Mechanisms of Ageing and Development
111 (1999) 175 – 188
www.elsevier.com/locate/mechagedev

Cell death of dopamine neurons in aging and
Parkinson’s disease
Makoto Naoi a,*, Wakako Maruyama b
a

Department of Brain Sciences, Institute of Applied Biochemistry, Yagi Memorial Park, Mitake,
Gifu 505 -0116, Japan
b
Laboratory of Biochemistry and Metabolism, Department of Basic Gerontology,
National Institute for Longe6ity Sciences, Obu, Aichi, Japan
Received 6 April 1999; received in revised form 7 June 1999; accepted 10 June 1999

Abstract
Dopamine neurons in the substantia nigra of human brain are selectively vulnerable and
the number decline by aging at 5–10% per decade. Enzymatic and non-enzymatic oxidation
of dopamine generates reactive oxygen species, which induces apoptotic cell death in
dopamine neurons. Parkinson’s disease (PD) is also caused by selective cell death of
dopamine neurons in this brain region. The pathogenesis of Parkinson’s disease remains to
be an enigma, but it was found that an endogenous MPTP-like neurotoxin, 1(R), 2(N)dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline [N-methyl(R)salsolinol, NM(R)Sal],
may be one of the pathogenic agents of PD. NM(R)Sal increases in cerebrospinal ﬂuid from
untreated parkinsonian patients, and two enzymes, a (R)salsolinol synthase and a neutral
N-methyltransferase, synthesize this neurotoxin in the nigro-striatum. The activity of a
neutral N-methyltransferase is signiﬁcantly higher in lymphocytes from parkinsonian patients than in control. The mechanism of cell death by this toxin was proved to be by the
induction of apoptosis, by use of dopaminergic SH-SY5Y cells. The apoptosis was suppressed by anti-oxidants, suggesting that the generation of reactive oxygen species may
initiate cellular death process. These results indicate that in aging and PD oxidative stress
induces degeneration of dopamine neurons, and the antioxidant therapy may delay the
decline of dopamine neurons in the brain. © 1999 Elsevier Science Ireland Ltd. All rights
reserved.
Keywords: Aging; Parkinson’s disease; N-Methyl(R)salsolinol; (R)Salsolinol N-methyltransferase; Oxidative stress
* Corresponding author. Tel.: +81-574-67-5500; fax: +81-574-67-5310.
E-mail address: appbio@mbf.sphere.ne.jp (M. Naoi)
0047-6374/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 0 4 7 - 6 3 7 4 ( 9 9 ) 0 0 0 6 4 - 0

