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Keywords:

  • Manganese;
  • L-DOPA;
  • Dopamine;
  • Autoxidation;
  • Apoptosis;
  • PC12 cells

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Assay of DA and its metabolites in cell lysate and incubation medium by HPLC with electrochemical detection
  5. RESULTS
  6. PC12 cell viability is greatly reduced when L-DOPA is associated with manganese
  7. L-DOPA + manganese cytotoxicity is reduced by antioxidants NAC and AA
  8. DA formation and metabolism are impaired when manganese is associated with L-DOPA : NAC protects
  9. l-DOPA-induced apoptosis is potentiated by manganese association : NAC protects
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

Abstract : L-DOPA and manganese both induce oxidative stress-mediated apoptosis in catecholaminergic PC12 cells. In this study, exposure of PC12 cells to 0.2 mM MnCl2 or 10-20 μM L-DOPA neither affected cell viability, determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, nor induced apoptosis, tested by flow cytometry, fluorescence microscopy, and the TUNEL technique. L-DOPA (50 μM) induced decreases in both cell viability and apoptosis. When 0.2 mM MnCl2 was associated with 10, 20, or 50 μM L-DOPA, a concentration-dependent decrease in cell viability was observed. Apoptotic cell death also occurred. In addition, manganese inhibited L-DOPA effects on dopamine (DA) metabolism (i.e., increases in DA and its acidic metabolite levels in both cell lysate and incubation medium). The antioxidant N-acetyl-L-cysteine significantly inhibited decreases in cell viability, apoptosis, and changes in DA metabolism induced by the manganese association with L-DOPA. An increase in autoxidation of L-DOPA and of newly formed DA is suggested as a mechanism of manganese action. These data show that agents that induce oxidative stress-mediated apoptosis in catecholaminergic cells may act synergistically.

Oxidative stress has been widely believed to be an important pathogenetic mechanism of neuronal death in Parkinson’s disease (PD) (Halliwell, 1992), although it is still not clear whether it is an initial event causing cell death or a consequence of the disease (Jellinger, 1999). L-DOPA is to date the drug of choice in PD therapy, and its use is cause for concern as L-DOPA can undergo autoxidation and enzymatic oxidation, generating reactive oxygen species (ROS), which may further load the preexisting condition of oxidative stress (Basma et al., 1995 ; Spencer et al., 1995). The latter has been suggested also as a mediator of apoptosis, because many of the chemical and physical treatments capable of inducing apoptosis are also known to induce oxidative stress (Sandstrom et al., 1994). Both dopamine (DA) and L-DOPA have been shown to induce oxidative stress-mediated apoptosis in cultured neuronal cells, and inappropriate DA-and/or L-DOPA-induced activation of apoptosis might have a role in neuronal death in PD (Ziv et al., 1994, 1997 ; Walkinshaw and Waters, 1995 ; Hastings et al., 1996). Apoptotic degeneration of nigral dopaminergic neurons in PD is still a controversial issue : The death of dopaminergic neurons has been reported to occur by apoptosis (Mochizuki et al., 1996). Anglade et al. (1997) claimed that even at the final stage of the disease, the dopaminergic neurons undergo the active process of cell death, whereas Banati et al. (1998) failed to detect apoptosis in the substantia nigra of PD patients.

Chronic manganese intoxication in humans can induce an early psychotic disorder that is later followed by permanent degenerative damage in the nigrostriatal system, resulting in a PD-like syndrome (Donaldson, 1987). Manganese may stimulate DA autoxidation within the dopaminergic neuron, a process accompanied by an increase in the formation of quinones (Graham, 1984 ; Florence and Stauber, 1989 ; Shen and Dryhurst, 1998) and protein-bound cysteinyl DA and cysteinyl dihydroxyphenylacetic acid (DOPAC) (Hastings et al., 1996). Oxidative stress has been proposed to be responsible for manganese-induced neuronal damage (Sun et al., 1993). Consistently, it has been hypothesized that a diminution of cellular protective mechanisms, such as glutathione levels, glutathione peroxidase activity (Liccione and Maines, 1988 ; Desole et al., 1997a ; Rabinovich and Hastings, 1998), and ascorbic acid (AA) levels (Desole et al., 1995 ; Hastings et al., 1996), might play a permissive role in manganese neurotoxicity. We first reported that manganese induces apoptosis in PC12 cells ; apoptosis was inhibited by the antioxidants N-acetyl-L-cysteine (NAC) and AA (Desole et al., 1997b). Hirata et al. (1998) confirmed that manganese induces apoptosis in PC12 cells and demonstrated that apoptosis is transcription dependent with an impairment of mitochondrial function and is blocked in Bcl-2-overexpressed cells.

