Levodopa modulating effects of inducible nitric oxide synthase

and reactive oxygen species in glioma cells

Mark K. Soliman, Elizabeth Mazzio, Karam F.A. Soliman*

College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL 32307, USA

Received 27 March 2002; accepted 24 July 2002

Abstract

Neurological injury and Parkinson disease (PD) are often associated with the increase of nitric oxide (NO) and

free radicals from resident glial cells in the brain. In vitro, exposure to L-3-4-dihydroxyphenylalanine (L-DOPA),

one of the main therapeutic agents for the treatment of PD, can lead to neurotoxicity. In this study,

lipopolysaccharide (LPS) and interferon-gamma (IFN-g) were used to stimulate C6 glioma cells in the presence of

varying concentrations of L-DOPA (1 AM-1 mM). The results indicated a slight augmentation of NO2� production

at low concentrations of L-DOPA (<100 AM) and complete inhibition of NO2� at higher concentrations (500 AM, 1

mM), (p < 0.001). Western blot analysis corroborated that L-DOPA effects on iNOS was at the level of its protein

expression. Total reactive oxygen species (ROS) were detected using 2V, 7V-dichlorofluorescein diacetate

fluorescence dye (2V, 7V-DCFC) and there was an increase of intensity with the increasing concentrations of L-

DOPA. Furthermore, large amounts of superoxide (O2�) and hydrogen peroxide (H2O2) were generated from the

autoxidation of L-DOPA. C6 cells contain high levels of catalase, with inadequate levels of superoxide dismutase

(SOD); therefore, there was an accumulation of O2�, tantamount to elevation in 2V7V-DCFC intensity. Simultaneous

accumulation of O2� and NO2

� would propel formation of peroxynitrite (ONOO-). SOD completely attenuated the

autoxidation of L-DOPA and significantly reversed the inhibitory effects on iNOS at high concentrations. The data

obtained confirmed that the observed effects on iNOS were not due to the activation of the D1 or h1 adrenergic

receptors by L-DOPA. It was concluded from this study that L-DOPA contributed to the modulation of iNOS and

to the increase of O2� production in the stimulated glioma cells in vitro.

D 2002 Elsevier Science Inc. All rights reserved.

Keywords: Levodopa; Nitric oxide; C6 glioma cells; iNOS

0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved.

PII: S0024 -3205 (02 )02204 -X

* Corresponding author. Tel.: +850-599-3306; fax: +850-599-3667.

E-mail address: Karam.Soliman@famu.edu (K.F.A. Soliman).

www.elsevier.com/locate/lifescie

Life Sciences 72 (2002) 185–198

Introduction

One of the major therapeutic agents used for the treatment of Parkinson’s disease (PD) is L-3-4-

dihydroxyphenylalanine (L-DOPA). L-DOPA is molecularly unstable and can rapidly autoxidize in the

presence of oxygen or light to yield toxic free radicals. Therefore, there has been considerable concern that

this drugmay accelerate neuronal loss in the substantia nigra. In vitro exposure to L-DOPAwas found to be

toxic in several neuronal cell lines including PC12 and SH-SY5Y cells [1–3]. L-DOPA through its

autoxidation and by enzymatic oxidation can produce semiquinones, O-quinone derivatives and several

free radicals such as hydrogen peroxide (H2O2) and superoxide (O2�). The produced free radicals can lead

to oxidative stress and cellular damage. In addition, free radical formation can cause lipid peroxidation of

membranes and mitochondrial dysfunction leading to cell injury and death [4,5].

On the other hand, glial cells may contribute to Parkinson’s disease since they are a potential source of

neurotoxic factors. Glial cells may transform inactive compounds to active neurotoxins, as in the case of

MPTP, which is converted to MPP+ by astrocytes [6]. Moreover, cytokine activation of glial cells often

leads to neurotoxicity and neurodegeneration [7–11]. The elevation of neurotoxic oxygen free radicals and

NO generated from glia [7,8] has been implicated in the pathology of PD [8].

In general, research on L-DOPA has largely targeted the control of symptoms and not the pathological

degeneration that occurs with time. Although there is substantial evidence to support toxicity of L-DOPA

in vitro, studies have failed to demonstrate in vivo that levodopa can exert neurotoxicity to nigral cells in

human, rat and mouse models [12]. Although there is a need to examine the toxicity of L-DOPA, there is

meager data available regarding the effects of L-DOPA on glial cell free radical production during

inflammation. Therefore, the current study was designed to investigate the effect of L-DOPA on nitric

oxide (NO), inducible nitric oxide synthase (iNOS) expression and free radical handling by the glioma

cells.