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1. Introduction
Dopamine neurons in the substantia nigra of human brains are known to be
selectively vulnerable and neuronal loss with advancing age was estimated to be
more than one third between the age of 20 and 90 years (McGeer et al., 1988).
There is a linear fallout of dopamine neurons with aging at a rate of 5–10% per
decade (Fearnley and Lees, 1991), and the limited number of the cells causes
dysfunction in cognition and motor movement. The vulnerability of the dopamine
neurons is considered to be due to the oxidative stress caused by increased
generation of reactive oxygen species (ROS) and reduced capacity of anti-oxidant
system. The enzymatic oxidation of dopamine generates hydrogen peroxide, which
yields more cytotoxic hydroxyl radicals in the presence of iron (II), which is rich in
the substantia nigra (Youdim, 1988). Non-enzymatic oxidation of dopamine produces superoxide, which reacts with nitric oxide to produce peroxynitrite, a most
stable and potent cytotoxin, and a quinone, which binds to thiol groups and
denatures biologically active protein. Another metabolite of dopamine is 6-hydroxydopamine, a dopaminergic neurotoxin (Seiden and Vosmer, 1984). In these ways,
the evidences to connect the oxidative stress to the deterioration of dopamine
neurons have been accumulated. 8-Hydroxy-2%-deoxyguanosine is elevated in the
substantia nigra (Sanchez-Ramos et al., 1994), and carbonyl levels of protein, an
indicator of protein oxidation, increase in tyrosine hydroxylase, a rate-limiting
enzyme of dopamine synthesis with aging (De La Cruz et al., 1996). In the basal
ganglia and substantia nigra mutation and deletion of mitochondrial DNA are
more abundantly induced by oxidative stress than in cortex and they increase with
aging (Corral-Debrinski et al., 1992).
Similarities have been drawn between senility and Parkinson’s disease (PD) based
on the similar depletion of dopamine neurons and clinical features (Teravainen and
Calne, 1983). On the other hand, aging has been considered to play a role in the
pathogenesis of PD. In PD the clinical signs are detected when 50% of nigral
neurons and 80% of striatal dopamine are lost (Marsden, 1990), and the velocity
and the intensity of the neuronal loss are more marked than those during
physiological aging. Therefore, PD was once proposed to be a form of accelerated
aging (Mann and Yates, 1983). However, it is now considered that other pathological process is involved in the pathogenesis of PD (Kish et al., 1992), because the
depletion of dopamine neurons in the substantia nigra is different in aging and PD
(Hassler, 1938). There has been an increasing body of evidences to indicate the
involvement of neurotoxins to the deterioration of nigro-striatal dopamine system.
An endogenous neurotoxin, 1(R), 2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline [Nmethyl(R)salsolinol, NM(R)Sal], was found to induce parkinsonism in rats (Naoi et al., 1996a). The biochemical, pathological and behavioral
features were proved to be appropriate for an animal model of PD.
The enantio-selective occurrence of NM(R)Sal in the human brain (Maruyama,
et al., 1997a), cerebrospinal ﬂuid (CSF) (Maruyama et al., 1996a) and intraventricular ﬂuid (Maruyama et al., 1996b) was found. These results suggest that it should
be synthesized enzymatically in the brain, and actually NM(R)Sal is synthesized by

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177

two step enzyme reactions from dopamine, as shown in Fig. 1. (R)Sal is
synthesized from dopamine and acetaldehyde by a (R)salsolinol synthase (Naoi et
al., 1996b) and NM(R)Sal by an N-methyltransferase (Maruyama et al., 1992; Naoi
et al., 1997a). The selective occurrence of NM(R)Sal in the nigro-striatum
(Maruyama et al., 1996a) is considered to be due to high activity of N-methyltransferase in this brain region (Maruyama et al., 1992). NM(R)Sal is oxidized into
1,2-dimethyl-6,7-dihydroxyisoquinolinium ion (DMDHIQ+) by enzymatic (Naoi et
al., 1995) or non-enzymatic oxidation (Maruyama et al., 1995a,b), that simultaneously generates hydroxyl radicals. The potent cytotoxicity of DMDHIQ+ was
proved to be ascribed to the reduction of ATP synthesis via mitochondrial
respiratory enzymes (Takahashi et al., 1997; Morikawa et al., 1998).
To clarify whether this NM(R)Sal is involved in the pathogenesis of PD,
NM(R)Sal and the enzymes relating its metabolism were analyzed in clinical
samples from parkinsonian patients. This paper describes the increase in NM(R)Sal
level in the CSF from parkinsonian patients (Maruyama et al., 1996a) and changes
in the enzymes related to its metabolism in PD (Naoi et al., 1998a). Finally the cell
death of dopamine neurons by NM(R)Sal proved to be induced by the apoptotic
death process, as shown by use of a single cell gel electrophoresis (Comet) assay and
human dopaminergic neuroblastoma SH-SYSY cells (Maruyama et al., 1997b,c).
The oxidation of NM(R)Sal was conﬁrmed to generate hydroxyl radicals and
induce apoptosis in the cells. These results are discussed in relation to the selective
depletion of dopamine neurons in the substantia nigra in PD, compared to that
during advancing aging.

