www.elsevier.com/locate/brainres

Brain Research 1004 (2004) 29–44

Research report

The role of oxidative stress, impaired glycolysis and mitochondrial

respiratory redox failure in the cytotoxic effects of 6-hydroxydopamine

in vitro

Elizabeth A. Mazzio, Renee R. Reams, Karam F.A. Soliman*

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

Accepted 11 December 2003

Abstract

The neurotoxin, 6-hydroxydopamine (6-OHDA) has been implicated in the neurodegenerative process of Parkinson’s disease. The current

study was designed to elucidate the toxicological effects of 6-OHDA on energy metabolism in neuroblastoma (N-2A) cells. The toxicity of 6-

OHDA corresponds to the total collapse of anaerobic/aerobic cell function, unlike other mitochondrial toxins such as MPP+ that target specific

loss of aerobic metabolism. The toxicity of 6-OHDA paralleled the loss of mitochondrial oxygen (O2) consumption (MOC), glycolytic activity,

ATP, H+ ion gradients, membrane potential and accumulation of the autoxidative product, hydrogen peroxide (H2O2). Removing H2O2 with

nonenzymatic stoichiometric scavengers, such as carboxylic acids, glutathione and catalase yielded partial protection. The rapid removal of

H2O2 with pyruvate or catalase restored only anaerobic glycolysis, but did not reverse the loss of MOC, indicating mitochondrial impairment is

independent of H2O2. The H2O2 generated by 6-OHDA contributed toward the loss of anaerobic glycolysis through lipid peroxidation and lactic

acid dehydrogenase inhibition. The ability of 6-OHDA to maintain oxidized cytochrome c (CYT-C-OX) in its reduced form (CYT-C-RED),

appears to play a role in mitochondrial impairment. The reduction of CYT-C by 6-OHDA, was extensive, occurred within minutes, preceded

formation of H2O2 and was unaffected by catalase or superoxide dismutase. At similar concentrations, 6-OHDA readily altered the valence state

of iron [Fe(III)] to Fe(II), which would also theoretically sustain CYT-C in its reduced form. In isolated mitochondria, 6-OHDA had negligible

effects on complex I, inhibited complex II and interfered with complex III by maintaining the substrate, CYT-C in a reduced state. 6-OHDA

caused a transient and potent surge in isolated cytochrome oxidase (complex IV) activity, with rapid recovery as a result of 6-OHDA recycling

CYT-C-OX to CYT-C-RED. Typical mitochondrial toxins such as MPP+, azide and antimycin appeared to inhibit the catalytic activity of ETC

enzymes. In contrast, 6-OHDA alters the redox of the cytochromes, resulting in loss of substrate availability and obstruction of oxidation–

reduction events. Complete cytoprotection against 6-OHDA toxicity and restoredMOCwas achieved by combining catalase with CYT-C (horse

heart). In summary, CYT-C reducing properties are unique to catecholamine neurotransmitters, and may play a significant role in selective

vulnerability of dopaminergic neurons to mitochondrial insults.

D 2004 Elsevier B.V. All rights reserved.

Theme: Disorders of the nervous system

Topic: Neurotoxicity

Keywords: 6-Hydroxydopamine; Glycolysis; Mitochondria; Complex; Dopamine; DOPAC; Parkinson’s disease; Hydrogen peroxide

1. Introduction

Parkinson’s disease pathology involves neurological de-

generation within the substantia nigra (SN), accompanied by

oxidative stress and mitochondrial anomalies [10,40]. A

contributing factor toward oxidative stress is the endoge-

0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2003.12.034

* Corresponding author. Tel.: +1-850-599-3306; fax: +1-850-599-

3667.

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

nous production of 6-hydroxydopamine (6-OHDA), a toxic

oxidative metabolite of dopamine (DA). 6-OHDA is known

to contribute to the production of hydrogen peroxide (H2O2)

and other reactive oxygen species (ROS), capable of

destroying cellular structural and functional apparatus with-

in the SN [5,12]. Similarly, the toxic effects of 6-OHDA

correspond to lipid peroxidation, loss of endogenous gluta-

thione and neuronal apoptosis as demonstrated by nuclear

condensation, DNA degradation [12] and the expression of

early response gene proteins c-Fos, c-Jun [33].

E.A. Mazzio et al. / Brain Research 1004 (2004) 29–4430

Glutathione plays a significant role in antagonizing the

toxic effects of 6-OHDA, and its depletion with buthionine-

(S,R)-sulfoximine potentiates toxicological damage [35],

indicating a role for H2O2 in the neurodegenerative mecha-

nism of this toxin [24]. In vivo, intrastriatal injections of 6-

OHDA can induce extensive nigrostriatal lesions that occur

due to oxidative stress [23,29]. The lesions are similar to

those that occur in response to systemic administration 1-

methyl-4-phenyl-1,2,3-6-tetrahydropyridine (MPTP) [21].

While, MPTP is not a direct prooxidant, it is converted to

the active toxin, 1-methyl-4-phenylpyridinium (MPP+),

resulting in inhibition of mitochondrial complex I [37]. It is

thought that MPP+ can trigger the rapid efflux of DA from

nerve terminals [32], potentially leading to the oxidation of

DA, which can form 6-OHDA [9,17]. Once formed, 6-

OHDA can induce a cyclic cascade of toxicity parallel to

accumulation of ROS produced through the release of free

iron from bound ferritin [9], H2O2, and the production of

hydroxyl radicals, which further perpetuate oxidation of DA

into 6-OHDA [38].

Although a number of studies have demonstrated detri-

mental effects of 6-OHDA in vitro and in vivo involving the

ROS medicated component, it is unclear as to what extent

mitochondrial impairment or free radicals each contribute to

the damage. Moreover, with the exception of few reports

investigating complex I as a target, it is still uncertain as to

how 6-OHDA directly impairs the function of the mito-

chondria [5,24].

Preliminary data from our laboratory reveal that damage

to the mitochondria by 6-OHDA is independent of ROS. We

consistently found that antioxidants such as glutathione and

pyruvate protect against 6-OHDAwithout reversing the loss

of mitochondrial respiration. Conversely, these antioxidants

reverse the loss of mitochondrial O2 consumption parallel to

cytoprotection against toxicity of H2O2, the primary autox-

idative product of 6-OHDA [25]. Moreover, we have found

that 6-OHDA and catecholamines are unique from other

neurotransmitters, having potent chemical reducing effects

on cytochrome c (CYT-C). 6-OHDA is known to reduce

Fe(III) to Fe(II), and similar events may be occurring within

the heme core of CYT-C (14). If the reduction of CYT-C by

6-OHDA occurs in the mitochondria, this could potentially

obstruct electron flow through complexes II–IV. Yet, there

has not been an investigation to examine if CYT-C reduc-

tion by either 6-OHDA or catecholamines contribute to the

loss of oxidation–reduction events through the electron

transport chain (ETC). Past research defining a role for

CYT-C in the toxicity of 6-OHDA, has been limited to the

investigation of apoptotic signaling involving the release of

mitochondrial CYT-C, activation of caspase-3 and -9 and

DNA fragmentation [15,39].

We hypothesize that 6-OHDA may be contributing to the

failure of mitochondrial respiration, through preventing

availability of oxidized CYT-C as a substrate carrier for

electrons, primarily through altering the redox state of Fe in

complex III. Complex III (cytochrome c–coenzyme Q oxi-

doreductase) contains cytochromes b and c, and accepts

electrons from ubiquinol in complex II. 6-OHDA may be

interfering with availability of CYT-C-OX in complex III,

rendering loss of electron transfer down the ETC to its

terminal receptor O2 in complex IV (cytochrome oxidase).

Therefore, the present study was designed to elucidate the

mechanism of 6-OHDA toxicity as it relates to ROS, effects

on CYT-C, impaired aerobic/anaerobic metabolism, mito-

chondrial oxidative phosphorylation and complexes I, II, III,

IV activities.