It has been shown (Parenti et al., 1986) that manganese neurotoxicity in rats (assessed by decrease in striatal DA content) is worsened by L-DOPA treatment. To see whether manganese could in turn worsen L-DOPA neurotoxicity, we investigated the effects of exposure of PC12 cells to increasing concentrations of L-DOPA alone or associated with a subtoxic manganese concentration (i.e., unable to induce impairment of DA metabolism, decrease in cell viability, and apoptosis). The results indicate that manganese enhances L-DOPA cytotoxic and apoptotic effects on PC12 cells.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Assay of DA and its metabolites in cell lysate and incubation medium by HPLC with electrochemical detection
  5. RESULTS
  6. PC12 cell viability is greatly reduced when L-DOPA is associated with manganese
  7. L-DOPA + manganese cytotoxicity is reduced by antioxidants NAC and AA
  8. DA formation and metabolism are impaired when manganese is associated with L-DOPA : NAC protects
  9. l-DOPA-induced apoptosis is potentiated by manganese association : NAC protects
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

Sources of compounds

L-DOPA and NAC were purchased from Sigma-Aldrich (Milan, Italy), AA from Fisher Scientific (Fair Lawn, NJ, U.S.A.), and manganese chloride (MnCl2) from Merck (Darmstadt, Germany).

Cell culture and drug concentrations

All experiments were performed on PC12 cells during their exponential phase of growth. Cells were maintained in 100-cm2 plastic tissue culture dishes in an atmosphere of 5% CO2/95% air in Dulbecco’s modified Eagle’s medium containing 10% horse serum and 5% calf serum. Cells were exposed to increasing concentrations of L-DOPA (10, 20, 50, 100 μM) alone or associated with a fixed MnCl2 concentration (0.2 mM) for 8-24 or 48 h. NAC and AA concentrations were chosen on the basis of previous work (Desole et al., 1997a,b). For each experiment, 140 × 103 PC12 cells/cm2 were plated and treated 24 h later (time 0). Experiments were done in triplicate.

Assessment of cell viability

Cells were processed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after 24-48 h of incubation. In the MTT assay, viable cells convert the soluble dye MTT to insoluble (in aqueous medium) blue formazan crystals. At the start of each experiment and at selected intervals thereafter, cell number was determined in triplicate wells. In brief, 1 mg of MTT [200 μl of a 5-mg/ml stock solution in phosphate-buffered saline (PBS)] was added per milliliter of medium, and the cultures were allowed to incubate at 37°C for 4 h. The MTT was removed, and the cells were rinsed with PBS and centrifuged at 4,000 rpm for 20 min. Thereafter, the supernatant was discarded, and the pellet was dissolved in 2 ml of isopropanol ; after centrifugation at 4,000 rpm for 5 min, the color was read at 600 nm using a Bauty Diagnostic microplate reader. A standard curve was constructed utilizing different concentrations of cells at the start of every experiment. In preliminary experiments, neither 0.1 mM NAC nor 0.1 mM AA had interference with the MTT assay.

Assay of DA and its metabolites in cell lysate and incubation medium by HPLC with electrochemical detection

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Assay of DA and its metabolites in cell lysate and incubation medium by HPLC with electrochemical detection
  5. RESULTS
  6. PC12 cell viability is greatly reduced when L-DOPA is associated with manganese
  7. L-DOPA + manganese cytotoxicity is reduced by antioxidants NAC and AA
  8. DA formation and metabolism are impaired when manganese is associated with L-DOPA : NAC protects
  9. l-DOPA-induced apoptosis is potentiated by manganese association : NAC protects
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

DA, DOPAC, and homovanillic acid (HVA) were determined in the cell lysate and incubation medium by HPLC with electrochemical detection according to the method previously described (Desole et al., 1997a). In brief, cells were lysed in 1% metaphosphoric acid containing 1 mM EDTA. After centrifugation (17,500 g for 10 min at 4°C), the supernatant was filtered, and a 10-μl aliquot was immediately injected into the HPLC system for DA, DOPAC, and HVA determinations. The assay was done with a high-pressure Varian 9001 pump with a Rheodyne injector, 15 cm × 4.6 mm i.d. column (TSK-ODS-80 TM), electrochemical detector (BAS LC-4B), and integrator (Spectra-Physics SP 4290). The mobile phase was 0.1 M citric acid, 0.1 M K2HPO4, 1 mM EDTA, 5% methanol, and 70 mg/L sodium octylsulfate (pH 3.0) ; the flow rate was 1.2 ml/min.

Neurochemical values in cell lysate were expressed as nanomoles per milligram of protein. Tissue protein concentration was determined using the method of Lowry et al. (1951).

Apoptosis

Apoptosis was determined by the following methods.