Fig. 1. The metabolic pathway of 1(R), 2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline
[N-methyl(R)salsolinol, NM(R)Sal] in the human brain. (R)Sal is synthesized from dopamine by a
synthase and N-methylated into NM(R)Sal by a methyltransferase. The oxidation of NM(R)Sal is
non-enzymatic and enzymatic by an oxidase sensitive to semicarbazide, but not by moncamine oxidase.

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2. Materials and methods

2.1. Chemicals
NM(R)Sal and the derivatives were synthesized according to Teitel et al. (1972).
Cycloheximide, ethidinium bromide, retinoic acid, butylated hydroxyanisole,
(− ) deprenyl and superoxide dismutase (SOD) were purchased from Sigma (St.
Louis, MO); a-tocopherol, catalase, agarose (low melting-temperature), reduced
glutathione (GSH), n-propyl gallate, a-tocopherol, mannitol, N-acetylcysteine and
other reagents were purchased from Nacalai Tesque (Kyoto, Japan).

2.2. Subjects for analysis of CSF and methods
The lumbar CSF samples from 16 patients with newly diagnosed and untreated
PD and from 29 control subjects without neurological disorders were used for the
analysis. As a disease control, CSF from ﬁve patients with multiple system atrophy
(MSA) was analyzed. All the patients were fully informed on the risks and potential
beneﬁts of the CSF examination. The Ethical Committee of Iwate Medical University approved the protocol of this study.
Enantiomers of Sal and NMSal, dopamine and homovanillic acid (HVA) were
analyzed by high-performance liquid chromatography (HPLC) as reported previously (Maruyama, et al., 1997d).

2.3. Analysis of (R)Sal and NM(R)Sal deri6ati6es in human brain
Ten control human brains without neuropsychiatric disorders were obtained and
stored at −80°C until analysis. Four brain regions, frontal cortex, caudate,
putamen and substantia nigra, were punched out and the content of dopamine, the
(R)- and (S)-enantiomers of Sal and NMSal, and the isoquinolinium ion were
analyzed. The enantiomeric separation of (R)- and (S)-Sal and NMSal was
performed using HPLC-electrochemical detection (ECD) with b-cyclodextrinbound column as reported (Maruyama et al., 1996a). 1,2-DMDHIQ+ was
quantiﬁed by HPLC with ﬂuorometric detection (Naoi et al., 1995). Differences
between two groups were compared by the Mann–Whitney U test, and between
three or more groups by two way analysis of variance (ANOVA). A P value less
than 0.05 was regarded as statistically signiﬁcant. Correlation was evaluated by
Pearson product-moment correlation coefﬁcients.

2.4. Analysis of N-methyltransferase acti6ity in lymphocytes
The activity of an N-methyltransferase which catalysis N-methylation of (R)Sal
was analyzed in human brain samples (Naoi et al., 1997a) and lymphocytes (Naoi
et al., 1998a), using (R)Sal as a substrate and S-adenosyl-L-methionine as a methyl
donor (Naoi et al., 1997a). Lymphocytes prepared from 56 idiopathic parkinsonian
patients and 24 control patients were used for the measurement of the enzyme

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179

activity. The Ethical Committee of Aichi Medical University approved the protocols for the examination of lymphocytes, and the patients were fully informed about
the risks and the beneﬁts of the examination. The activity of (R)Sal N-methyltransferases with the optimal pH at alkaline and neutral, and NM(R)Sal oxidase was
analyzed as reported (Naoi et al., 1998a).

2.5. Single cell gel electrophoresis (comet) assay
SH-SY5Y cells incubated with catechol isoquinolines were subjected to the comet
8
assay as reported previously (Ostling and Hohanson, 1984; Kasamatsu et al., 1996;
Maruyama et al., 1997b). Two hundred comet images were randomly selected, and
the tail length (length of DNA tail from the trailing edge of nucleus) and the comet
length (nucleus plus migrated DNA tail) were measured on a video camera screen
connected to a ﬂuorescence microscope. To evaluate DNA damage, the cells with
the comet length longer than 30 mm were classiﬁed to be positive for DNA damage.
The number of DNA-damaged cells was expressed as percentages of the total, and
also the tail length was compared. Statistical signiﬁcance was assessed by ANOVA
for the tail length. All differences with PB 0.05 were considered to be statistically
signiﬁcant.