2. Materials and methods

Murine brain neuroblastoma (N-2A) cells were obtained

from American Type Culture Collection (Manassas, VA).

Dulbecco’s modified Eagle medium (DMEM), L-glutamine,

fetal bovine serum (FBS)-heat inactivated, Hank’s balanced

salt solution (HBSS), phosphate buffered saline (PBS) and

penicillin/streptomycin were supplied by Fischer Scientific,

Mediatech (Pittsburgh, PA). CYT-C, 6-OHDA and all other

chemicals and supplies were purchased from Sigma (St.

Louis, MO).

2.1. Cell culture

Murine brain neuroblastoma (N-2A) cells exhibit neuronal

morphology, and are vulnerable to the toxic effects of

mitochondrial toxins, ROS and 6-OHDA [26,28]. Briefly,

cells were grown in DMEM+phenol red, supplemented with

10% FBS (v/v), 4 mM L-glutamine, penicillin (100 U/ml)/

streptomycin (0.1 mg/ml) and 20 AM of sodium pyruvate.

The cells were cultured under stable conditions maintained at

temperature of 37 jC and 5% CO2 in atmosphere. Every 2–5

days, cells were subcultured, and for experiments, the cells

were plated at approximately 0.5� 106 cells/ml in 96-well

plates. The experimental plating media consisted of DMEM

(without phenol red), supplemented with 1.8% FBS (v/v),

penicillin (100 U/ml)/streptomycin (0.1 mg/ml), 4 mM L-

glutamine and 20 AM sodium pyruvate. A stock solution of

each experimental compound was prepared in HBSS con-

taining 5 mM (N-[2-hydroxyethylpiperazine]-NV-[2-ethane-sulfonic acid]) (HEPES), adjusted to a pH of 7.4. Six

concentrations of each treatment were prepared to span a

1000-fold dilution range. Solutions of 6-OHDA were pre-

pared fresh daily, immediately prior to use. In all experiments,

the cells were exposed to experimental treatments f 2 h prior

to addition of 6-OHDA, and incubated for 24 h at 37 jC in 5%

CO2/atmosphere.

2.2. Measurements of cell viability

Resazurin (Almar blue) indicator dye was used to assess

cell viability as previously described [11]. Briefly, a work-

ing solution of resazurin (0.5 mg/ml) was prepared in sterile

PBS and added to the samples for 6–8 h. Blanks and

E.A. Mazzio et al. / Brain Research 1004 (2004) 29–44 31

controls were run simultaneously to account for interference

or interplate variability. Quantitative analysis of dye con-

version was measured on a Cambridge microplate fluorom-

eter—Model 7620, version 5.02 (Cambridge Technologies,

Watertown, MA)—with settings fixed at [550/580] [excita-

tion/emission] wavelengths.

Loss of cell viability by 6-OHDA was confirmed with

fluorescein diacetate (FD) after 24 h, as previously de-

scribed [19,26]. Briefly, FD (final working concentration:

20 Ag/ml) was incubated with the samples at 37 jC for 20

min. Images were acquired using a Nikon Eclipse TE300

inverted microscope with a SPOT cooled CCD color digital

camera (Diagnostic Instruments, Sterling Hts., MI) using a

blue filter cube. The images were further captured by SPOT

Imaging Software Version 2.2.

2.3. Measurements of cell death

Propidium iodide (PI) was used to observe cell death

after 24 h incubation with 6-OHDA [19,26]. Briefly, PI

(final working concentration: 7 Ag/ml) was added to the

samples and returned to the incubator for 15 min. Samples

were evaluated using a Nikon Eclipse TE300 inverted

microscope with a SPOT cooled CCD color digital camera

(Diagnostic Instruments). Images were captured by SPOT

Imaging Software Version 2.2.

2.4. Measurements of endoplasmic reticulum and intracel-

lular organelle membrane potentials

Endoplasmic reticulum (ER), mitochondria and intracel-

lular organelle transmembrane potentials were determined

using 3,3V-dihexyloxacarbocyanine iodide [DiOC6(3)] after

24 h incubation with 6-OHDA [26,34]. Briefly, DiOC6(3)

(final working concentration: 4 Ag/ml) was added to the

samples and incubated at 37 jC for 15 min. Samples were

evaluated on a Nikon Eclipse TE300 inverted microscope

with a SPOT cooled CCD color digital camera (Diagnostic

Instruments) using a blue filter cube. DiOC6(3) is typically

quantified at approximately 480 nm excitation and 600 nm

emission. Images were captured by SPOT Imaging Software

Version 2.2.

2.5. Measurements of intracellular pH

2,7V-bis(2-carboxyethyl)-5(6) carboxy-fluorescein ace-

toxymethyl ester (BCECF-AM) was used to measure cyto-

solic pH after 24 h incubation with 6-OHDA [26,31]. Briefly,

the stock solution of BCECF-AM was prepared in ethanol (1

mg/ml) and stored at 0 jC. The reagent (final working

concentration 7 Ag/ml) was added to the samples and incu-

bated at 37 jC for 15 min. Samples were evaluated utilizing a

Nikon Eclipse TE300 inverted microscope with a SPOT

cooled CCD color digital camera (Diagnostic Instruments)

using a blue filter cube. BCECF-AM is typically quantified at

490 nm (blue) excitation and 535 nm/emissions. Images were

captured using SPOT Imaging Software Version 2.2.

2.6. Hydrogen peroxide quantifications

Hydrogen peroxide was quantified during the autoxida-

tion of 6-OHDA using a peroxidase-linked continuous assay

as previously described [16,26]. To assess nonenzymatic

scavenging capability, H2O2 (500 AM) was prepared in PBS

and incubated with experimental compounds for 30 min.

The chromogenic solution was prepared in PBS + 2 mM

HEPES at a pH of 7.4 with final working concentration of 1

mM vanillic acid, 500 AM 4-aminoantipyrine and horserad-

ish peroxidase (4 U/ml). The chromogenic reagent was

added to the samples, and incubated for 10 min at 37 jC.Controls and blanks were run simultaneously to account for

interference and a standard curve was established with H2O2

(1–500 AM). The data were quantified at 490 nm on a

Cambridge UV microplate spectrophotometer—Model

7520, version 5.02 (Cambridge Technologies).

2.7. O2 consumption determinations

Mitochondrial O2 consumption (MOC) is indicative of

cell respiration. MOC was analyzed using a Clark electrode

after 24 h incubation with 6-OHDAF experimental com-

pounds (Hansatech Instruments, Norfolk, England) [39].

Briefly, the electrode was calibrated with air-saturated

deionized water and deionized water-containing sodium

dithionite. The samples or control blanks were loaded into

a chamber jacket at 25 jC. After instrument stabilization

and equilibration, a reading was taken. The data were

acquired as nM of O2/ml and converted to % control.

2.8. Determination of cytochrome c reduction

The reduction of CYT-C (derived from horse heart, 130

AM) was determined monitoring the increase in optical

density (O.D.) at 550 nm on a multiplate UV spectropho-

tometer. Solutions of CYT-C were prepared in experimental

plating medium. The oxidation–reduction range for CYT-C

was established by monitoring the absorbance change from

CYT-C-OX to CYT-C-RED by 3 mM of ascorbic acid.