Terminal deoxynucleotidyl transferase (TdT)-mediated 2′-deoxyuridine-5′-triphosphate (dUTP) nick end-labeling (TUNEL) technique. This technique permits the specific labeling of the 3-′OH of DNA breaks with modified nucleotides by TdT. For nicked DNA end-labeling experiments, cells were plated at a density of 70 × 103 cells/cm2 on coverslips (100 × 20 mm). After washing with PBS, the cultures were fixed in 4% buffered paraformaldeyde (pH 7.4), permeabilized with 0.1% Triton X-100, and incubated with fluorescein-dUTP and TdT. Thereafter, cells were stained by an antifluorescein antibody conjugated with horseradish peroxidase. Subsequently, cells were incubated with diaminobenzidine substrate to produce a dark brown precipitate. Stained cells were analyzed under light microscopy.

Flow cytometry analysis. Cells were trypsinized, harvested by centrifugation, and washed in PBS ; thereafter, cells were fixed in 70% ethanol and stored at -20°C prior to analysis. Before staining with 4′,6-diamidine-2-phenylindole (DAPI ; 3 μg/ml), fixed cells were again centrifuged and rehydrated in Tris-HCl buffer (0.1 M Tris, 0.1 M NaCl, pH 7.5). The cellular DNA content was measured using a Partec PAS II flow cytometer. Calculation of the percentage of apoptotic cells was based on the cumulative frequency curves of the appropriate DNA histograms.

Fluorescence microscopy. Fluorescence microscopy (Nikon Optiphot) followed 3 μg/ml DAPI fluorochromization of fixed cells. Cells were judged to be apoptotic on the basis of chromatin condensation and nuclear fragmentation.

Statistical analysis

Data were evaluated using ANOVA with the Student-Newman-Keuls post hoc t test. In some instances, Pearson’s correlation coefficient (r) was also calculated. The null hypothesis was rejected when p < 0.05.

PC12 cell viability is greatly reduced when L-DOPA is associated with manganese

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Assay of DA and its metabolites in cell lysate and incubation medium by HPLC with electrochemical detection
  5. RESULTS
  6. PC12 cell viability is greatly reduced when L-DOPA is associated with manganese
  7. L-DOPA + manganese cytotoxicity is reduced by antioxidants NAC and AA
  8. DA formation and metabolism are impaired when manganese is associated with L-DOPA : NAC protects
  9. l-DOPA-induced apoptosis is potentiated by manganese association : NAC protects
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

PC12 cells were exposed to varying concentrations of L-DOPA to choose concentrations that did not affect cell viability. Figure 1 shows that L-DOPA in concentrations of 10 and 20 μM did not decrease cell viability after 24 h (Fig. 1A) or 48 h (Fig. 1B) of incubation. A slight but significant decrease (by 11%) in cell viability was observed when PC12 cells were exposed to 50 μM L-DOPA for 48 h. Exposure to 100 μM L-DOPA resulted in a greater decrease (by ~30%) at both time points.

image

Figure 1. Effects of manganese and L-DOPA, alone or associated, on PC12 cell viability. PC12 cells in culture were incubated in drug-free medium or medium containing either MnCl2 (0.2 mM), increasing L-DOPA concentrations (10, 20, 50, 100 μM), or MnCl2 (0.2 mM) associated with L-DOPA (10-20 or 50 μM). At the start of each experiment, 140 × 103 cells/cm2 were plated and treated 24 h later (time 0) ; after 24 h (A) or 48 h (B), viable cell number was determined in triplicate wells. Cell viability was established by the MTT assay, in which viable cells convert the soluble dye MTT to insoluble (in aqueous medium) blue formazan crystals. A standard curve was constructed utilizing different concentrations of cells at the start of each experiment. Results are given as percent of control PC12 cells (incubated in drug-free medium) and represent the means ± SEM of three experiments. *p < 0.05 compared with controls ; **p < 0.05 compared with corresponding L-DOPA concentrations ; Student-Newman-Keuls t test.

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When a fixed concentration of MnCl2 (0.2 mM), which did not affect cell viability, was associated with 10-20 and 50 μM L-DOPA, a significant concentration-dependent (r = +0.98, p < 0.05) decrease in cell viability was observed at both incubation time points (Fig. 1).

L-DOPA + manganese cytotoxicity is reduced by antioxidants NAC and AA

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Assay of DA and its metabolites in cell lysate and incubation medium by HPLC with electrochemical detection
  5. RESULTS
  6. PC12 cell viability is greatly reduced when L-DOPA is associated with manganese
  7. L-DOPA + manganese cytotoxicity is reduced by antioxidants NAC and AA
  8. DA formation and metabolism are impaired when manganese is associated with L-DOPA : NAC protects
  9. l-DOPA-induced apoptosis is potentiated by manganese association : NAC protects
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