3. Results

3.1. Analysis of NM(R)Sal in the CSF from parkinsonian patients
Only the (R)-enantiomer of NMSal was detected in CSF from control, patients
with PD and MSA, and the level of (S)-enantiomer was below the detection limit
(B0.01 nM). The concentration in the control group was not affected by the age
from 22 to 76 years (r =0.141) or by the sex [male; 4.39 9 1.73 nM, female;
4.899 2.79 nM (mean 9S.D.)]. In most of CSF from control and patients with
MSA the NM(R)Sal level was lower than 6 nM. On the other hand, in 12 PD
patients out of 16 the level was higher than 6 nM. NM(R)Sal level in the PD
patients was signiﬁcantly higher than that either in control (PB 0.0001) or patients
with MSA (P B0.0022) (Table 1). The concentration of (R)Sal and free dopamine,
the metabolic precursors of NM(R)Sal, were under 0.1 nM in CSF. To estimate the
biosynthesis rate of this isoquinoline from dopamine, the concentration of
NM(R)Sal was compared with that of HVA, a major metabolise of dopamine. The
ratio also signiﬁcantly increased in PD patients compared with control (PB 0.0002)
or MSA patients (P B 0.0455). The HVA level was reduced in the CSF of PD and
MSA, but the difference from control was not statistically signiﬁcant. These results
suggest that NM(R)Sal level in CSF and probably also in the brain may be
determined by the activity of enzymes related to its synthesis and metabolism.

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Table 1
Analysis of cerebrospinal ﬂuid (CSF) prepared from control and patients with Parkinson’s disease
(PD) or multiple system atrophy (MSA)a
PD patients
(n= 16)
NM(R)Sal (nM)
HVA (nM)
NM(R)Sal/HVA

Control
(n =29)

MSA patients
(n =5)

8.32 92.89*
(4.54–15.7)
141 975
(17.4–259.8)
0.0869 0.067**
(0.028–0.261)

4.53 9 2.08
(0.62–11.9)
180 9 92
(57.6–422.0)
0.034 9 0.030
(0.012–0.162)

3.59 91.52
(1.33–5.25)
156 975
(74.6–259.0)
0.034 90.029
(0.017–0.070

a

The concentration of Sal, NMSal, dopamine and homovanillic acid (HVA) were analyzed by
high-performance liquid chromatography (HPLC)-electrochemical detection (ECD) and (R)- and (S)enantiomer of Sal and NMSal were quantitatively determined with a cyclodextrin-bonded chiral column.
The values represent mean 9S.D. Range of the value is shown in parenthesis. The values of three groups
were analyzed for signiﬁcant difference nonparametrically by the Kruskal–Wallis test and the difference
between two groups was examined by the Mann–Whitney U test separately. n =number of subjects.
* PB0.001 compared with control and MSA patients.
** PB0.001 compared with control and PB0.05 compared with MSA patients.

3.2. Analysis of salsolinol deri6ati6es in human brain
From control human brains (n= 10), four brain regions, frontal cortex, caudate,
putamen and substantia nigra, were analyzed for NMSal and related compounds.
In human brain only the (R)-enantiomer of Sal and NMSal were detected. The
distribution of (R)Sal, dopamine, NM(R)Sal and the isoquinolinium ion was
shown in Fig. 2. (R)Sal occurs in the brain non-selectively, whereas NM(R)Sal
accumulates in the nigro-striatal system and the isoquinolinium ion is detected only
in the substantia nigra. There was no correlation of the contents of salsolinol
derivatives with the age. These results suggest the in situ enantioselective synthesis
of (R)Sal in the brain. The high activity of a N-methyltransferase in dopamine
neurons may account for the accumulation of NM(R)Sal in the nigro-striatum, as
shown by use of in vivo microdialysis in rat brain (Maruyama et al., 1992). The
accumulation of DMDHIQ+ in the substantia nigra may be due to the binding of
the isoquinolinium ion to neuromelanin, as proved by in vitro (Naoi et al., 1994)
and in vivo experiments (Naoi et al., 1996a).