Materials and methods

Materials

C6 rat glioma cells were obtained from American Type Culture Collection (Manassas, VA, USA).

Dulbecco’s modified Eagle medium (DMEM), L-glutamine, fetal bovine serum – heat inactivated (FBS),

phosphate buffered saline (PBS), Hank’s balanced salt solution (HBSS) and penicillin/streptomycin were

supplied by Fischer Scientific, Mediatech (Pittsburgh, PA). Tris (hydroxymethyl) aminomethane hydro-

chloride (Tris)/glycine 10 � buffer, sodium dodecyl sulfate (SDS) buffer, Laemmli sample buffer, gels

and ready gel blotting – (0.2 Am nitrocellulose) were purchased from Biorad (Hercules, CA, USA). All

other chemicals and supplies were purchased from Sigma Chemical (St. Louis, MO, USA).

Cell culture

C6 glioma cells were grown in DMEM with phenol red supplemented with 10% FBS (v/v), 4 mM L-

glutamine and penicillin/streptomycin (100 Units/0.1 mg per ml). The culture medium was changed

every 2–3 days, and cells were trypsinized (sterile trypsin 0.25%/ethylenediaminetetraacetic acid

M.K. Soliman et al. / Life Sciences 72 (2002) 185–198186

(EDTA)/0.1% in HBSS) and subcultured at confluence every 3–5 days. Experimental plating media

consisted of DMEM minus phenol red, 1.8% FBS (v/v), penicillin/streptomycin (100 Units/0.1 mg per

ml) and 4 mM L-glutamine. Cells (100 Al), were plated at 1.0 � 106 cells/ml in 96 well plates. A stock

solution for each experimental compound was prepared in HBSS + 10 mM (N-(2-hydroxyethylpiper-

azine-NV-(2-ethanesulfonic acid]) (HEPES) and adjusted to a pH of 7.4. Six concentrations of L-DOPA

were prepared fresh daily, to span a 1000-fold dilution range.

Nitrite determination

Quantification of nitrite (NO2�) was determined by a spectrophotometric procedure [13]. Briefly, the

Griess reagent was prepared by mixing an equal volume of 1% sulfanilamide in 0.5 N HCl and 0.1%

N-(1-naphthyl) ethylenediamine in deionized water. The Griess reagent was added directly to the cell

suspension and incubated under reduced light at room temperature for 10 minutes. Samples were

analyzed at 550 nm on a UV microplate spectrophotometer – model 7600, version 5.02, Cambridge

Technologies (Watertown, MA, USA). Controls and blanks were measured simultaneously, and

subtracted from the final value to eliminate interference. A standard curve was generated from a

range of dilutions of sodium nitrite (1–100 AM) prepared in the plating medium. Protein level of each

sample was determined using the Lowry method [14]. The data were calculated as nM NO�2/mg

protein.

iNOS Western blot analysis

Western blot analysis was done using a previously described method [15] with minor modifications.

The supernatant for each sample was removed and discarded. The cell monolayer was washed with

PBS and placed in a lysis buffer. The lysis buffer consisted of 5% glycerol, 1 mM sucrose, 200 AMphenylmethylsulfonyl fluoride, 10 mM Tris, 5 Ag/ml pepstatin A, 1 mM EDTA, 10 Ag/ml apoprotin,

10 Ag/ml leupeptin, 2 mM DL-dithiothreitol, 3 mM urea prepared in 18 megaV water. Samples were

stored at � 80 jC for 24 H and lysed by freeze thaw. The cell lysate was placed in Laemmli sample

buffer containing 3% mercaptoethanol. Samples were then boiled for 5 minutes and centrifuged at

13,000 g for 5 minutes. The supernatant was removed for Western blot and protein analysis. Proteins

were separated on a 10% SDS – polyacrylamide gel using the buffer system of Laemmli and

transferred to nitrocellulose at 125 V for 1 hour in Towbin-SDS transfer buffer 25 mM Tris, 192 mM

glycine and 20% methanol. After transfer, the blot was washed once with PBS containing 0.05%

Tween 20 (TTBS).

