Inhibitory Effects of Cigarette Smoke on Glial Inducible Nitric Oxide Synthase and Lack of...

NeuroToxicology 26 (2005) 49–62

Inhibitory Effects of Cigarette Smoke onGlial Inducible Nitric Oxide Synthase and

Lack of Protective Properties AgainstOxidative Neurotoxins In Vitro

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

* Corre

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0161-813

doi:10.10

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

Received 18 March 2004; accepted 14 July 2004

Available online 28 August 2004

Abstract

Epidemiological studies consistently report an inverse correlation between cigarette smoking and associated risk for

Parkinson’s disease (PD). The degeneration of dopaminergic neurons may involve the toxic metabolic products of glial

cell monoamine oxidase (MAO) and inducible nitric oxide synthase (iNOS). This study evaluates the direct protective

effects of cigarette smoke (CS) against potential neurotoxic products of MAO, such as 1-methyl-4-phenylpyridinium

(MPP+), 6-hydroxydopamine (6-OHDA) and hydrogen peroxide (H2O2) in brain neuroblastoma. Moreover, the effects of

CS were also evaluated on endotoxin/cytokine activated glioma iNOS protein expression and MAO enzyme activity.

Cigarette smoke condensates (CSCs) were acquired from Marlboro 20 Class A and Kentucky 2R4F reference research

(2R4F) cigarettes. The CSCs did not protect against 6-OHDA or H2O2 toxicity in neuroblastoma, and exhibited a very

mild protective effect [�10%] against MPP+. Neither CSC demonstrated antioxidant capability, but conversely

contained high concentration of NO2�

. Paradoxically, in glioma cells, iNOS protein expression and endogenous

enzymatic NO2� production were significantly blocked by both CSCs. Both CSCs also inhibited glioma MAO-A and

MAO-B [1.4.3.4]. Kinetic analysis indicated that 2R4F–CSC displayed competitive inhibition and the Marlboro–CSC

exerted potent competitive and non-competitive inhibition. In conclusion, these data suggest that cigarette smoke does not

appear to directly protect against the toxicity of the selected neurotoxins. In contrast, CS exerts pronounced effects on

glia, whereby its presence can simultaneously attenuate cytokine induction of iNOS and MAO.

# 2004 Elsevier Inc. All rights reserved.

Keywords: Parkinson’s disease; Smoking; Cigarettes; MPP+; Nitric oxide; Monoamine oxidase

INTRODUCTION

While reports indicate that smoking is a major risk

factor for developing coronary heart disease, diabetes

and lung cancer (Skurnik and Shoenfeld, 1998), smo-

kers are 50% less likely to develop Parkinson’s disease

(PD) (Fratiglioni and Wang, 2000). Epidemiological

studies consistently report a positive correlation

sponding author. Tel.: +1 850 599 3306;

50 599 3667.

dress: [email protected] (Karam F.A. Soliman).

X/$ – see front matter # 2004 Elsevier Inc. All rights reserv

16/j.neuro.2004.07.005

between cigarette smoking and a reduced risk for

developing PD (De Michele et al., 1996; Gorell et

al., 1999; Fratiglioni and Wang, 2000; Preux et al.,

2000; Hernan et al., 2001; Behari et al., 2001; Her-

ishanu et al., 2001). This relationship is evident in both

men and women, and is strengthened with longevity of

smoking and number of cigarettes smoked per day

(Gorell et al., 1999; Hernan et al., 2001). Moreover, the

correlation is consistently corroborated in studies

employing multivariate regression and in many parts

of the world, such as India (Behari et al., 2001), Israel

(Herishanu et al., 2001), France (Preux et al., 2000) and

ed.

E.A. Mazzio et al. / NeuroToxicology 26 (2005) 49–6250

Italy (De Michele et al., 1996). Although the relation-

ship between smoking and the reduced incidence of PD

is extensively documented, very little is known about

the exact mechanism responsible for this effect. More-

over, there are several lines of thought as to how

cigarette smoke (CS) may be neuroprotective, such

as activation of neuronal nicotinic acetycholine recep-

tors (nAChRs), and inhibition of monoamine oxidase

(MAO) in glial cells.

Neuronal AChR are located throughout the CNS,

and play an integral role in neurotransmission release.

While there are a wide variety of characteristic homol-

ogy between subunits that comprise AChR, the a3b2

and a4b2 subunits are highly expressed in striatal

dopaminergic neurons, playing a critical role in mod-

ulating synaptic dopamine (DA) (Jones et al., 2001).

