 130 µM EDTA, 100 µM 1-octanesulfonic acid
sodium salt, and 23% methanol. The fluorescence at 370 nm
was measured with excitation at 300 nm. For the experiments
with enzyme inhibitors, several concentrations (final concentra-
tion of 1, 10, and 100 µM) of clorgyline (MAO-A inhibitor) or
deprenyl (MAO-B inhibitor) were added to the mixture (200 µL)
of APTP (1.25 mM) and cell homogenate (0.48 mg of protein).
Resu lts a n d Discu ssion
Syn th eses of N-Am in o Der iva tives of P yr id in es
a n d Isoqu in olin es. Structures of the test compounds
employed in this study are shown in Table 1. Iodide salt
of APP+ was prepared by amination of 4-phenylpyridine
with hydroxylamine-O-sulfonic acid. Since sufficient
spectral data of iodide salt of APP+ were not available
(12), the results are presented in Experimental Proce-
dures. Reduction of iodide salt of APP+ with NaBH4
yielded APTP. Similarly, EPTP hydrochloride (13) was
prepared by reduction of iodide salt of EPP+ with NaBH4

1252 Chem. Res. Toxicol., Vol. 11, No. 11, 1998
Communications
than the latter, and this ratio was almost the same as
that for MPP+ versus APP+. Even with isoquinoline
derivatives, ATIQ was cytotoxic as well as the AIQ+ and
MIQ+, while MTIQ was not, as well as MPTP.
The mechanism proposed for the cytotoxicity of MPTP
suggests that the MPP+ formed reduces ATP synthesis
via inhibition of Complex I (9) or R-ketoglutarate dehy-
drogenase (20). Also, the isoquinolines, MTIQ and MIQ+,
were reported to inhibit Complex I (21), but they were
very weakly cytotoxic to PC 12 cells, as reported here and
previously by others (22). On the other hand, our novel
amino analogues of MPTP and MPP+ were found to be
much more cytotoxic than the isoquinolines. Although
we have not examined whether APTP and APP+ can
inhibit Complex I activity in mitochondria, the reduction
of ATP synthesis may be a mechanism for their cytotox-
icity. In addition to the reduction of ATP, the hydroxy
radical formation is proposed to be an another cytotoxic
mechanism of MPTP (23). The radical formation is
reported to result in induction of apoptosis in dopamin-
ergic cells (24, 25). Further investigation along this line
with APTP and APP+ is in progress.
F igu r e 2. Formation of APP+ from APTP in homogenates of
PC12 cells. Two hundred microliters of 4 mM potassium
phosphate buffer (pH 7.4) containing 0.38 or 1.25 mM APTP
and increasing amounts of PC12 cell homogenate (0.10-0.95
mg of protein) was incubated at 37 °C for 1 h. The amount of
APP+ formed was quantitated by HPLC with a fluorospectrom-
eter: 0.38 (b) and 1.25 mM APTP (O).
Ta ble 2. Effect of MAO In h ibitor s on th e F or m a tion of
AP P + fr om AP TP by P C12 Cell Hom ogen a tesa
In conclusion, APP+, the 1-amino analogue of neuro-
toxic MPP+, was cytotoxic to dopaminergic PC12 cells.
APTP, a precursor of APP+, was also cytotoxic to PC12
cells after it was oxidized to APP+. This was different
from the results with the corresponding 1-methyl ana-
logue, MPTP, which was not cytotoxic to PC12 cells. This
was due to the fact that the substrate specificity of MAO
is different for APTP; i.e., APTP can be oxidized by both
MAO-A and -B (26) while MPTP only by MAO-B.
amount of APP+
(pmol/60 min/mg protein)
inhibitor
yieldb
none
506 ( 8
529 ( 2
528 ( 65
403 ( 3
236 ( 5
238 ( 74
59 ( 2
100
105
104
80
47
47
deprenyl (1 µM)
deprenyl (10 µM)
deprenyl (100 µM)
clorgyline (1 µM)
clorgyline (10 µM)
clorgyline (100 µM)
12
a
Clorgyline (MAO-A inhibitor) or deprenyl (MAO-B inhibitor)
was added to the reaction mixture (200 µL) containing APTP (1.25
mM) and cell homogenates (0.48 mg of protein). The mixture was
incubated for 1 h, and the amount of APP+ was quantitated as
Ack n ow led gm en t. We thank Professor Emeritus Y.
Kawazoe of Nagoya City University for his encourage-
ment. We also thank Miss R. Itoh for the cytotoxicity
assays in the intial stage of this work.
b
described in the legend of Figure 2. Relative value.
showed strong cytotoxicity as described below.
Refer en ces
Oxid a t ion of AP TP t o AP P + b y Typ e A Mon o-
a m in e Oxid a se. To determine whether APTP is oxi-
dized to APP+ by MAO-A in PC12 cells, APTP was
incubated with a homogenate of PC12 cells, and the
amount of APP+ formed was quantitated using an HPLC
system equipped with a fluorospectrometer. As shown
in Figure 2, the formation of APP+ increased linearly with
increasing amounts of cell homogenates. In the presence
of the MAO-A inhibitor, clorgyline, the formation of APP+
was inhibited (Table 2), while in the presence of the
MAO-Binhibtor, deprenyl, no inhibiton was observed at
concentrations of 1 and 10 µM, although a slight inhibi-
tion was seen at 100 µM. These results strongly indicate
that APTP was oxidized to APP+ by MAO-A in PC12
cells. The marked cytotoxicity of APTP is due to the
efficient incorporation of APTP into the cells and its
oxidation to the active form APP+ by MAO-A. The fact
that APTP requires much more time than APP+ to show
its full cytotoxicity (Figure 1A,C) supports this idea.
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