For determination of iNOS, 2 hr protocol for immunostaining developed by R&D Systems

(Minneapolis, MN, USA) was used. Briefly, the blot was thoroughly dried. The primary antibody

used was specific for iNOS, macNOS and was developed in rabbit using the synthetic peptide

FSYGAKKGSALEEPKATRL corresponding to the C-terminus of iNOS of mouse macrophage origin

(amino acids 1126–1144) conjugated to keyhole limpet hemocyanin as the immunogen, Sigma

Chemical (St. Louis, MO, USA). This sequence is highly conserved in rat macNOS. Whole antiserum

was fractionated and then further purified by ion exchange chromatography to provide the IgG fraction

of antiserum that was free of other rabbit serum proteins, Sigma Chemical (St. Louis, MO, USA). The

1j antibody was diluted 1:6000 in a diluent buffer containing 1% bovine serum albumin (Fraction V)

in TTBS and 0.2% sodium azide. 20 ml of diluted primary antibody was added to the membrane and

M.K. Soliman et al. / Life Sciences 72 (2002) 185–198 187

incubated on a rocker/shaker at room temperature for 1 hour. The membrane was washed and 20 ml of

diluted secondary antibody (anti-rabbit IgG (whole molecule) peroxidase conjugate) 1:4000 was added

and incubated on a rocker/shaker at room temperature for 1 hour. After a final wash, peroxidase was

detected with SIGMA FASTk DAB (3,3V-diaminobenzidine tetrahydrochloride) with a metal enhancer

cobalt chloride.

Superoxide assay

Production of superoxide radical was assayed using cytochrome C reduction [16]. Briefly, after

experimental treatment, sample supernatant was added to an equal volume of cytochrome C (from

horse heart) prepared in PBS – (final working concentration: 160 AM). Samples were then

incubated for 35 minutes at 37 jC in 5% CO2/atmosphere. Samples were analyzed at 550 nm

on a UV Microplate Spectrophotometer – Model 7600, version 5.02, Cambridge Technologies

(Watertown, MA, USA). Protein concentration was determined by the Lowry method (Lowry et al.,

1951) and the data were calculated as nM O�2/mg protein or volume equivalent for autoxidation

studies.

Hydrogen peroxide assay

Hydrogen Peroxide (H2O2) was assessed by the modification of a previously described method [17].

After experimental treatment, sample supernatants were assessed for H2O2 content based on a peroxidase

linked continuous assay. The chromogenic solution contained (final working concentration) 1 mM

vanillic acid, 500 AM 4-aminoantipyrine and horseradish peroxidase (4 units/ml) in PBS with 2 mM of

HEPES (pH 7.4). The chromogenic reagent was added to each sample and incubated for 10 minutes at

37 jC. Samples were analyzed at 490 nm on a UV Microplate Spectrophotometer – Model 7600,

version 5.02, Cambridge Technologies (Watertown, MA, USA). Controls and blanks were measured

simultaneously, and subtracted from the final value to eliminate interference. Protein was assessed by the

Lowry method [14]. Final data were expressed as nM H2O2/mg protein or volume equivalent for

autoxidation studies.

Cell viability assessment

Cell viability was assessed by resazurin oxidoreduction indicator dye [18]. A working solution of

resazurin was prepared in PBS minus phenol red (0.5 mg/ml). Reduction of the dye by viable cells

reduces the amount of oxidized form and increases the amount of its bright red fluorescent

intermediate (resorufin). The dye solution was added (15% v/v equivalent) to the samples. Cultures

were returned to the incubator for 6–8 H. Quantitative analysis of dye conversion was measured on

a Cambridge microplate fluorometer – model 7620-version 5.02, Cambridge Technologies (Water-

town, MA, USA) set at (550/580] (excitation/emission].

Total reactive oxygen species determination

Quantification of ROS was determined using a fluorescence procedure with 2V, 7V-dichlorofluoresceindiacetate (2V, 7V-DCFC) [19]. In this assay, a stock solution of 2V, 7V-DCFC was prepared in absolute

M.K. Soliman et al. / Life Sciences 72 (2002) 185–198188

ethanol (10 mg/ml) and stored at 0 jC. 40 Al of the stock solution was diluted in 10 ml of PBS to achieve

a working concentration of 40 Ag/ml. The dye solution was added (15% v/v equivalent) to the original

culture medium. Cultures were returned to the incubator for 1 H. Samples were analyzed on a Nikon

Eclipse TE300 inverted microscope with a SPOT cooled CCD color digital camera (Diagnostic

Instruments, Sterling Hts, MI, USA). Images were captured by SPOT imaging software version 2.2

and analyzed using IP Lab Scientific Processing software (Scanalytics, Fairfax, VA, USA).