3.3. Relationship of the NM(R)Sal concentration and the acti6ity of the
synthesizing enzymes in the brain regions
The activity of a (R)Sal synthase, a neutral and alkaline (R)Sal N-methyltransferase, and NM(R)Sal oxidase were analyzed in the brain sample prepared from the
frontal cortex, caudate and putamen (Naoi et al., 1997a). The strong positive
correlation was found between the activity of a neutral (R)Sal N-methyltransferase
in the caudate-putamen and the content of DMDHIQ+ in the substantia nigra

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181

(Fig. 3). Neither the activity of other enzymes nor that of a neutral N-methyltransferase in other brain regions correlated with the level of any NMSal derivatives.
The activities of these enzymes were not affected by the age in any brain regions.
The results suggest that NM(R)Sal synthesized in the striatum is transported by
retrograde axonal ﬂow to the substantia nigra, and oxidized there or on the way to
produce the isoquinolinium ion.

3.4. Analysis of the enzyme acti6ities related to the NM(R)Sal metabolism in
parkinsonian lymphocytes
Lymphocytes from parkinsonian patients and control were analyzed for the
activity of a (R)Sal N-methyltransferase and an NM(R)Sal oxidase (Naoi et al.,

Fig. 2. The distribution of 1(R), 2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline [Nmethyl(R)salsolinol, NM(R)Sal] and related compounds in brain regions. From ten control brains
without history of neurological and psychiatric disorders, frontal lobe, caudate, putamen and substantia
nigra were punched out. Dopamine, (R)Sal, NM(R)Sal were quantitatively analyzed by high-performance liquid chromatography (HPLC)-multi-electrochemical detection (ECD) with a chiral column.
DMDHIQ+ was analyzed by HPLC-ﬂuorometric detection; *P\ 0.01.

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M. Naoi, W. Maruyama / Mechanisms of Ageing and De6elopment 111 (1999) 175–188

Fig. 3. Correlation of DMDHIQ+ content in the substantia nigra with the activity of a neutral
N-methyltransferase in the caudate. DMDHIQ+ was measured with high-performance liquid chromatography (HPLC)-ﬂuoriometric detection and the activity of an N-methyltransferase by HPLC-electrochemical detection (ECD) as described in the Section 2. PB 0.001.

1998a). In lymphocytes the activity of N-methyltransferase showed the two peaks
around pH 7.0 and 8.0. In control samples, the enzyme activity at pH 7.0 and 8.0
were almost the same values. In lymphocytes from parkinsonian patients the
activity of a neutral N-methyltransferase measured at pH 7.0 was signiﬁcantly
higher than that in control, whereas the activity of an alkaline N-methyltransferase
assayed as pH 8.0 was almost the same as that of control.
The levels of the neutral (R)Sal N-methyltransferase activity were measured in
the lymphocytes from 24 control subjects and 56 parkinsonian patients, and the
results are summarized in Table 2. The activity in parkinsonian lymphocytes was
signiﬁcantly higher than in the controls: 100.29 81.8 and 18.99 15.0 pmol/min per
mg protein, respectively [mean9S.D.]. This difference is statistically signiﬁcant;
P= 0.0001. The activity of a (R)Sal synthase was not detectable in the lymphocyte
sample. The substrate speciﬁcity of a neutral N-methyltransferase was restricted to
(R)Sal, whereas (S)Sal was not a substrate of this enzyme. (R)Sal synthase activity
was not detected in lymphocytes. As summarized in Table 2, the activity of an
alkaline (R)Sal N-methyltransferase measured at pH 8.0 and that of NM(R)Sal
oxidase were the same in the control and parkinsonian lymphocytes. The age and
sex did not affect the activity of these enzymes in the lymphocytes. Considering that
the activity of this enzyme in the striatum determines the level of cytotoxic