2.9. Measurements of mitochondrial respiratory chain

activity

Mitochondrial activities were determined using a modi-

fication of a method described elsewhere [6,41]. Briefly, N-

2A cell pellets were washed in PBS and then dispersed in

ice-cold mitochondrial isolation buffer consisting of HEPES

(20 mM), pH 7.4, MgCl2 (1.5 mM) KCl (10 mM), ethyl-

enediaminetetraacetic acid (EDTA) (1 mM), dithiothreitol (1

mM), ethylene glycol-bis(h-aminoethyl ether)-N,N,NV,NV-tetraacetic acid (1 mM), phenylmethylsulfonyl fluoride (1

mM), leupeptin (10 AM), aprotinin (10 AM) and sucrose

E.A. Mazzio et al. / Brain Research 1004 (2004) 29–4432

(250 mM). The homogenate was chilled, disrupted and

centrifuged at 500� g for 10 min at 4 jC. The mitochondria

were pelleted by centrifugation at 9400� g for 10 min at 4

jC.The protein concentration of the mitochondrial homog-

enate was adjusted so that the final working concentration

was equal to f 200 Ag/ml. Protein was determined with the

Lowry procedure, using bovine albumin as a standard (0–

100 mg/dl) [22]. Existing protocols for mitochondrial assays

utilize substrate reaction mixtures at a stabilized pH of 7.4.

However, it was our observation that optimal enzyme

conditions for complexes II, III and IV in validation controls

were observed at a pH of f 5.6. This anomaly may be due

to the unusual nature of immortal cell lines that allow them

to thrive under acidic conditions. Control blanks were

established for all experimental compounds, for each com-

plex activity assay, to identify potential reactivity between

inhibitors and substrates, in the absence of mitochondrial

homogenates.

2.10. Complex I activity determinations

NADH:ubiquinone oxidoreductase activity was deter-

mined by quantifying the decrease in UV absorbance that

accompanied the oxidation of NADH at 340 nm. The

reaction mixture consisted of sodium phosphate (8 mM),

MgSO4 (10 mM), NaCl (136 mM), KH2PO4 (1.5 mM),

CaCl2 (1 mM), HEPES (25 mM) and KCl (2.5 mM) in

deionized H20. The buffer contained 2.5 mg/ml bovine

serum albumin, 50 Ag/ml antimycin A, sodium azide 4

mM, 50 AM decylubiquinone and flavin adenine dinucleo-

tide (FAD)/flavin mononucleotide (FMN) 25 AM. The final

pH was readjusted to 7.4. The reaction was initiated by the

addition of 500 AM NADH. A standard curve was estab-

lished for NADH along with an NAD+ nonresponse control

(1–2500 AM).

2.11. Complex II activity determinations

Succinate:2,6-dichlorophenolindophenol (DCPIP) oxido-

reductase activity was quantified by measuring the decrease

in UV absorbance due to the reduction of 2,6-DCPIP at 600

nm. The reaction mixture consisted of sodium phosphate (8

mM), NaCl (136 mM), KH2PO4 (1.5 mM), MgSO4 (10

mM), CaCl2 (1 mM), HEPES (25 mM) and KCl (2.5 mM)

in deionized H20. The buffer contained 2.5 mg/ml bovine

serum albumin, 12 mM succinic acid, 50 AM decylubiqui-

none, 25 AM FAD/FMN, 75 AM DCPIP, 50 Ag/ml anti-

mycin A and sodium azide 4 mM. The final pH was

readjusted to 5.6. DCPIP reduction was monitored over

time. A standard curve was established using DCPIP

(1–100 AM).

2.12. Complex III activity determinations

Ubiquinol:cytochrome c oxidoreductase was determined

by monitoring the reduction of oxidized cytochrome c at

550 nm. The reaction mixture consisted of sodium phos-

phate (8 mM), NaCl (136 mM), KH2PO4 (1.5 mM), CaCl2(1 mM), MgSO4 (10 mM), HEPES (25 mM) and KCl (2.5

mM) in deionized H20. The reaction mixture contained 2.5

mg/ml bovine serum albumin, 12 mM succinic acid, 50 AMdecylubiquinone, 25 AM FAD/FMN, 4 mM sodium azide

and 125 AM cytochrome c. The final pH was readjusted to

5.6. The oxidation–reduction state of cytochrome c was

established with ascorbic acid.

2.13. Complex IV activity determinations

Cytochrome c oxidase was determined by monitoring the

oxidation of reduced cytochrome c at 550 nm. The reaction

mixture consisted of sodium phosphate (8 mM), NaCl (136

mM), KH2PO4 (1.5 mM), MgSO4 (10 mM), CaCl2 (1 mM),

HEPES (25 mM) and KCl (2.5 mM) in deionized H20. The

reaction mixture contained 2.5 mg/ml bovine serum albu-

min, 50 AM decylubiquinone, 25 AM FAD/FMN, 50 Ag/ml

antimycin and 15 mM malonate. Cytochrome c 125 AM was

added in the presence of 3 mM ascorbic acid. The final pH

was readjusted to 5.6. Oxidation was monitored over time.

2.14. Determinations of ATP levels

ATP was assessed by firefly bioluminescence as previ-

ously described [39]. Briefly, samples were frozen in a

lysing buffer and stored at � 80 jC. A luciferase–luciferin

solution in glycine buffer (Sigma) was reconstituted with

sterile deionized water, 0.04 mg/ml luciferase, 0.26 mM

luciferin, 19.5 mM MgSO4, 1.95 mM EDTA, 195 mM

glycine and 324 mM Tris–HCl (pH 7.4). The ATP reagent

was added and light units were converted to CPM and

quantified on a Beckman LS 6500 Scintillation Counter

(Beckman, and Schaumburg, IL, USA). The data were

converted to % control.

2.15. Determinations of lactic acid levels

Lactic acid was quantified using a colorimetric enzymatic

assay (Procedure No. 735, Sigma). The base reagent was

mixed with a chromogenic solution prepared in PBS + 2

mM HEPES, pH 7.4, containing (final working concentra-

tions) 1 mM vanillic acid, 500 AM 4-aminoantipyrine and

horseradish peroxidase (4 U/ml). The reagent was added to

each sample and incubated for 15 min at 37 jC. Lactate wasquantified at 490 nm on a UV microplate spectrophotome-

ter. A lactic acid standard curve was generated by preparing

a dilution of lactic acid (10 AM–10 mM) in ultralow serum

plating media minus phenol red. Although control blanks

were run to eliminate potential interference effects of 6-

OHDA on chromogenic reagent reactivity for H2O2, to

ensure accuracy, HPLC quantification of lactic acid was

also examined for MPP+ and 6-OHDA—IC50 samples (500

and 175 AM), respectively. Briefly, lactate was quantified

using an Interaction Ion-300 anion exchange column on a

E.A. Mazzio et al. / Brain Research 1004 (2004) 29–44 33

Waters 2487/Millennium 32 version 3.20, system controller

with 2690 Separations Module and UV detector (210 nm).

The mobile phase consisted of 0.01 N H2SO4, the flow rate

was set at 0.4 ml/min and column temperature 40 jC.

2.16. Measurements of LDH activity

Pyruvic acid was converted to lactate in the presence of

lactic acid dehydrogenase (LDH) (from rabbit muscle, type

V-S [EC 1.1.1.27]), at a concentration of 1 U/ml in the

Fig. 1. (A) The effect of 6-OHDA on N-2A cell viability, ATP, lactic acid pro

atmosphere. The data are expressed as % live control and are plotted as the meanFcontrol was determined by a one-way ANOVA, followed by a Tukey mean compari

of lactate was also corroborated with HPLC analysis during 6-OHDA (175 AM) tox

acid production after 24 h incubation at 37 jC in 5% CO2/atmosphere. The data a

Significance of difference between the treatment groups and the control was deter

hoc test, *P< 0.001. Quantification of lactate was corroborated with HPLC analy

presence of adenosine, 2V,5V-diphospahte (ADP) (1.5 mM),

h-NADH (1 mM) +magnesium sulfate (MgSO4) (5 mM)

prepared in buffered distilled water, pH 7.4. Dose-depen-

dent enzyme activity in the presence of varying concentra-

tion of the substrate (pyruvate) was determined by

quantifying formation of lactic acid as described above.