Likewise A-85380 is a marker of a4b2 subunit, and

both loss of A-85380 and I125-a conotoxin MII (a3

and a6 subunits specific) binding is observed in the

human striatum, parallel to a loss of dopaminergic

neurons in human PD, Lewy body dementia and after

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

(MPTP) administration in monkeys (Pimlott et al.,

2004; Kulak et al., 2002). Other reports corroborate

the a3 and a6 subunit to be highly expressed in

nigrostriatal dopaminergic neurons and nerve term-

inals (Quik and Kulak, 2002; Quik et al., 2000) and

the loss of binding is associated with degeneration of

the nigrostriatal-mesolimbic pathways after MPTP

administration in monkeys (Quik et al., 2001; Quik

and Jeyarasasingam, 2000). Although, the mechanism

of action for AChR in neuroprotection is not clearly

understood, these findings support an inverse relation-

ship between loss of substantia nigra (SN) nicotinic

receptors and PD severity.

Similarly, human PD patients exhibit a loss of

striatal nicotine binding, where tobacco use may aug-

ment receptor density (Court et al., 2000). Nicotine,

can increase nicotinic receptor density in dopaminergic

neurons (Jeyarasasingam et al., 2001), and both pre/

post intermittent or chronic administration of subcu-

taneous nicotine (< 2 mg/kg day), can partially protect

against the loss of striatal DA, and dopaminergic nerve

terminals induced by 6-hydroxydopamine (6-OHDA)

in rat SN (Costa et al., 2001; Ryan et al., 2001) and the

loss of SN neurons induced by MPTP administration in

C57Bl/6 mice (Parain et al., 2003; Ross and Petrovitch,

2001). These studies suggest a pertinent role for

nAChRs in protection against SN damage, because

either nAChRs receptor antagonists or a4 nAChRs

knock out mice are associated with the loss of protec-

tive effects of nicotine. Other studies also support that

upregulation of nicotinic receptors in the basal ganglia

can provide partial protection against dopaminergic

neurodegenerative processes (Le Novere et al., 1996;

Balfour and Fagerstrom, 1996). In humans, cigarette

smoking or administration of nicotine can activate

nAChRs leading to an increase of striatal dopaminergic

activity, effects that correlate with attenuation of tre-

mor, rigidity and bradykinesia and improved cognitive

function in PD patients (Kelton et al., 2000; Quik and

Kulak, 2002).

While previous research has focused on defining a

therapeutic role for nicotine, its protection in animal

models is only partial, and it is well known that

cigarette smoke contains thousands of other chemicals

(Rustemeier et al., 2002). The effects of nicotine may

be significantly different from that of whole smoke. For

example, nicotine is not a known MAO inhibitor

(Berlin et al., 2000). Yet, it is believed that the pro-

tective effects of cigarette smoke largely involve con-

stant downregulation of MAO activity within the CNS

(Carr and Basham, 1991; Fowler et al., 1998; Volkow et

al., 1999). In the human brain, MAO-B is abundant in

glia (Squires, 1997), and its activity plays a critical role

in DA metabolism within the CNS. Routine MAO

activity can produce toxic products, such as hydrogen

peroxide (H2O2), ammonia, aldehydes, reactive oxy-

gen species (Pizzinat et al., 1999; Youdim and Lavie,

1994; Venarucci et al., 1999) and 3,4-dihydroxyphe-

nylacetaldehyde/3,4-dihydroxyphenylglycolaldehyde

which can condense with H2O2 to form �OH radicals

(Li et al., 2001; Tabner et al., 2002). Further oxidation

of DA by H2O2 and other radicals can lead to formation

of 6-OHDA, a potent neurotoxin (Blum et al., 2001).

DA can also condense with acetaldehydes, to yield

toxic substances and upon further methylation can

produce potent neurotoxic substances, such as

N(methyl)-R-salsolinol (Maruyama, 2001). Moreover,

MAO can convert endogenous toxic precursors, such

as 1,2,3,4-tetrahydroisoquinoline (TIQs) and 1,2,3,4-

tetrahydro-b-carboline (THbCs) into toxic cationic

species, structurally similar to 1-methyl-4-phenylpyr-

idinium (MPP+) (Soto-Otero et al., 1998, 2001). It is

also important to note that the effects of MAO inhibi-

tors in antagonizing toxicity of dopaminergic neuro-

toxins, may be due to unique chemical properties of

selegiline, rasagiline or propargylamines that can

effectively block apoptosis through impeding opening

of the mitochondrial permeability transition pore

(Maruyama, 2001; Maruyama et al., 2001; Naoi and

Maruyama, 2001; Naoi et al., 2003).