We have previously shown that manganese cytotoxicity is antagonized by the antioxidants NAC and AA (Desole et al., 1997b) ; in addition, a mechanism of autoxidation into highly ROS has been recognized in L-DOPA cytotoxicity (Basma et al., 1995). Therefore, 0.1 mM NAC or 0.1 mM AA was added to PC12 cell culture exposed to 20 or 50 μM L-DOPA associated with 0.2 mM MnCl2. Figure 2 shows that NAC and, to a lesser extent, AA both antagonized the L-DOPA + MnCl2-induced decrease in cell viability. Specifically, at both time point exposures, NAC protected PC12 cells completely from the lower (20 μM + 0.2 mM) and partially from the higher (50 μM + 0.2 mM) L-DOPA + MnCl2 concentrations, whereas AA partially protected the cells only from the lower one.

image

Figure 2. Effects of NAC or AA on manganese + L-DOPA-induced decrease in PC12 cell viability. At the start of each experiment, 140 × 103 cells/cm2 were plated and exposed 24 h later (time 0) to 0.2 mM MnCl2 + 20 or 50 μM L-DOPA alone or associated with 0.1 mM NAC or 0.1 mM AA. After 24 h (A) or 48 h (B), viable cell number was determined in triplicate wells. Cell viability was established by the MTT assay, in which viable cells convert the soluble dye MTT to insoluble (in aqueous medium) blue formazan crystals. Neither 0.2 mM MnCl2 nor 20 μM L-DOPA, respectively, associated with NAC or AA, had an effect on cell viability. Results are the means ± SEM of three experiments. *p < 0.05 compared with controls ; **p < 0.05 compared with corresponding L-DOPA concentrations ; Student-Newman-Keuls t test.

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DA formation and metabolism are impaired when manganese is associated with L-DOPA : NAC protects

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Assay of DA and its metabolites in cell lysate and incubation medium by HPLC with electrochemical detection
  5. RESULTS
  6. PC12 cell viability is greatly reduced when L-DOPA is associated with manganese
  7. L-DOPA + manganese cytotoxicity is reduced by antioxidants NAC and AA
  8. DA formation and metabolism are impaired when manganese is associated with L-DOPA : NAC protects
  9. l-DOPA-induced apoptosis is potentiated by manganese association : NAC protects
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

PC12 cell exposure to nontoxic concentrations of 20 μM L-DOPA results in an increase in the amount of DA and its metabolites DOPAC and HVA both in the cell lysate and in the incubation culture medium (Basma et al., 1995). We have previously shown (Desole et al., 1997a) that manganese induces an early decrease in tissue DA and DOPAC in glutathione-deficient PC12 cells. Therefore, experiments were performed to investigate the effects of manganese association with L-DOPA on DA formation and metabolism in PC12 cells in culture. The protective effect of NAC was also investigated. AA was not tested because AA, when added to PC12 cell culture (Desole et al., 1997a), is readily taken up by cells, is in part oxidized to dehydroascorbic acid (DHAA), and produces a marked increase in noradrenaline (NA) levels, owing to the well-known role of AA as cofactor in DA β-hydroxylation, according to the following reaction : DA + O2 + AA [RIGHTWARDS ARROW] NA + DHAA + H2O.

Tissue and incubation medium concentrations of DA, DOPAC, and HVA were determined after 8- and 24-h exposure to 20 or 50 μM L-DOPA alone or associated with 0.2 mM MnCl2. As shown in Table 1, 8-h exposure resulted in a significant and concentration-related increase in tissue DA (+64 and +120%, respectively) and DOPAC + HVA (+62 and +158%, respectively) levels as compared with controls. Changes in DA concentrations in the incubation medium reached statistical significance with neither 20 nor 50 μM L-DOPA, whereas DOPAC + HVA concentrations increased by ~49 and 101%, respectively ; statistical significance, however, was reached only with 50 μM L-DOPA. MnCl2 alone at 0.2 mM did not significantly modify DA or DOPAC + HVA levels either in cell lysate or in incubation medium (Table 1). MnCl2 at 0.2 mM, when associated with 20 and 50 μM L-DOPA, inhibited all the above L-DOPA-induced increases in DA and DOPAC + HVA concentrations, both in cell lysate and in incubation medium. For example, addition of 50 μM L-DOPA resulted in increases in the amounts of DA and DOPAC + HVA in the cell lysate of 120 and 158%, respectively, compared with controls ; MnCl2 association significantly inhibited the increases in both DA and DOPAC + HVA levels, which were in the control range. Likewise, the increase in DOPAC + HVA concentration in the incubation medium (+47% compared with controls) was completely inhibited by MnCl2 association.

Table 1. Effects of NAC on manganese- and L-DOPA-induced changes in DA and DOPAC + HVA concentrations in PC12 tissue and incubation medium after 8-h exposureAt the start of each experiment, 140 × 103 PC12 cells/cm2 were plated and treated 24 h later (time 0). After the desired incubation period, the medium was aspirated from each well and stored, and the cells were collected in metaphosphoric acid. Samples were subsequently analyzed for levels of DA and its metabolites DOPAC and HVA in cell lysates and incubation medium. Results are the means ± SEM of three experiments performed in triplicate.
 Tissue content (nmol/mg of protein) Medium concentration (μM)
TreatmentDADOPAC + HVADADOPAC + HVA
  1. ap < 0.05 compared with controls ;

  2. bp < 0.05 compared with 20 μM L-DOPA ;

  3. cp < 0.05 compared with corresponding L-DOPA concentrations ;

  4. dp < 0.05 compared with corresponding L-DOPA + MnCl2 concentrations ; Student-Newman-Keuls t test.