In order to assess the effects of H2O2 on LDH, the

peroxidase-linked reaction was not used due to potential

interferences. LDH inhibition was established by monitor-

ing the oxidation of NADH by LDH at 340 nm on a UV

duction and O2 consumption after 24 h incubation at 37 jC in 5% CO2/

S.E.M. (n= 4). Significance of difference between treatment groups and the

son post hoc test. All groups [*] P< 0.001 or single groups *. Quantification

icity (right panel). (B) The effect of MPP+ on N-2A cell viability and lactic

re expressed as % live control and are plotted as the meanF S.E.M. (n= 4).

mined by a one-way ANOVA, followed by a Tukey mean comparison post

sis during MPP+ (500 AM) toxicity (right panel).

E.A. Mazzio et al. / Brain Research 1004 (2004) 29–4434

spectrophotometer. Negative control blanks were estab-

lished in the presence of H2O2 containing the substrate

solution minus the enzyme.

2.17. Measurements of ferritin–Fe+2 release

The release of free Fe+ 2 from horse spleen bound

ferritin type I (15 mg/ml) was determined by monitoring

the release of ferrous iron in the presence of various

concentrations of 6-OHDA [1]. The ferrous iron reactant

(FIR) solution was prepared and kept from light. The FIR

consisted of 1,10-phenanthroline (1 mg/ml) dissolved in

deionized water. Separate solutions of hydroxylamine hy-

drochloride and sodium acetate (100 mg/ml) were prepared

in deionized water. To a sample volume (81% total vol-

ume), the reagent was added (19% of total volume) and

consisted of total volume (1% hydroxylamine solution, 8%

Fig. 2. (A) Autoxidative H2O2 produced by 6-OHDA in the absence or presence of

represent H2O2 produced (nM/mg protein or volume equivalent) and are plotted as

groups and the zero control was determined by a one-way ANOVA, followed by a T

OHDA on oxidized CYT-C (130 AM) in the presence of catalase [2500 U/ml] or ca

% CYT-C-RED relative to ascorbic acid controls (100% reduced). The data are p

treatment groups and the control was determined by a one-way ANOVA, followed

indicates no statistical differences between the groups.

sodium acetate solution, 10% phenathroline). After 15 min

at 25 jC, Fe+ 2 was quantified at 550 nm on a UV

microplate spectrophotometer. A standard curve for Fe+ 2

(3 AM–3 mM) was established both in the presence or

absence of ferritin. It was critical that controls for all

experimental compound dilutions consisted of identical

samples containing ferritin + modified FIR (hydroxyl-

amine/sodium acetate minus 1,10-phenanthroline).

2.18. Data analyses

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 ANOVA, followed by

a Tukey post hoc means comparison test or Student’s t-test.

N-2A cells over 24 h incubation at 37 jC in 5% CO2/atmosphere. The data

the meanF S.E.M. (n= 4). Significance of difference between the treatment

ukey mean comparison post hoc test. *P< 0.001. (B) Reducing effects of 6-

talase [1250 U/ml] + superoxide dismutase [1250 U/ml]. The data represents

lotted as the meanF S.E.M. (n= 4). Significance of difference between the

by a Tukey mean comparison post hoc test. *P < 0.001. A two-way ANOVA

E.A. Mazzio et al. / Brain Research 1004 (2004) 29–44 35

3. Results

3.1. Toxic profile of 6-OHDA toxicity

N-2A cells were incubated with varying concentration of

6-OHDA for 24 h, leading to complete metabolic collapse

as indicated by the reduction in lactic acid, ATP and whole

cell O2 consumption (Fig. 1A). The decrease in lactate

associated with toxicity of 6-OHDA is unique from other

mitochondrial toxins such as MPP+, that typically lead to an

increase of lactic acid due to increased compensatory

anaerobic glycolysis (Fig. 1B). In Fig. 1, lactic acid profiles

are demonstrated in the graph (left panel) and corroborated

with HPLC analysis (right panel).

3.2. Chemical properties of 6-OHDA

Two distinct chemical properties of 6-OHDA, that occur

independent of a biological system include autoxidative

generation of H2O2 in aqueous solution (Fig. 2A) and

reduction of CYT-C (Fig. 2B). Fig. 2A demonstrates that

under experimental cell culture conditions, 6-OHDA can

produce significant amount of H2O2. Furthermore, N2A

cells contain limited antioxidant capability and are ineffec-

tive in removing a large portion of the H2O2 at 24 h. The

Fig. 3. Fluorescence photographic analysis of 6-OHDA effects on membrane pot

activity after 24 h incubation at 37 jC in 5% CO2/atmosphere.

reduction of CYT-C by 6-OHDA occurred within minutes

and is independent of H2O2 or superoxide (Fig. 2B). The

loss of cell viability in the presence of varying concentra-

tion of 6-OHDA corresponds to CYT-C reduction, H2O2

production, and the loss of intracellular organelle potential,

H+ ion gradients, a reduction in live cell esterase activity

and increased propidium iodide nuclear staining, as de-

monstrated in Fig. 3. Thiobarbituric reactive substances

were measured during 6-OHDA toxicity at 50, 250 and 500

AM as a measure of lipid peroxidation. The data indicate

that cell death corresponded to increased concentrations of

malondialdehyde, 1.3F 1.2, 8.6F 2.1 and 21.1F 2.4 nM/

mg protein, respectively. These data indicate that H2O2

formed by 6-OHDA can lead to lipid peroxidation, poten-

tially contributing to the observed loss of cell membrane

integrity.

3.3. Screening for cytoprotection against 6-OHDA

In order to elucidate potential cytoprotective com-

pounds, over a hundred compounds were screened against

6-OHDA (175 AM) toxicity. Experimental compounds

included superoxide dismutase, plant-derived polyphenolic

antioxidants, vitamins, amino acids, energy intermediates,

protein kinase inhibitors, calcium channel blockers, nitric

ential, H+ concentration, nucleic acid staining and loss of live cell esterase

Fig. 4. (A–E) The cytoprotective effects of carboxylic acid intermediates and glutathione against 6-OHDA (175 AM) toxicity at 24 h incubation. The data in

Panels A and C represent cell viability as % live control and are expressed as the meanF S.E.M. (n= 4). The data in Panels B and D represent corresponding

antioxidant effects, quantified by ability to trap H2O2 (500 AM) 30 min after incubation at room temperature. The data represent residual AM H2O2 remaining

and are expressed as the meanF S.E.M. (n= 4). The significance of difference between treatment and control groups were determined by a one-way ANOVA,

followed by a Tukey mean comparison post hoc test. *P < 0.001. (E) The effects of pyruvate (10 mM) and catalase (2500 U/ml) on restoring the loss of

mitochondrial O2 consumption (MOC) and viability by either 6-OHDA (175 AM) or H2O2 (500 AM) at 24 h incubation � 37 jC in 5% CO2/atmosphere. The

data values as % live control, and are expressed as the meanF S.E.M. (n= 4). The significance of difference between treatment and control groups (in the

presence of toxins) were determined by a one-way ANOVA, followed by a Tukey mean comparison post hoc test. *P < 0.001.

E.A. Mazzio et al. / Brain Research 1004 (2004) 29–4436

Fig. 5. (A and B) The effects of H2O2 on isolated LDH [EC 1.1.1.27]

enzyme activity assessed in the presence of ADP, h-NADH+Mg-

SO4F substrate. Panel A is the control at 15 min, analyzed with increasing

concentration of substrate. Panel B represents LDH activity + 1 mM

pyruvateF 500 AM H2O2 over time. The data are plotted as the

meanF S.E.M. (n= 4). Significance of difference between the treatment

groups and the control was determined by a one-way ANOVA, followed by

a Tukey mean comparison post hoc test. *P< 0.001. A two-way ANOVA

indicates no statistical differences between the groups in Panel B.