While there have been reports investigating a role

for CS regarding neuroprotective changes in trophic

E.A. Mazzio et al. / NeuroToxicology 26 (2005) 49–62 51

factors (Maggio et al., 1997), nicotinic receptors or

MAO, there is little to no research evaluating the

effects of cigarette smoke on CNS inflammation or

cytokine activated glial cell inducible NOS (iNOS), an

enzyme that plays a significant contributing role in the

pathological destruction associated with degenerative

diseases (Goodwin et al., 1995; Peng et al., 1996; Clark

et al., 1996), including Alzheimer’s disease (Luth et al.,

2002) multiple sclerosis (Broholm et al., 2004) and PD

(Hyun et al., 2002). While there are few to no studies

examining the effects of CS on iNOS in glia, previous

research has focused on defining a role for endothelial

nitric oxide synthase (eNOS), due to associated hyper-

tension and coronary artery disease with smoking

(Tsuchiya et al., 2002). In addition, the potential anti-

oxidant effects of cigarette smoke against oxidative

neurotoxins are not known, although the effects of

nicotine against oxidative stress incurred by 6-OHDA

or H2O2 has been evaluated, and found ineffective

(Linert et al., 1999). Further, there is meager data

investigating effects of whole cigarette smoke directly

against the toxicity of MPP+ in vitro. Therefore, the

aim of this study is to analyze the in vitro effects of

cigarette smoke directly against toxic products of

MAO [MPP+, 6-hydroxydopamine (6-OHDA) and

hydrogen peroxide], and to investigate the effects of

cigarette smoke on glial cell inflammatory induction of

NOS, production of NO2� and MAO enzyme function.

EXPERIMENTAL PROCEDURES

Materials

Rat glioma (C6) cells and neuroblastoma cells

(N2A) were obtained from American Type Culture

Collection (Manassas, VA, USA). Dulbecco’s Modi-

fied Eagle Medium (DMEM), L-glutamine, fetal

bovine serum, heat inactivated (FBS), Hank’s Balanced

Salt Solution (HBSS) and penicillin/streptomycin were

supplied by Fischer Scientific, Mediatech (Pittsburgh,

PA, USA). Rat interferon-gamma was purchased from

Gibco (Grand Island, NY, USA) and all other chemi-

cals and supplies were purchased from Sigma Chemi-

cal (St. Louis, MO, USA).

Cigarette Smoke Condensate

Cigarette smoke condensate was obtained from

Marlboro 20 Class A cigarettes (MAR) manufactured

by Philip Morris Inc. (Richmond, VA, USA) and 2R4F

reference research cigarettes (2R4F) obtained from the

Smoking and Health Institute of the University of

Kentucky (Lexington, KY, USA). CSCs are often

collected using a smoking machine, adhering to a

standard Federal Trade Commission puffing regimen

consisting of a 35 ml puff volume every 60 s for a 2 s

duration, or slight variations (Foy et al., 2004). How-

ever, there are a number of studies that employ differ-

ent protocols for in vitro studies, including bubbling

cigarette smoke directly into buffered saline or DMEM

(Yamaguchi et al., 2004). The procedure in this study,

is a modification of a number of existing methods.

Briefly, cigarette smoke was collected using a Drum-

mond pipetter and a sterile 50 ml serological pipette,

modified to fit the filtered end of a cigarette. For each

MAR cigarette, a 50 ml puff of smoke was obtained

once every minute for 6 min. For each 2R4F cigarette,

a 50 ml puff of smoke was obtained every minute and a

half for 6 min. The MAR cigarettes were smoked to

within 0.9 � 0.23 mm of the edge of the filter, and the

2R4F cigarettes were smoke to within 0.8 � 0.12 mm

of the edge of the filter. Each 50 ml volume of smoke

was collected into a sterile cell culture flask, containing

a small hole only allowing for an incoming stream of

cigarette smoke, and immediately capped. The smoke

from five cigarettes was collected, and dissolved in

1 ml of pure 100% absolute ethanol. The flask was

rinsed by gentle shaking until the yellow-golden brown

residual on the sides of the wall was dissolved in

solution. A 1:10 dilution was achieved with sterile

HBSS containing 5 mM (N-[2-hydroxyethylpipera-

zine-N0-[2-ethanesulfonic acid]) (HEPES) at pH of

7.4. Six dilutions of each condensate were prepared

in sterile buffered HBSS to span a 1000 fold dilution

range. For experiments, the various CSC dilutions were

added to 96 well plates with the highest concentration

being 20% solution in DMEM experimental medium

(v/v). Ethanol controls were established, comprising

�2% final volume at the highest concentration of CSC

dilutions. This protocol allowed for the collection of

both water soluble and lipophilic chemicals derived

from cigarette smoke, eliminates solvent extraction

from filters with varying consistencies, and contributes

toward sterilization of CS by directly dissolving the

smoke into pure ethanol, in preparation for in vitro

studies.