None5.66 ± 0.580.31 ± 0.020.15 ± 0.033.76 ± 0.32
MnCl2 (0.2 mM) 5.14 ± 0.540.30 ± 0.310.13 ± 0.012.19 ± 0.63
L-DOPA (20 μM) 9.29 ± 0.20a0.50 ± 0.03a0.23 ± 0.054.11 ± 0.31
L-DOPA (50 μM) 12.47 ± 0.66a,b0.80 ± 0.02a,b0.29 ± 0.025.54 ± 0.61a
L-DOPA (20 μM) + MnCl2 (0.2 mM) 5.60 ± 0.31c0.24 ± 0.01c0.13 ± 0.0012.37 ± 0.34
L-DOPA (50 μM) + MnCl2 (0.2 mM) 6.36 ± 0.66c0.29 ± 0.03c0.14 ± 0.022.78 ± 0.55c
L-DOPA (20 μM) + MnCl2 (0.2 mM) + NAC (0.1 mM) 8.46 ± 0.66a,d0.35 ± 0.01c,d0.23 ± 0.063.57 ± 0.58
L-DOPA (50 μM) + MnCl2 (0.2 mM) + NAC (0.1 mM) 10.69 ± 0.97a,d0.46 ± 0.05a,c,d0.24 ± 0.054.14 ± 0.39

The addition of 0.1 mM NAC to L-DOPA + MnCl2 resulted in an almost complete recovery of the L-DOPA-increasing effect in both DA and DOPAC + HVA levels in cell lysate. For instance, the NAC addition to 50 μM L-DOPA + 0.2 mM MnCl2 concentration significantly increased the DA and DOPAC + HVA levels by 68 and 59%, respectively, as compared with the corresponding L-DOPA + MnCl2 concentration.

The data in Table 2 show that a 24-h exposure to 20 or 50 μM L-DOPA resulted in a significant increase in the amount of DA and DOPAC + HVA compared with controls, in both the cell lysate and the incubation medium. For instance, 50 μM L-DOPA increased DA levels in cell lysate and incubation medium by 33.5 and 117%, respectively ; likewise, the DOPAC + HVA levels in cell lysate and incubation medium were, respectively, increased by 30 and 126%. MnCl2 alone did not modify DA or DOPAC + HVA levels in either the cell lysate or the incubation medium. The addition of MnCl2 to both L-DOPA concentrations inhibited the increasing effect of L-DOPA on DA and DOPAC + HVA levels in both the cell lysate and the incubation medium ; in addition, DA content in the cell lysate was also significantly lowered compared with controls (-38% after exposure to 20 μM L-DOPA + 0.2 mM MnCl2 and -32% after exposure to 50 μM L-DOPA + 0.2 mM MnCl2).

Table 2. Effects of NAC on manganese- and l-DOPA-induced changes in DA and DOPAC + HVA content in PC12 tissue and incubation medium after 24-h exposureAt the start of each experiment, 140 × 103 PC12 cells/cm2 were plated and treated 24 h later (time 0). After the desired incubation period, the medium was aspirated from each well and stored, and the cells were collected in metaphosphoric acid. Samples were subsequently analyzed for levels of l-DOPA, DA and its metabolites DOPAC and HVA in cell lysates and incubation medium. Results are the means ± SEM of three experiments performed in triplicate.
 Tissue content (nmol/mg of protein) Medium concentration (μM)
TreatmentDADOPAC + HVADADOPAC + HVA
  1. ap < 0.05 compared with controls ;

  2. bp < 0.05 compared with corresponding l-DOPA concentrations ;

  3. cp < 0.05 compared with corresponding l-DOPA + MnCl2 concentrations ; Student-Newman-Keuls t test.