E.A. Mazzio et al. / Brain Research 1004 (2004) 29–44 37

oxide synthase inhibitors, and electrolytes. The results

revealed that only five compounds were protective

against 6-OHDA. These included the tricarboxylic acid

intermediates: pyruvate, oxaloacetate, a-ketoglutarate, re-

duced glutathione (GSH) and N-acetyl-L-cysteine (NAC)

(Fig. 4A and C). The cytoprotection observed by these

compounds paralleled their nonenzymatic stoichiometric

scavenging potency against H2O2 (500 AM) (Fig. 4B and

D). In order to ensure the antioxidant effects of carbox-

ylic acids were not due to the effects of acidity on the

chromogenic peroxidase enzyme system, neutralized car-

boxylic acid solutions were also analyzed for radical

trapping. The results were identical, indicating that pro-

tective properties are related to the direct antioxidant

capabilities of these acids. These data clearly indicate a

role for H2O2 in the toxicity of 6-OHDA. Fig. 4E

corroborates protective effects of pyruvate specifically

against H2O2 or 6-OHDA. However, these data indicate

that protection against the toxicity of H2O2 by pyruvate

and catalase, are associated with restored function to the

mitochondria. This is vastly different from toxicity of 6-

OHDA, where pyruvate and catalase are protective

through restoring anaerobic metabolism, evident by the

sustained loss of respiration. These data suggest that

mitochondrial impairment by 6-OHDA is independent of

ROS production.

The loss of anaerobic glycolysis appears to be the direct

result of an action caused by H2O2. While H2O2 can

induce loss of membrane integrity as demonstrated in

Fig. 3, H2O2 may also attenuate LDH activity (Fig. 5).

Fig. 5A is a control for the LDH assay, with increasing

concentration of pyruvate at 15 min. Fig. 5B quantifies

LDH activity with 1 mM pyruvateFH2O2 (500 AM) over

19 min. The data show significant reduction in LDH

activity by peroxide.

3.4. Restoration of anaerobic mitochondrial function by

addition of catalase and CYT-C

To define a specific role for H2O2 in cell death, we used

catalase rather than other antioxidants, such as GSH that

could potentially contribute to protective effects through

another mechanism. Due to the profound effects of 6-

OHDA on reduction of CYT-C, toxicity assays were

established to elucidate if exogenous CYT-C can provide

any protection in the absence or presence of catalase. The

effects of catalaseFCYT-C against 6-OHDA (175 AM)

toxicity after 24 h incubation are presented in Fig. 6A. The

right-hand panel displays the extent of H2O2 (500 AM)

metabolized within 1 min with varying concentration of

catalase. The left-hand panel demonstrates cell viability in

the presence of 6-OHDA with varying concentration of

catalaseF 62.5 AM of CYT-C. A remarkable synergistic

effect was evident, when combined 12.5 U/ml of catalase

and 62.5 AM CYT-C yielded 100% cytoprotection. Fig. 6B

demonstrates the effects of catalase and/or CYT-C on

restoring mitochondrial function during cytoprotection

against 6-OHDA (175 AM) after 24 h incubation. Fig.

6B, (right panel) demonstrates that the cytoprotection

observed in the presence of catalase does not restore

mitochondrial O2 consumption. Conversely, there is a

greater decline in cell respiration indicating efficient an-

aerobic compensatory survival. The data in Fig. 6B (left

panel) clearly indicate that while CYT-C alone leads to

E.A. Mazzio et al. / Brain Research 1004 (2004) 29–4438

Fig. 7. Mitochondrial respiratory chain activity controls. Individual complex assays were acquired in the presence of mitochondrial isolates in the presence of

respective inhibitors. Each assay was monitored with variation in time and concentration. The Vmax and product formed at Vmax (nM/mg protein) was

established for each complex. The data represent activity [nM/mg protein of mitochondrial homogenate/time at 37 jC] and are plotted as the meanF S.E.M.

(n= 4). Significance of difference between the treatment groups and the control was determined by a one-way ANOVA, followed by a Tukey mean comparison

post hoc test. *P< 0.001.

E.A. Mazzio et al. / Brain Research 1004 (2004) 29–44 39

slight protection against mitochondrial impairment, in

combination with catalase, mitochondrial O2 consumption

is completely restored to 100% of controls. The synergic

effects on O2 consumption as demonstrated in Fig. 6B at

62.5 AM CYT-C + 1250 U/ml catalase correspond to cell

viability in Fig. 6A at 62.5 AM CYT-C + 1250 U/ml

catalase. In order to examine the contribution of each

component toward the cytoprotection against 6-OHDA,

cells were incubated for 24 h with either 125 AM CYT-

C or 2500 U/ml of catalase, with variation in 6-OHDA

(Fig. 6C). Independently, both are cytoprotective; however,

neither protected against the toxicity of MPP+ (500 AM)

(Fig. 6D). These data clearly indicate that removing H2O2

Fig. 6. (A) Dual protective effects of catalase and CYT-C against 6-OHDA (175 Awere treated with varying concentrations of catalaseF cytochrome C [62.5 AM]

displayed (right). The data represent viability as % live control (left) and catalase m

meanF S.E.M. (n= 4). Significance of difference between the treatment groups

followed by a Tukey mean comparison post hoc test. *P< 0.001. (B) Effects of var

induced loss of mitochondrial O2 consumption at 24 h incubation � 37 jC in 5%

function during cytoprotection is displayed (right panel). All data represent O2 co

Significance of difference between the treatment groups and the 6-OHDA treated

comparison post hoc test. *P < 0.001. (C) Independent protective effects of catala

incubation. N-2A cells were treated with varying concentrations of 6-OHDA+ cat

the meanF S.E.M. (n= 4). Significance of difference between and within the grou

groups were statistically different from the PBS control. *P< 0.0001. (D) Lack of

h incubation � 37 jC in 5% CO2/atmosphere. The data represent cell viability

significance of difference between treatment and control groups were determined b

No significant differences were found.

partially reduces toxicity through restoring anaerobic func-

tion. 6-OHDA continues to exert inhibitory effects on O2

consumption, through a mechanism involving CYT-C.

They both play a significant contributing role to the toxic

effects of 6-OHDA.

3.5. The effects of 6-OHDA on complexes I–IV activity in

mitochondrial isolates

In the next set of experiments, mitochondrial isolates

were analyzed for the effects of 6-OHDA on electron

transport. A method validation control was established

for this cell line for each complex (Fig. 7). 6-OHDA had

M) toxicity at 24 h incubation � 37 jC in 5% CO2/atmosphere. N-2A cells

(left panel). The effects of catalase on scavenging 500 AM H2O2 are also

etabolism/min as % zero control 500 AM (right). The data are plotted as the

and the 6-OHDA treated control was determined by a one-way ANOVA,

ying concentration of CYT-CF catalase [1250 U/ml] on 6-OHDA (175 AM)

CO2/atmosphere (left panel). The lack of catalase to restore mitochondrial

nsumption as % live controls and are plotted as the meanF S.E.M. (n= 4).

control was determined by a one-way ANOVA, followed by a Tukey mean

se (2500 U/ml) and CYT-C (125 AM) against 6-OHDA toxicity at 24 h of

alase or CYT-C. The data represent viability as % control and are plotted as

ps was determined by a two-way ANOVA. The catalase and CYT-C treated

cytoprotection by CYT-C or catalase against MPP+ (500 AM) toxicity at 24

as % live control, and are expressed as the meanF S.E.M. (n= 4). The

y a one-way ANOVA, followed by a Tukey mean comparison post hoc test.

E.A. Mazzio et al. / Brain Research 1004 (2004) 29–4440

Fig. 9. (A) Demonstrates the effect of 6-OHDA on the release of Fe+ 2

from ferritin. The data are expressed as Fe+ 2 release (nM/mg ferritin)

and plotted as the meanF S.E.M. (n= 4). (B) Demonstrates there are no

cytoprotective effects of EDTA against 6-OHDA (175 AM) toxicity after

24 h incubation at 37 jC in 5% CO2/atmosphere. The data represents

cell viability as % live control, and are expressed as the meanF S.E.M.