Catechols oxidation

The oxidative pathway for L-DOPAwas assessed by quantifying color formation over time. Samples

were analyzed at 550 nm on a UV Microplate Spectrophotometer – Model 7600, version 5.02,

Cambridge Technologies (Watertown, MA, USA).

Statistical analysis

Statistical analysis was performed using Graphpad Prism (version 3.0)-Graphpad Software (San

Diego, CA, USA). Data were expressed as the meanF S.E.M. for each group. Significance of difference

between the groups was assessed using a one-way or two-way analysis of variance (ANOVA), followed

by a Tukey post-hoc means comparison test.

Results

C6 glioma cells were incubated in the presence of varying concentration of L-DOPA (1 AM–1 mM) FLPS: Escherichia coli 0111:B4 (6 Ag/ml):IFN-g (100 units/ml). Samples were returned to the incubator

for 24–36 H at 37 jC in 5% CO2/atmosphere. The tests were monitored with timed controls, to ensure

a desired level of NO2� production had been achieved. Treatment with L-DOPA resulted in a slight

elevation of NO2� at low doses and inhibition of detected NO2

� at higher doses (p < 0.001) (Fig. 1).

Western blot analysis of iNOS corroborated the regulation of the NOS enzyme. At 500 AM and 1 mM

L-DOPA, there were slight to moderate cytotoxic effects, however iNOS expression was completely

blocked at 1 mM (p < 0.001). These effects may or may not be due to the toxic concentration of L-

DOPA.

ROS were detected using 2V,7V-DCFC fluorescence dye and there was an increase of intensity with

increasing concentrations of L-DOPA from 32F 0.216 to 217F 0.208 intensity/pixel, for control and 1

mM L-DOPA, respectively (Fig. 2a,b). In order to determine which oxygen species were present,

quantification of both H2O2 and O2� radicals were performed independently. The autoxidation of 1 mM

L-DOPA resulted in 271.8F 3.5 nM/O2� (medium equivalent) and 173.9F 1.6 nM/mg protein

(stimulated cells) (Fig. 3). There was a large amount of H2O2 generated from autoxidation of L-DOPA

(Fig. 4). However, in an equal volume of cells with L-DOPA, most of the H2O2 was catabolized by

endogenous antioxidant enzymes in the cells. These data, indicated that the primary reactive oxygen

species that paralleled 2V, 7V-DCFC fluorescence was the O2� radical and, that the accumulation of O2

�

may contribute to the attenuation of NOS. Therefore, L-DOPA autoxidation in LPS/IFN-g treated cells

was assessed in the presence of superoxide dismutase enzyme (SOD) (75 Units/ml) (Fig. 5).

Interestingly, the data indicate complete inhibition of L-DOPA autoxidation in the presence of SOD.

M.K. Soliman et al. / Life Sciences 72 (2002) 185–198 189

It appears that endogenous catalase in the glioma combined with exogenous SOD, completely

prevented L-DOPA oxidation. The effects of SOD on L-DOPA inhibition of iNOS in LPS/IFN-g treated

cells were evaluated (Fig. 6). Interestingly, the effects of L-DOPA are mediated in part through the

generation of either O2�.or oxidation products of L-DOPA. In order to ensure that iNOS inhibition was

not due to activation of the D1 or h1 receptor, the effects of SCH 23390 and atenolol were evaluated

Fig. 1. The effects of L-DOPA on iNOS. C6 cells + LPS/IFN-gamma were treated with varying concentration of L-DOPA. The

data represents NO2� and viability, [% control] and are expressed as the meanF S.E.M., (n = 4). Significance of difference

between treatment and control was determined by a one-way ANOVA, followed by a Tukey mean comparison post hoc test,

(NO2�)* = p < 0.001 (viability) a = p < 0.001. The corresponding western blot analysis is displayed underneath the graphic data

and represents iNOS protein expression.

M.K. Soliman et al. / Life Sciences 72 (2002) 185–198190

Fig. 2. a. C6 glioma cells + LPS/IFN-gamma were treated with varying levels of L-DOPA. Each photo represents ROS detected

at each dose using 2V,7V-DCFC fluorescence dye. Photos were captured by a Nikon Eclipse TE300 inverted microscope with a

SPOT cooled CCD color digital camera. b. 2V,7V-DCFC fluorescence intensity was analyzed using IP Lab Scientific Processing

Software. The data are expressed as the meanF S.E.M. for each photograph as shown in Fig. 2a. Significance of difference

from the controls was determined by a one-way ANOVA, followed by a Tukey mean comparison post hoc test. * = p < 0.001.