None7.47 ± 0.540.30 ± 0.540.18 ± 0.034.30 ± 0.31
MnCl2 (0.2 mM) 5.99 ± 0.870.28 ± 0.010.17 ± 0.024.12 ± 0.87
l-DOPA (20 μM) 9.78 ± 0.26a0.37 ± 0.01a0.33 ± 0.02a7.28 ± 0.71a
l-DOPA (50 μM) 9.97 ± 0.18a0.38 ± 0.02a0.39 ± 0.02a9.72 ± 1.16a
l-DOPA (20 μM) + MnCl2 (0.2 mM) 4.62 ± 0.02a,b0.21 ± 0.01a,b0.18 ± 0.04b3.20 ± 0.57b
l-DOPA (50 μM) + MnCl2 (0.2 mM) 5.01 ± 0.19a,b0.25 ± 0.01b0.17 ± 0.03b2.48 ± 0.71b
l-DOPA (20 μM) + MnCl2 (0.2 mM) + NAC (0.1 mM) 6.72 ± 0.53b,c0.33 ± 0.02c0.30 ± 0.02a,c6.57 ± 0.88c
l-DOPA (50 μM) + MnCl2 (0.2 mM) + NAC (0.1 mM) 6.93 ± 0.51b,c0.35 ± 0.03c0.34 ± 0.07a,c7.37 ± 0.60c

The addition of 0.1 mM NAC to L-DOPA + MnCl2 (Table 2) resulted in a significant recovery of the L-DOPA-increasing effect on DA and DOPAC + HVA levels in both cell lysate and incubation medium. For instance, the NAC addition to 20 μM L-DOPA + 0.2 mM MnCl2 concentration significantly increased by 45 and 57%, respectively, the DA and DOPAC + HVA levels in the cell lysate compared with the corresponding L-DOPA + MnCl2 concentration. Likewise, DA and DOPAC + HVA concentrations in the incubation medium significantly increased by 67 and 105%, respectively, compared with the corresponding l-DOPA + MnCl2 concentration.

l-DOPA-induced apoptosis is potentiated by manganese association : NAC protects

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Assay of DA and its metabolites in cell lysate and incubation medium by HPLC with electrochemical detection
  5. RESULTS
  6. PC12 cell viability is greatly reduced when L-DOPA is associated with manganese
  7. L-DOPA + manganese cytotoxicity is reduced by antioxidants NAC and AA
  8. DA formation and metabolism are impaired when manganese is associated with L-DOPA : NAC protects
  9. l-DOPA-induced apoptosis is potentiated by manganese association : NAC protects
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

Experiments were performed to investigate whether the cellular damage induced by l-DOPA plus manganese might be characteristic of changes leading to apoptosis. PC12 cells were therefore exposed for 48 h to increasing concentrations of l-DOPA (10-20 and 50 μM) alone or associated with 0.2 mM manganese ; the latter was also tested alone. Three independent histological methods were used to evaluate whether PC12 cells died via apoptosis : TUNEL ; flow cytometry analysis ; and fluorescence microscopy following DAPI fluorochromization of fixed cells.

Neither 0.2 mM manganese nor 10 and 20 μMl-DOPA induced nuclear changes characteristic of apoptosis (Fig. 3). However, the higher l-DOPA concentration (50 μM) did induce apoptotic nuclear changes, as revealed by TUNEL (Fig. 4), fluorescence microscopy, and flow cytometry (Fig. 5). The percentage of apoptotic cells ranged from 10 to 20%. When 0.2 mM manganese was associated with l-DOPA, apoptotic nuclear changes were induced also by the lower concentrations of l-DOPA tested (10 and 20 μM) (Figs. 3-5Figs. 3-5Figs. 3-5). The number of apoptotic cells ranged from 8-10 to 30-40%, respectively.

image

Figure 3. Fluorescence micrographs of PC12 cell nuclei from untreated cultures (A) and from cultures exposed for 48 h to 0.2 mM MnCl2 + 10 μMl-DOPA (B) or 0.2 mM MnCl2 + 50 μMl-DOPA + 0.1 mM NAC (C). Cells were stained with the DNA-specific fluorescent dye DAPI (3 μg/ml). Nuclear changes are typical of apoptosis.

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image

Figure 4. Micrographs of PC12 cells from untreated cultures (A) and from cultures exposed for 48 h to 0.2 mM MnCl2 + 20 μMl-DOPA (B) or 0.2 mM MnCl2 + 50 μMl-DOPA + 0.1 mM NAC (C). Dark cells are apoptotic. DNA strand breaks were labeled by the TUNEL technique.

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image

Figure 5. Flow cytometric histograms of PC12 cells after 48-h exposure to 0.2 mM MnCl2 or 20 or 50 μM L-DOPA. After incubation, cells were harvested and stained with DAPI. DNA content was analyzed by a Partec PAS II flow cytometer : x-axis, DNA content ; y-axis, number of cells. Neither 0.2 mM MnCl2 nor L-DOPA induced apoptotic cell death. The percentage of apoptotic cells after exposure to 50 μM L-DOPA was ~15%.