(n= 4). The significance of difference between treatment and control

groups were determined by a one-way ANOVA, followed by a Tukey

mean comparison post hoc test. No significant differences were found.

E.A. Mazzio et al. / Brain Research 1004 (2004) 29–44 41

mild potentiating effects on complex I (Fig. 8A). However,

6-OHDA had extensive reductive capacity, both reducing

available substrate in complex II and reducing activity

(Fig. 8B). The effects of 6-OHDA on DCPIP in the

controls (mitochondrial homogenate) were not complete,

indicating a significant reduction in complex II. The right-

hand panel represents actual complex II activity, after

accounting for controls by subtracting the effects of 6-

OHDA on the substrate blanks. Reduction of complex III

substrate, (oxidized CYT-C) in the control blank was

complete (Fig. 8C). Complex III activity at 90 min was

determined by subtracting the control blank from results in

the presence of mitochondrial homogenate. The adverse

effects on complex III appear to be due to the loss of

oxidized substrate incurred by the presence of 6-OHDA. 6-

OHDA caused a potent surge in complex IV activity,

converting reduced CYT-C to its oxidized form with in the

first 15 min (Fig. 8D). However, within 60 min, any

oxidized CYT-C was eventually reduced by 6-OHDA.

These data indicate that 6-OHDA significantly alters the

capacity of the cytochromes by maintaining them in a

reduced state. A control was established to determine the

direct effects of H2O2 on mitochondrial complex activities.

Although H2O2 can oxidize CYT-C in the opposite fashion

to 6-OHDA, in the presence of antioxidants, 6-OHDA will

continue to maintain CYT-C in a reduced state and

potentially impair mitochondrial function.

3.6. The effects of 6-OHDA on Fe+2 release from bound

ferritin

The effects of 6-OHDA on the release of Fe+ 2 from

bound ferritin were assessed (Fig. 9A). Despite the release

of free Fe+ 2, EDTAwas not cytoprotective against 6-OHDA

toxicity (Fig. 9B), indicating metal-catalyzed oxidative

stress may not play a significant role in vitro.

In summary, the reduction of CYT-C and H2O2 produc-

tion from autoxidation, as demonstrated in this study con-

stitute chemical properties of 6-OHDA independent of

Fig. 8. The individual effects of 6-OHDA on mitochondrial complexes I, II, III and IV. Part A demonstrates the effect of 6-OHDA on complex I activity in

isolated mitochondria. The data represent activity [nM NADH oxidized/mg protein/20 min at 37 jC] and are plotted as the meanF S.E.M. (n= 4). Significance

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

*P < 0.001. The effects of 6-OHDA on complex II activity in isolated mitochondria are demonstrated in Part B. The data represent activity [DCPIP reduced] as

% control. A control for the effect of 6-OHDA on DCPIP reduction in the absence of mitochondrial homogenate is displayed (left panel). Complex II activity

was analyzed subtracting from the effects of 6-OHDA on DCPIP. The data are plotted as the meanF S.E.M. (n= 4). Significance of difference between the

treatment groups and the control was determined by a one-way ANOVA, followed by a Tukey mean comparison post hoc test. *P< 0.001. Part C demonstrates

the effect of 6-OHDA on complex III activity in isolated mitochondria (right panel). The data represent activity [CYT-C-RED] as % control (approx. 420 nM/

mg proteins) for 90 min at 37 jC. A control for the effect of 6-OHDA on CYT-C reduction in the absence of mitochondrial homogenate is displayed (left

panel). Complex III activity was analyzed subtracting from the effects of 6-OHDA on CYT-C. The data are plotted as the meanF S.E.M. (n= 4). Significance

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

*P < 0.001. Part D demonstrates the effect of 6-OHDA on complex IV activity in isolated mitochondria. The data represent activity [CYT-C-OX] as % control

(approx. 420 nM/mg proteins) for 60 min at 37 jC. The data reveal two phases. Within 15 min, there was a very rapid and robust increase in oxidized CYT-C

by 6-OHDA (left panel). However, these effects dissipated rapidly by 60 and 90 min due to the inherent reducing nature of 6-OHDA (right). The data are

plotted as the meanF S.E.M. (n= 4). Significance of difference between the treatment groups and the control was determined by a one-way ANOVA, followed

by a Tukey mean comparison post hoc test. *P< 0.001.

E.A. Mazzio et al. / Brain Research 1004 (2004) 29–4442

biological systems. Both components appear to contribute to

the loss of aerobic and anaerobic function, respectively in

living cells.

4. Discussion

The data in this study demonstrate that 6-OHDA

toxicity is associated with simultaneous collapse of anaer-

obic and aerobic metabolism. Moreover, this study dem-

onstrates that H2O2 produced from autoxidation may

contribute to loss of anaerobic glycolysis, possibly through

inhibiting lactic acid dehydrogenase. The reductive prop-

erties of 6-OHDA on CYT-C in the presence of catalase

are potent, and may be contributing to impaired mitochon-

drial function. The complete loss of available CYT-C-OX

in the ETC would block proton translocation and electron

transfer at complex III. Fig. 9A corroborates previous

findings in that 6-OHDA can reduce Fe(III) to Fe(II),

initiating the release of free Fe from its bound form. Fig.

2B indicates a similar pattern, where the reduction of Fe

could lead to the stabilization of mitochondrial CYT-C-

RED. The data suggest that the toxic effects of 6-OHDA

may involve impaired oxidation–reduction events at com-

plex III (cytochrome c reductase) by preventing electrons

received from complex II, and those transferred to the

terminal receptor, being O2 in complex IV. These events

could potentially contribute to aerobic metabolic respirato-

ry failure and the loss of MOC, as clearly demonstrated in

this study.

The toxicity of 6-OHDA was associated with reduction

in lactic acid, ATP and whole-cell O2 consumption,

indicating complete metabolic collapse. The reduction in

lactate in the presence of 6-OHDA is unique from the

toxicological profiles observed with other mitochondrial

toxins. Typically, toxins such MPP+, target specific

damage to aerobic cell function and correspond to an

increase rather than a decrease in lactate as demonstrated

in Fig. 1B. The reduction in lactate during 6-OHDA

toxicity signifies destruction to functional anaerobic gly-

colysis. The loss of glycolysis appears to involve H2O2

contributing to a number of factors including LDH

inhibition and lipid peroxidation. Furthermore, a symbi-

otic relationship could exist between the two, where

structural damage could render anaerobic production of

ATP ineffective in driving membrane Na+,K+-ATPase

pumps to sustain voltage. Membrane depolarization could

prompt the abnormal influx of calcium ions through

voltage-linked channels, leading to excitotoxicity. How-

ever, data from our lab have indicated that the buffering

of intracellular calcium, or blocking Ca+ 2 ionic receptor-

linked excitatory channels do not spare 6-OHDA toxicity,

suggesting oxidative stress to be a predominant factor in

cellular injury.

Oxidative stress associated with 6-OHDA is thought to

contribute to morphological neurodegenerative lesions

[30]. This and previous research corroborate that partial

cytoprotection against 6-OHDA can be achieved with

antioxidants [5]. Our findings show significant protective

effects by the carboxylic acids. Oxaloacetate and a-

ketoglutarate are structurally similar to pyruvate, an

energy intermediate that can rapidly dissipate H2O2

[3,42] by converting it to water through decarboxylation

[13,27]. Pyruvic acid can also sustain energy by fueling

aerobic and anaerobic metabolism in neurons and other

tissue, indicating its powerful dual protective role [7,18].

