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 - 0 176 M. Naoi, W. Maruyama / Mechanisms of Ageing and De6elopment 111 (1999) 175–188 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 M. Naoi, W. Maruyama / Mechanisms of Ageing and De6elopment 111 (1999) 175–188 177 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. 178 M. Naoi, W. Maruyama / Mechanisms of Ageing and De6elopment 111 (1999) 175–188 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 M. Naoi, W. Maruyama / Mechanisms of Ageing and De6elopment 111 (1999) 175–188 179 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. 180 M. Naoi, W. Maruyama / Mechanisms of Ageing and De6elopment 111 (1999) 175–188 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 M. Naoi, W. Maruyama / Mechanisms of Ageing and De6elopment 111 (1999) 175–188 181 (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. 182 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 M. Naoi, W. Maruyama / Mechanisms of Ageing and De6elopment 111 (1999) 175–188 183 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. 184 M. Naoi, W. Maruyama / Mechanisms of Ageing and De6elopment 111 (1999) 175–188 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). M. Naoi, W. Maruyama / Mechanisms of Ageing and De6elopment 111 (1999) 175–188 185 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. 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