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The addition of 0.1 mM NAC to all l-DOPA + MnCl2 concentrations tested resulted in an almost complete inhibition of nuclear changes characteristic of apoptosis (Fig. 6).

image

Figure 6. Flow cytometric histograms of PC12 cells after 48-h exposure to 0.2 mM MnCl2 + 10, 20, or 50 μM L-DOPA. The bottom histogram refers to PC12 cells after 48-h exposure to 0.2 mM MnCl2 + 50 μM L-DOPA associated with 0.1 mM NAC. After incubation, cells were harvested and stained with DAPI. DNA content was analyzed by a Partec PAS II flow cytometer : x-axis, DNA content ; y-axis, number of cells. The percentage of apoptotic cells after exposure to 0.2 mM MnCl2 + 10, 20, or 50 μM L-DOPA was, respectively, 8, 40, and 40%. No apoptotic cells were detected in this experiment after exposure to 0.2 mM MnCl2 + 50 μM L-DOPA associated with 0.1 mM NAC.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Assay of DA and its metabolites in cell lysate and incubation medium by HPLC with electrochemical detection
  5. RESULTS
  6. PC12 cell viability is greatly reduced when L-DOPA is associated with manganese
  7. L-DOPA + manganese cytotoxicity is reduced by antioxidants NAC and AA
  8. DA formation and metabolism are impaired when manganese is associated with L-DOPA : NAC protects
  9. l-DOPA-induced apoptosis is potentiated by manganese association : NAC protects
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES

The present study was designed to evaluate whether manganese could worsen l-DOPA neurotoxicity. The rationale of the experiments is based on the fact that oxidative stress has been recognized as a mechanism of both l-DOPA-induced (Walkinshaw and Waters, 1995) and manganese-induced (Desole et al., 1997b) apoptosis. l-DOPA cytotoxicity was assessed in PC12 cells, a reliable model for studying neurotoxic agents (Basma et al., 1995), by means of the following parameters : cell viability, DA metabolism, and nuclear changes leading to apoptosis. Our findings show that manganese enhanced l-DOPA cytotoxicity, as concentrations of l-DOPA (10-20 μM) that were ineffective greatly impaired cell viability and induced apoptosis following manganese association. In addition, manganese inhibited the l-DOPA-induced increase in DA and metabolite levels both in the cell lysate and in the incubation medium. All these manganese effects were inhibited by the antioxidants NAC and, to a lesser extent (cell viability), AA. The fact that antioxidants inhibited manganese effects suggests an oxidative mechanism. Florence and Stauber (1989), to explain the significant manganese-induced increase in DA oxidation, proposed a mechanism involving a manganese (II)/(III) redox couple and a semiquinone free radical intermediate. In addition, they found that an electrophilic compound, like AA and DHAA, effectively inhibited DA oxidation.

The mechanism of l-DOPA toxicity in PC12 cells has been elucidated by Basma et al. (1995) and Walkinshaw and Waters (1995) : The increase in ROS formation arises from l-DOPA autoxidation rather than from enzymatic oxidative metabolism of endogenous DA or DA formed from l-DOPA added to the incubation medium, as monoamine oxidase inhibitors did not affect l-DOPA cytotoxicity. According to Parsons (1985), the first step of l-DOPA oxidation occurs in the incubation medium, leading to formation of superoxide, which in turn generates highly reactive hydroxyl radicals via the trace metalcatalyzed Fenton-type reaction (Haber and Weiss, 1934). In addition, l-DOPA decreases catalase activity in neuronal cell culture (Han and Cohen, 1996), an effect that may be of relevance to l-DOPA cytotoxicity in vitro, as the latter is reduced by catalase (Basma et al., 1995). The autoxidation of L-DOPA in vitro generates also a semiquinone free radical, which is further oxidized to o-quinone ; the quinones give an orange-brown color to incubation medium containing L-DOPA (Basma et al., 1995). However, quinones are not essential for L-DOPA cytotoxicity (Basma et al., 1995). The above orange-brown color was observed also in the present study.

The enabling effect of manganese may occur either outside or inside the cell or both. The first and simplest explanation is that manganese, as a transition metal (Florence and Stauber, 1989 ; Miller et al., 1990 ; Klegeris et al., 1995 ; Shen and Dryhurst, 1998), may act as a catalyst outside the cell in promoting L-DOPA autoxidation. However, the link between the manganese-induced increase in L-DOPA autoxidation and apoptotic cell death remains unclear. Toxic byproducts of manganese-induced DA autoxidation (Florence and Stauber, 1989) and/or manganese itself in its trivalent form (III) (Alinovi et al., 1996) might lower the triggering level for inducing apoptosis. The role of newly formed DA autoxidation is supported by the following consideration. The higher L-DOPA concentration (50 μM) induced both apoptosis and increases in DA and metabolite levels in both cell lysate and incubation medium. Thus, we might admit that the ROS generated from L-DOPA autoxidation were enough for triggering apoptosis without affecting the increase in DA synthesis and metabolism in nonapoptotic cells. Following manganese addition, apoptosis occurred also with the lower L-DOPA concentrations (10-20 μM), but DA and metabolite levels were decreased in both cell lysate (even below the control values) and incubation medium. A decrease in DA and DOPAC in the incubation medium of PC12 cells exposed to manganese has been shown by Alinovi et al. (1996). Thus, in the present study, the decrease in DA levels following L-DOPA + manganese exposure could be a consequence of either a decreased supply of L-DOPA, owing to its greater autoxidation catalyzed by manganese, or a nonenzymatic oxidation of DA, promoted by the metal (Shen and Dryhurst, 1998) or both.