NAC and GSH are also cytoprotective in vitro [4] and in

vivo against striatal degeneration induced by 6-OHDA

[2,36]. While H2O2 may play a significant role in neuro-

degeneration, 6-OHDA can also release free iron from

ferritin [9]. The combination of H2O2 with available

divalent metals could lead to the production of the

hydroxyl radical, which is biologically destructive. The

data in this study demonstrated that metal-catalyzed

oxidative stress does not play a role in vitro, where

EDTA did not offer cytoprotection. Ferritin is localized

primarily in glia and oligodendrocytes [8] indicating these

cells may play a more predominant role in the toxic

cascade of events that occur in vivo. For this reason,

administration of EDTA or desferrioxamine can attenuate

6-OHDA toxicity in vivo, where neurodegeneration may

be multifaceted [20].

The mechanism of mitochondrial impairment by 6-

OHDA appears to be unique from typical mitochondrial

toxins. Toxic substances such as MPP+, malonate, anti-

mycin, and sodium azide led to catalytic inactivation of

complexes I, II, III and IV, halting mitochondrial respira-

tion. This is corroborated by the data presented in this

study, where these toxins did not affect the redox of

enzyme substrates in blank control groups (Fig. 7, data

on control groups not shown). In contrast, control blanks

for 6-OHDA exhibited reduction of oxidized substrate, in

the absence of mitochondria homogenate (Fig. 8, control

blanks shown). Furthermore, addition of CYT-C and cat-

alase did not offer cytoprotection against the toxicity of

MPP+ (Fig. 6D), because its mechanism does not involve

altering the redox state of the cytochromes or production

of ROS. Unlike 6-OHDA, protection against MPP+ is

achieved by driving anaerobic glycolysis with additional

glucose [24,39]. On the other hand, complete cytoprotec-

tion against 6-OHDA was achieved with CYT-C + catalase

(Fig. 6A–C), indicating that reductive properties of 6-

OHDA may play a paramount role in mitochondrial

impairment.

A hypothetical schematic for how 6-OHDA may

impact mitochondrial function is displayed in Fig. 10.

We found no direct inhibitory effects on complex I in

contrast to previous research [14] and noted inhibition of

complex II activity by 6-OHDA. The mechanism for this

is uncertain and future research will be required to

examine if 6-OHDA is reducing either the CYT-b or

ubiquinone content of complex II. Complex III activity

Fig. 10. A theoretical diagram describing mitochondrial impairment by 6-OHDA on individual complexes through alteration of the oxidation state of

cytochromes. Three boxes denote where CYT-C reduction by 6-OHDA could block the flow of electrons and obstruct complexes III– IV function.

Abbreviations: cytochrome (Cyt), ubiquinone (CoQ), oxygen (O2), water (H20).

E.A. Mazzio et al. / Brain Research 1004 (2004) 29–44 43

was obstructed, because the substrate [CYT-C-OX] was

completely reduced by 6-OHDA. In a similar fashion, in

complex IV, while the substrate (CYT-C-RED) was

oxidized by cytochrome oxidase, the product was rapidly

reduced by 6-OHDA. In total, our findings although

preliminary, suggest that 6-OHDA slightly augments

complex I, inhibits complex II, and indirectly inhibits

(not through enzyme inhibition) complexes III and IV by

maintaining components of the ETC in a reduced state,

thereby preventing normal oxidation–reduction events.

While the evidence in this paper suggests this hypothesis,

it can not be excluded that the protection observed by

CYT-C on MOC in whole cells may have involved an

extracellular mechanism. Future research will be required

to corroborate these findings.

In summary, the ability of catecholamines such as DA

and 6-OHDA to reduce CYT-C constitutes a unique

chemical property inherent to this class of neurotransmit-

ters. These data provide preliminary evidence that cate-

cholamines may impair OXPHOS through altering the

redox state of CYT-C, leading to indirect inhibitory effects

on complexes II–IV. Antioxidants, while protective, pri-

marily antagonize cellular damage and loss of anaerobic

glycolysis.

Acknowledgements

This work was supported by grants received from the

National Institutes of Health (NCRR 03020 and GM 08111).

Special thanks goes to Bruce Smith, at Florida State

University, Analytical Laboratories, Biological Sciences,

Tallahassee, FL for assisting with HPLC organic acid

analysis and Mr. Rick Wolfe from Southern Micro Instru-

ments, Marietta, GA, USA.

References

[1] M. Aubailly, R. Santus, S. Salmon, Ferrous ion release from fer-

ritin by ultraviolet-A radiations, Photochem. Photobiol. 54 (1991)

769–773.

[2] J.C. Bensadoun, O. Mirochnitchenko, M. Inouye, R. Aebischer,

A.D. Zurn, Attenuation of 6-OHDA-induced neurotoxicity in glu-

tathione peroxidase transgenic mice, Eur. J. Neurosci. 10 (1998)

3231–3236.

[3] J.F. Bilodeau, S. Blanchette, N. Cormier, M. Sirard, Reactive

oxygen species-mediated loss of bovine sperm motility in egg

yolk Tris extender: protection by pyruvate, metal chelators and

bovine liver or oviductal fluid catalase, Theriogenology 57

(2002) 1105–1122.

[4] D. Blum, S. Torch, M.F. Nissou, A.L. Benabid, J.M. Verna, Extracel-

lular toxicity of 6-hydroxydopamine on PC12 cells, Neurosci. Lett.

283 (2000) 193–196.

[5] D. Blum, S. Torch, N. Lambeng, M. Nissou, A.L. Benabid, R. Sadoul,

J.M. Verna, Molecular pathways involved in the neurotoxicity of 6-

OHDA, dopamine and MPTP: contribution to the apoptotic theory in

Parkinson’s disease, Prog. Neurobiol. 65 (2001) 135–172.

[6] A.M. Brusque, R. Borba Rosa, P.F. Schuck, K.B. Dalcin, C.A.

Ribeiro, C.G. Silva, C.M. Wannmacher, C.S. Dutra-Filho, A.T.

Wyse, P. Briones, M. Wajner, Inhibition of the mitochondrial

respiratory chain complex activities in rat cerebral cortex by

methylmalonic acid, Neurochem. Int. 40 (2002) 593–601.

[7] D. Clough, R. Bunger, Protection by pyruvate against inhibition of

Na+, K(+)-ATPase by a free radical generating system containing t-

butylhydroperoxide, Life Sci. 57 (1995) 931–943.

[8] J.R. Connor, S.L. Menzies, S.M. St Martin, E.J. Mufson, Cellular

distribution of transferrin, ferritin, and iron in normal and aged

human brains, J. Neurosci. Res. 27 (4) (1990 Dec.) 595–611.

[9] K.L. Double, M. Maywald, M. Schmittel, R. Riederer, M. Gerlach, In

vitro studies of ferritin iron release and neurotoxicity, J. Neurochem.

70 (1998) 2492–2499.

E.A. Mazzio et al. / Brain Research 1004 (2004) 29–4444

[10] M. Ebadi, P. Govitrapong, S. Sharma, D. Muralikrishnan, S. Shavali,

L. Pellett, R. Schafer, C. Albano, J. Eken, Ubiquinone (coenzyme

q10) and mitochondria in oxidative stress of Parkinson’s disease, Biol.

Signals Recept. 10 (2001) 224–253.

[11] S.M. Evans, A. Casartelli, E. Herreros, D.T. Minnick, C. Day, E.

George, C. Westmoreland, Development of a high throughput in

vitro toxicity screen predictive of high acute in vivo toxic poten-

tial, Toxicol. In Vitro 15 (2001) 579–584.

[12] M.F. Galindo, J. Jordan, C. Gonzalez-Garcia, V. Cena, Chromaffin

cell death induced by 6-hydroxydopamine is independent of mito-

chondrial swelling and caspase activation, J. Neurochem. 84 (2003)

1066–1073.

[13] A.R. Giandomenico, G.E. Cerniglia, J.E. Biaglow, C.W. Stevens,

C.J. Koch, The importance of sodium pyruvate in assessing dam-

age produced by hydrogen peroxide, Free Radic. Biol. Med. 23

(1997) 426–434.