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183

DMDHIQ+ in the substantia nigra (Naoi et al., 1997a), the activity may increase
in the caudate-putamen of parkinsonian brain, resulting in the increase of the
neurotoxin in the substantia nigra.

3.5. Induction of apoptosis by NM(R)Sal in dopaminergic SH-SY5Y cells
In the SH-SY5Y cells incubated with NM(R)Sal, DNA took the form of a ‘head’
and a migrated ‘tail’ composed of DNA fragmented in smaller size, whereas control
did not show such comet image. The migration distance of DNA, from the comet
head to the tip of the tail, was signiﬁcantly longer in the NM(R)Sal-treated cells
than in control. Fig. 4 shows the histogram of the migration distance of DNA in
the cells treated with (R)- and (S)-enantiomers of NMSal. The mean head-tail
distances of control and cells incubated with (R)- and (S)Sal or DMDHIQ+ were
distributed between 10 and 25 mm (mean 9S.D., 11.2 9 0.02 mm). This is consistent
with intact nuclei with undamaged DNA, whereas those of the cells incubated with
NM(R)Sal were larger than 45 mm and did not overlap with that of the former cell
groups. The tail-length of the NM(S)Sal-treated cells was rather variable giving
values in the range 10 – 45 mm. The tail-length of 45 mm or longer were taken to
indicate extensive apoptosis. An inhibitor of protein synthesis, cycloheximide, and
an inhibitor of RNA synthesis, actinomycin D, suppressed DNA damage by
NM(R)Sal, suggesting that the DNA damage induced by NM(R)Sal is apoptotic.
Anti-oxidants and anti-oxidative enzymes suppressed the DNA damage (Table
3). Pretreatment with catalase, reduced glutathione, (− )deprenyl or semicarbazide
protected the cells from the DNA damage. The effects of radical scavengers on
DNA damage by NM(R)Sal were examined using differentiated SH-SY5Y cells.
The pre-incubation of the cells with mannitol, N-acetylcysteine, n-propyl gallate,
Tris and butylated hydroxyanisole suppressed the DNA damage, but a-tocopherol
did not. These results suggest that hydroxyl radicals are the major reactive oxygen
species to induce apoptosis in the cells.
Table 2
Activity of a neutral N-methyltransferase and related enzymes in lymphocytes
Enzyme activity in lymphocytes prepared from
Parkinsonian patients
(n= 56)

Control
(n =24)

Neutral N-methyltransferase* (pmol/min per mg protein)
100.2981.8

18.9 915.0

Alkaline N-methyltransferase* (pmol/min per mg protein)
41.8 917.3

25.0 923.0

N-Methyl(R)salsolinol oxidase (pmol/min per mg protein)
2.15 92.43

1.38 9 2.23

* The activities of neutral and alkaline (R)Sal N-methyltransferase were measured at pH 7.0 and 8.0,
respectively. The value represents the mean 9 S.D.

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Fig. 4. Frequency distribution of the DNA migration distance in SH-SY5Y cells incubated with the (R)and (S)-enantiomer of NMSal. SH-SY5Y cells were incubated with or without 1 mM NMSal at 37°C
for 3 h, then subjected to a Comet assay. The migration distance was measured as described in the text.
The distribution of the cells with a DNA image with a given migration distance was expressed as the
percentages of the total 200 cells. Each column represents the mean value of four experiments.