ROS formation from increased enzymatic oxidative metabolism of DA seems not to be involved in the L-DOPA mechanism of toxicity (Basma et al., 1995 ; Walkinshaw and Waters, 1995) ; however, in the present study, a manganese-induced DA autoxidation could be taken into account, at least in part, to explain manganese’s enabling effect on L-DOPA toxicity. This hypothesis is supported by the restoring effect of NAC on cellular and incubation medium DA levels. NAC may act as a direct inhibitor of both L-DOPA and DA autoxidation and/or by its proglutathione properties (Lai and Yu, 1997). Thus, the protective effect of NAC may occur both in the incubation medium and inside the cell. A crude assessment of the NAC effect outside the cell can be made on the basis of the orange-brown color of the incubation medium following the addition of L-DOPA : This color is indicative, according to Basma et al. (1995), of L-DOPA quinone generation. As the color intensity was greatly attenuated following NAC addition, it may be possible that NAC inhibits L-DOPA autoxidation in the incubation medium. The inhibition of L-DOPA quinone formation in mesencephalic culture by antioxidants has also been reported (Pardo et al., 1995). Inside the cell, NAC, acting as a glutathione precursor, would restore glutathione levels. It has been shown that oxidation of DA and L-DOPA leads to a very rapid loss of glutathione (Spencer et al., 1995) ; moreover, the cytotoxic effects of both DA (Rabinovich and Hastings, 1998) and manganese (Desole et al., 1997b) are increased if glutathione synthesis is compromised.

Oxidative stress has been suggested as a mediator of apoptosis (Sandstrom et al., 1994). Mitochondrial complex I inhibitors also are known to induce apoptosis (Hartley et al., 1994 ; Seaton et al., 1997), with a mechanism in which oxidative stress may play a role (Seaton et al., 1997). Manganese is sequestered in mitochondria (Liccione and Maines, 1988 ; Gavin et al., 1992) and inhibits oxidative phosphorylation (Gavin et al., 1992). Galvani et al. (1995) suggest that manganese toxicity to PC12 cells could be the result of either a direct or an indirect effect on complex I activity, mediated by oxidative stress. It has been shown that manganese-induced apoptosis is transcription dependent with an impairment of mitochondrial function and is blocked in Bcl-2-over expressed cells (Hirata et al., 1998). Nakao et al. (1997) demonstrated that inhibition of mitochondrial complex I activity by rotenone enhances selective toxicity of L-DOPA toward mesencephalic DA neurons in vitro ; moreover, prior metabolic inhibition of complex I activity renders DA cells susceptible to nontoxic L-DOPA concentrations. L-DOPA inhibits complex I in vitro, and the inhibition is prevented by antioxidants (Przedborski et al., 1995). In addition, L-DOPA and its metabolities promote oxidative DNA damage (Spencer et al., 1994). All these data, taken together, support the hypothesis that manganese inhibition of mitochondrial function may be taken into account as an additional mechanism in enhancing L-DOPA-induced apoptosis.

The results of this study show that agents whose neurotoxic effects recognize similar mechanisms may manifest their neurotoxic potential when associated. This may be of relevance to L-DOPA-induced apoptotic neuronal cell death. Following a toxic injury, necrotic neuronal death, as a passive cellular process, will occur irrespective of other factors ; on the contrary, apoptosis, as an active cellular process, may be conditioned by intracellular or extracellular factors (Walkinshaw and Waters, 1995) ; for instance, a decrease in the cellular defense mechanism would activate apoptotic cellular death in response to an oxidative insult to which a healthy cell would have been resistant. Thus, surviving dopaminergic neurons in parkinsonian patients, which are the target of both therapeutic and neurotoxic L-DOPA effects, might be predicted to be damaged and consequently might have a threshold for activation of an active apoptotic death lower than healthy neurons (Walkinshaw and Waters, 1995). The potential risk of apoptotic death may be manifested if an additional oxidative injury further lowers the threshold for apoptosis activation.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Assay of DA and its metabolites in cell lysate and incubation medium by HPLC with electrochemical detection
  5. RESULTS
  6. PC12 cell viability is greatly reduced when L-DOPA is associated with manganese
  7. L-DOPA + manganese cytotoxicity is reduced by antioxidants NAC and AA
  8. DA formation and metabolism are impaired when manganese is associated with L-DOPA : NAC protects
  9. l-DOPA-induced apoptosis is potentiated by manganese association : NAC protects
  10. DISCUSSION
  11. Acknowledgements
  12. REFERENCES
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