[14] Y. Glinka, K.F. Tipton, M.B. Youdim, Mechanism of inhibition of

mitochondrial respiratory complex I by 6-hydroxydopamine and its

prevention by desferrioxamine, Eur. J. Pharmacol. 351 (1998)

121–129.

[15] K.S. Ha, K.M. Kim, Y.G. Kwon, S.K. Bai, W.D. Nam, Y.M. Yoo,

P.K. Kim, H.T. Chung, T.R. Billiar, Y.M. Kim, Nitric oxide pre-

vents 6-hydroxydopamine-induced apoptosis in PC12 cells through

cGMP-dependent PI3 kinase/Akt activation, FASEB J. 17 (2003)

1036–1047.

[16] A. Holt, D.F. Sharman, G.B. Baker, M.M. Palcic, A continuous spec-

trophotometric assay for monoamine oxidase and related enzymes in

tissue homogenates, Anal. Biochem. 244 (1997) 384–392.

[17] J.S. Hothersall, A.L. Greenbaum, P. McLean, The functional signifi-

cance of the pentose phosphate pathway in synaptosomes: protection

against peroxidative damage by catecholamines and oxidants, J. Neu-

rochem. 39 (1982) 1325–1332.

[18] Y. Izumi, A.M. Benz, C.F. Zorumski, J.W. Olney, Effects of lactate

and pyruvate on glucose deprivation in rat hippocampal slices, Neuro-

Report 31 (1994) 617–620.

[19] K.H. Jones, J.A. Senft, An improved method to determine cell via-

bility by simultaneous staining with fluorescein diacetate-propidium

iodide, J. Histochem .Cytochem. 33 (1985) 77–79.

[20] H. Kabuto, L. Yokoi, E. Iwata-Ichikawa, N. Ogawa, EPC-K1, a hy-

droxyl radical scavenger, prevents 6-hydroxydopamine-induced do-

pamine depletion in the mouse striatum by up-regulation of catalase

activity, Neurochem. Res. 24 (1999) 1543–1548.

[21] J.W. Langston, P. Ballard, J.W. Tetrud, I. Irwin, Chronic parkinsonism

in humans due to a product of meperidine-analog synthesis, Science

219 (1983) 979–980.

[22] O.J. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein

measurement with Folin phenol reagent, J. Biol. Chem. 193

(1951) 265–275.

[23] M.J. Marti, J. Saura, R.E. Burke, V. Jackson-Lewis, A. Jimenez, M.

Bonastre, E. Tolosa, Striatal 6-hydroxydopamine induces apoptosis

of nigral neurons in the adult rat, Brain Res. 958 (2002) 185–191.

[24] E. Mazzio, K.F. Soliman, Pyruvic acid cytoprotection against 1-meth-

yl-4-phenylpyridinium, 6-hydroxydopamine and hydrogen peroxide

toxicities in vitro, Neurosci. Lett. 337 (2003) 77–80.

[25] E. Mazzio, K.F.A. Soliman, Cytoprotection of pyruvic acid and re-

duced beta-nicotinamide adenine dinucleotide against hydrogen per-

oxide toxicity in neuroblastoma cells, Neurochem. Res. 28 (2003)

733–741.

[26] E. Mazzio, K.F.A. Soliman, (+)-Glucose rescue against 1-methyl-4-

phenylpyridinium toxicity through anaerobic glycolysis in neuroblas-

toma cells, Brain Res. 962 (2003) 48–60.

[27] K.A. Nath, E.O. Ngo, R.P. Hebbel, A.J. Croatt, B. Zhou, L.M. Nutter,

Alpha-ketoacids scavenge H2O2 in vitro and in vivo and reduce

menadione-induced DNA injury and cytotoxicity, Am. J. Physiol.

268 (1995) C227–C236.

[28] M.F. Notter, I. Irwin, J.W. Langston, D.M. Gash, Neurotoxicity of

MPTP and MPP+ in vitro: characterization using specific cell lines,

Brain Res. 456 (1988) 254–262.

[29] Y. Oiwa, R. Sanchez-Pernaute, J. Harvey-White, K.S. Bankiewicz,

Progressive and extensive dopaminergic degeneration induced by

convection-enhanced delivery of 6-hydroxydopamine into the rat

striatum: a novel rodent model of Parkinson’s disease, J. Neurosurg.

98 (2003) 136–144.

[30] M. Rodriguez, P. Barroso-Chinea, P. Abdala, J. Obeso, T. Gonza-

lez-Hernandez, Dopamine cell degeneration induced by intraven-

tricular administration of 6-hydroxydopamine in the rat: similarities

with cell loss in Parkinson’s disease, Exp. Neurol. 169 (2001)

163–181.

[31] A. Sachinidis, I. Gouni-Berthold, C. Seul, S. Seewald, Y. Ko, U.

Schmitz, H. Vetter, Early intracellular signaling pathway of etha-

nol in vascular smooth muscle cells, Br. J. Pharmacol. 128 (1999)

1761–1771.

[32] M. Santiago, L. Granero, A. Machado, J. Cano, Complex I inhib-

itor effect on the nigral and striatal release of dopamine in the

presence and absence of nomifensine, Eur. J. Pharmacol. 280

(1995) 251–256.

[33] K. Seth, A.K. Agrawal, M.H. Aziz, A. Ahmad, Y. Shukla, N.

Mathur, P.K. Seth, Induced expression of early response genes/

oxidative injury in rat pheochromocytoma (PC12) cell line by 6-

hydroxydopamine: implication for Parkinson’s disease, Neurosci.

Lett. 330 (2002) 89–93.

[34] K. Seya, N. Ohkohchi, N. Watanabe, H. Shibuya, Y. Taguchi, S. Mori,

Rapid fluoremetric assay for mitochondrial proton adenosine triphos-

phatase activity for assessment of viability of liver graft tissue, J. Clin.

Lab. Anal. 8 (1994) 418–423.

[35] E. Shimizu, K. Hashimoto, N. Komatsu, M. Iyo, Roles of endogenous

glutathione levels on 6-hydroxydopamine-induced apoptotic neuronal

cell death in human neuroblastoma SK–N–SH cells, Neuropharma-

cology 43 (2002) 434–443.

[36] R. Soto-Otero, E. Mendez-Alvarez, A. Hermida-Ameijeiras, A.M.

Munoz-Patino, J.L. Labandeira-Garcia, Autoxidation and neurotoxic-

ity of 6-hydroxydopamine in the presence of some antioxidants: po-

tential implication in relation to the pathogenesis of Parkinson’s

disease, J. Neurochem. 74 (2000) 1605–1612.

[37] K. Suzuki, Y. Mizuno, M. Yoshida, Effects of 1-methyl-4-phenyl-

1,2,3,6-tetrahydropyridine (MPTP)-like compounds on mitochondrial

respiration, Adv. Neurol. 53 (1990) 215–218.

[38] K.J. Thompson, S. Shoham, J.R. Connor, Iron and neurodegenerative

disorders, Brain Res. Bull. 55 (2001) 155–164.

[39] A. Woodgate, G. MacGibbon, M. Walton, M. Dragunow, The toxicity

of 6-hydroxydopamine on PC12 and P19 cells, Brain Res. Mol. Brain

Res. 69 (1999) 84–92.

[40] Y. Zhang, V.L. Dawson, T.M. Dawson, Oxidative stress and genetics

in the pathogenesis of Parkinson’s disease, Neurobiol. Dis. 7 (2000)

240–250.

[41] J. Zhang, E.R. Block, J.M. Patel, Down-regulation of mitochondrial

cytochrome c oxidase in senescent porcine pulmonary artery endo-

thelial cells, Mech. Ageing Dev. 123 (2002) 1363–1374.

[42] F.Q. Zhou, Advantages of pyruvate over lactate in peritoneal dialysis

solutions, Acta Pharm. Sin. 22 (2001) 385–392.