M.K. Soliman et al. / Life Sciences 72 (2002) 185–198 191

Fig. 3. Autoxidation of L-DOPA into O2�. C6 cells + LPS/IFN-gamma or a blank medium control were treated with varying

concentration of L-DOPA. The data are expressed as nM-02/mg prot. or volume equivalent at 24 H and plotted as the meanFS.E.M., (n = 4). Significance of difference from the controls was determined by a one-way ANOVA, followed by a Tukey mean

comparison post hoc test, * = p < 0.001, (Medium), + = p < 0.001, (Cells).

Fig. 4. Autoxidation of L-DOPA into H2O2. C6 cells + LPS/IFN-gamma or a blank medium control were treated with varying

concentration of L-DOPA. The data are expressed as nM H2O2/mg protein or volume equivalent at 24 H and plotted as the

meanF S.E.M., (n = 4). Significance of difference from the controls was determined by a one-way ANOVA, followed by a

Tukey mean comparison post hoc test, * = p < 0.001, (Medium), + = p < 0.001, (Cells).

M.K. Soliman et al. / Life Sciences 72 (2002) 185–198192

Fig. 6. The effect of SOD on L-DOPA mediated iNOS inhibition. C6 cells + LPS/IFN-gammaF SOD (75 Units/ml) were treated

with varying concentration of L-DOPA. The data are expressed as NO2� [% Control] and plotted as the meanF S.E.M., (n = 4).

Significance of difference from the controls was determined by a two-way ANOVA. The level of significance between the groups

is * = p < 0.001.

Fig. 5. The effect of SOD on L-DOPA non-enzymatic oxidation. C6 cells + LPS/IFN-gammaF SOD (75 Units/ml) were treated

with varying concentration of L-DOPA. The data are expressed as O.D. at 550 nm and plotted as the meanF S.E.M., (n = 4).

Significance of difference from the controls was determined by a two-way ANOVA. The level of significance between the groups

is * = p < 0.001.

M.K. Soliman et al. / Life Sciences 72 (2002) 185–198 193

(Fig. 7). There were no statistical differences between receptor antagonisms on L-DOPA mediated iNOS

inhibition.

Discussion

The data obtained in the present study clearly indicated that L-DOPA could modulate free radicals in

cytokine/bacterial endotoxin stimulated glioma in vitro. L-DOPA can cross the blood brain barrier

through active transport by a neutral amino acid carrier, subject to competition by other amino acids such

as alanine, asparagine, glutamine, serine, and threonine [20]. Imaging using 18-fluoro-L-DOPA positron

emission tomography confirmed that the distribution of L-DOPA and subsequent metabolites are not

restricted to the striatal region, but also accumulate in other areas of the brain such as the hypothalamus,

brainstem and cortex [21]. The effects of L-DOPA administration on whole brain astrocytic function are

sparsely reported in the literature.

The administration of L-DOPA to PD patients might have some consequences since this drug can

increase free radicals. Typically, catechols can be oxidized in the presence of molecular oxygen to yield

semiquinones and O2�. The O2

� generated can further oxidize catechols to yield H2O2 [22]. L-DOPA and

other catechols can also propel oxidative stress, through initiating the release of iron from ferritin in the

brain, leading to metal catalyzed production of the hydroxyl (OH) radical [23]. Catecholamine derived

ROS and quinone formation contribute to the neurodegeneration associated with PD [24]. While there is

significant evidence to support a destructive role for ROS, such as H2O2, O2� and OH, research

examining a role for NO has been controversial. Evaluating the role for NO in PD has been primarily

focused on experimental models without L-DOPA use. Several studies indicated that both nNOS and

iNOS play a significant role in potentiating dopaminergic destruction in the presence of MPTP [25–28],

Fig. 7. The effect of DA1 and h1-adrenergic receptor antagonists on L-DOPA mediated iNOS inhibition. C6 cells + LPS/IFN-

gammaF SCH-23390 (1.5 mM) or atenolol (1 mM) were treated with varying concentration of L-DOPA. The data are

expressed as NO2� [% control] and plotted as the meanF S.E.M., (n = 4). Significance of difference from the controls was

determined by a two-way ANOVA. There were no statistical differences between groups.