4. Discussion
At present, the genetic marker of sporadic form of PD remains to be identiﬁed,
whereas that for familiar autosomal dominant parkinsonism was identiﬁed as the
mutation of a-synuclein gene (Polymeropoulos et al., 1997). More recently in
autosomal recessive juvenile parkinsonism, another mutation was identiﬁed in the
parkin gene (Kitada et al., 1998). However, the relation of these mutations to the
pathogenesis of the sporadic form of PD is an enigma. As described here,
neurotoxins may be one of the pathogenic factors in PD.
The selective neurotoxicity of NM(R)Sal to dopamine neurons is closely related
with its biosynthesis pathway in the brain (Maruyama and Naoi, 1998; Naoi et al.,
1997b, 1998b). The localization of the precursor dopamine and especially the
activity of a neutral N-methyltransferase in the striatum seem to determine the
speciﬁed localization of NM(R)Sal in the nigro-striatum, and an oxidation product,
DMDHIQ+ ion in the substantia nigra (Naoi et al., 1997a). The increase of the
activity of a neutral N-methyltransferase in the nigro-striatum of parkinsonian
brain is suggested by the increase in parkinsonian lymphocytes (Naoi et al., 1998a).

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185

The activity was not affected by the age, suggesting that in PD dopamine neurons
may be degenerated by a mechanism different from that in aging. At present the
mechanism of the increase remains to be clariﬁed, and genetic and environmental
factors should be involved in the change in the activity. The puriﬁcation and
characterization of the enzyme and the isolation of its cDNA will bring a new
development to understand the pathogenesis of PD.
The cell death by this neurotoxin is induced by intracellular apoptotic death
process. It is relevant with the clinical observation that apoptosis was detected in
the dopamine neurons in the substantia nigra of parkinsonian brains (Mochizuki et
al., 1996; Anglade et al., 1997). The generated hydroxyl radical from NM(R)Sal
(Maruyama et al., 1995a,b) seems to be essential to initiate the death program.
Deterioration of dopaminergic neurons in either PD or physiological aging is
hypothesized to involve oxidative stress (Jenner and Olanow, 1996). Even though
different ROS may be generated either by the oxidation of dopamine in aging or of
neurotoxins in PD, the oxidative stress is considered to be involved in apoptotic
death process as the ﬁnal inducer. These results suggest that anti-oxidative therapy
may protect the dopamine neurons from apoptosis and may delay the onset of the
dysfunction of extrapyramidal system in aging and age-related neurodegenerative
diseases.
Table 3
Effect of radical scavengers on DNA damage induced by NM(R)Sala
SH-SY5Y cells treated with

No. DNA damaged cells (%)

Control
NM(R)Sal (0.2 mM)

7.5 9 1.0*
29.0 9 8.24

Pretreated with
+mannitol (10 mM)
+N-acetylcysteine (500 mM)
+n-propyl gallate (5 mM)
+tocopherol (250 mM)
+Tris (10 mM)
+butylated hydroxyanisole (20 mM)

13.1 9 9.90*
7.3 9 4.24*
8.19 8.49*
21.0 9 12.7
6.9 9 1.41*
6.2 9 1.88*

a
Differentiated SH-SY5Y cells were treated with each radical scavenger for 20 min, and then with 0.2
mM NM(R)Sal for 3 h. The cells with the comet length longer than 25 mm were determined as DNA
damaged cells. Each value represents mean 9 S.D. of three independent experiments.
* PB0.05 compared to cells treated with NM(R)Sal alone by analysis of variance (ANOVA).

Acknowledgements
This work was supported by a Grant-in-Aid for Scientiﬁc Research on Priority
Area and for Exploratory Research from the Ministry of Education, Science and
Education, Japan, (M. N.), and the Grant for Longevity Sciences from the Ministry
of Health and Welfare, Japan (9C-03 for M.N. and 9C-02 for W.M.) and from

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Foundation for Comprehensive Research on Aging and Health of Japan (W.M.
and M.N.)

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