M.K. Soliman et al. / Life Sciences 72 (2002) 185–198194

whereas others find no evidence of this relationship [29]. It is believed that during brain injury, glial cell

iNOS releases large amounts of NO for extended periods. Moreover, the elevated levels of NO have

been correlated to the extensive injury associated with CNS viral infections [30], Alzheimer’s disease

[31] multiple sclerosis [32], ischemia and closed head injury [33–35]. However, as in the case with the

study of PD and NO, recent reports indicated a neuroprotective effect of NO during toxic conditions. It

has been reported that NO can act as an antioxidant and provide protection against neuronal injury

during encephalitis, ischemia and reperfusion [36,37], iron induced lipid peroxidation [38] and Ca+ 2

overload [39].

A precaution to consider regarding the findings of this study is that the concentrations of L-DOPA

required to mediate the downregulation of iNOS were extremely high. In the human body, L–DOPA has

a low bioavailability, is not lipid soluble, and an active transport process is required for its delivery from

intestine to the blood and from the blood to the brain [40]. In human subjects, a typical dose of Sinemet

(250 mg L-DOPA and 25 mg carbidopa), can lead to a peak L-DOPA plasma concentrations (21.7 Ag/ml) at 1 H and peak CSF concentrations (1.14 Ag/ml) at 8 H [41]. The concentration of L-DOPA required

in this study to downregulate iNOS was 500 AM, which is equal to 98.6 Ag/ml, significantly higher than

the applicable therapeutic concentration range.

From the data in this study, it appears that at the therapeutic range (< 100 AM), treatment with L-

DOPA resulted in the simultaneous elevation of both NO2� and O2, which together would lead to the

formation of peroxynitrite (ONOO-). Although C6 cells express MnSOD mRNA, the level of SOD was

inadequate to dispose of the vast O2� produced from L-DOPA autoxidation [42]. The dual presence of

both O2� and NO2

� can lead to a neurotoxic environment as elevated levels of ONOO�can mediate

destructive effects on thiols, amines and proteins and inhibitory effects on cellular respiration [43].

Moreover, the toxicity of astrocytic iNOS on neurons is attenuated with treatment of exogenous SOD,

indicating a paramount destructive role for ONOO�and inadequate SOD levels in glia [44]. On the other

hand, evidence from this study indicated that C6 glioma have a robust capacity to rapidly dispose of

H2O2 generated from the autoxidation of L-DOPA. These findings corroborate previous research [45].

Moreover, while L-DOPA can lead to accumulation of O2�, the addition of SOD resulted in higher levels

of NO2�. This may be due to either the toxic effects of L-DOPA or the reduced accumulation of ONOO�,

which would exert toxic effects on respiration.

Interestingly, previous research from our lab has discovered that dopamine (DA) and norepinephrine

can inhibit LPS/cytokine iNOS protein expression in glioma cells (data not published). Although DA is

thought to be autoxidized similar to L-DOPA, O2� generated from the autoxidation of DA, had no effect

on iNOS. The difference between the autoxidation of DA and L-DOPA defines a specific role of O2�

involvement in L-DOPA mediated effects. For example, the toxic effects of L-DOPA are mediated by

both H2O2 and O2�, as both SOD and catalase are protective against L-DOPA mediated toxicity in

neurons [1,46]. However, DA toxicity is only mediated via its autoxidative product H2O2, and despite

the elevation in O2�, SOD is not protective against this type of toxicity [1]. Similarly, O2

� generated by 6-

hydroxydopamine autoxidation is also not lethal, only H2O2 appears to be the substance responsible for

toxicity [47]. These research findings, indicate a specific modulatory component of the O2� generated

from L-DOPA, which is different from that generated by oxidation of typical catechols. Furthermore, it

has been reported that effects of DA on iNOS expression are receptor mediated, ruling out effects by

ROS [48].

It was concluded from this study that L-DOPA contributed to the modulation of iNOS and to the

increase of O2� in stimulated glioma cells in vitro. Collectively, the data presented here, along with other

M.K. Soliman et al. / Life Sciences 72 (2002) 185–198 195

research work, point out to the fact that prolong use of L-DOPA might be associated with the production

of neurotoxic substances that can speed the neurodegeneration process of the striatal tissue.

Acknowledgements

This work was supported by National Institutes of Health grants GM 08111 and RR03020.

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