Neuroblastoma Cell Culture

N2A cells are vulnerable to the toxic effects of

dopaminergic neurotoxins (Simmons and Notter,

1991; Mazzio and Soliman, 2003b). Briefly, N2A cells

were cultured in DMEM containing phenol red, 10%


FBS, 20 mM sodium pyruvate, 4 mM L-glutamine and

penicillin/streptomycin (100 Units/0.1 mg per ml).

Cells were maintained at 37 8C in 5% CO2/atmosphere.

The cells were sub-cultured every 2–5 days. Experi-

mental plating media consisted of DMEM without

phenol red, 1.8% FBS, penicillin/streptomycin (100

Units/0.1 mg per ml), 20 mM sodium pyruvate and

4 mM L-glutamine. For experiments, the cells were

plated at �0.5 � 106 cells/ml. N2A cells were treated

with CSCs for 2 h, prior to the addition of MPP+

(500 mM), H2O2 (500 mM) and 6-OHDA (100 mM),

and further incubated for 24 h at 37 8C, 5% CO2 in

atmosphere.

C6 Glioma Cell Culture

C6 glioma cells were cultured in DMEM containing

phenol red, 10% FBS, in 4 mM L-glutamine and peni-

cillin/streptomycin (100 Units/0.1 mg per ml). Cells

were grown at 37 8C in 5% CO2/atmosphere, trypsi-

nized (sterile trypsin 0.25%/EDTA 0.02% in HBSS)

and subcultured every 2–5 days. For experiments, the

plating media contained DMEM (without phenol red),

1.8% FBS, penicillin/streptomycin (100 Units/0.1 mg

per ml) and 4 mM L-glutamine. Cells were plated at

�1.0 � 106 cells/ml for both iNOS and MAO experi-

ments. For experiments, C6 cells were treated with

CSCs for 2 h, prior to the addition of lipopolysacchar-

ide (LPS) E. coli 0111:B4 (6 mg/ml) and interferon-

gamma (IFN-g) and further incubated for 24 h at 37 8C,

5% CO2 in atmosphere.

Monoamine Oxidase Activity and HydrogenPeroxide Determination

Monoamine oxidase activity was determined by

modification of a previous colorimetric method (Maz-

zio et al., 2003; Holt et al., 1997). Briefly, immediately

after plating, various concentrations of CSCs were

added to the cells, and the samples were immediately

frozen at �80 8C for 48 h. Cells were lysed by freeze-

thaw. Tyramine was prepared in HBSS containing

3 mM HEPES and adjusted to pH 7.4. Immediately

after the addition of the substrate, a chromogenic

reagent was added (final concentration: 1 mM vanillic

acid, 500 mM 4-aminoantipyrine and horseradish per-

oxidase (4 purpurogallin Units/ml) in HBSS. Samples

were then gently vortexed and returned to the incubator

for timed experiments. To examine scavenging abilities

of MAO inhibitors for the product—hydrogen peroxide

(H2O2), 250 mM of H2O2 was prepared in HBSS at a

pH of 7.4, and incubated for 30 min at RT. At the end of

30 min, the chromogenic reagent was added and data

were quantified at 490 nm on a UV Microplate Spec-

trophotometer—Model 7600, version 5.02, Cambridge

Technologies Inc., (Watertown, MA, USA). A standard

curve of H2O2 (1–500 mM) was prepared in serum free

plating medium. A standard curve for protein was

established using bovine albumin (1–100 mg/dl) and

protein was assessed by the Lowry method using a UV

Microplate Spectrophotometer at 550 nm (Lowry et al.,

1951). The data were expressed as nM product: H2O2/

mg protein.

Nitrite Determination

C6 cells were treated with various dilutions of CSC

� lipopolysaccharide (LPS) E. coli 0111:B4 (6 mg/ml)

and interferon-gamma (IFN-g) (100 Units/ml). Sam-

ples were returned to the incubator for 48 h. Quanti-

fication of NO2� in biological samples and blanks were

determined by a colorimetric assay (Mazzio et al.,

2003; Park and Murphy, 1994). The Griess reagent

was prepared by mixing equal volume (1:1) of 1%

sulfanilamide in 0.5 N HCl and 0.1% N-(1-naphthyl)

ethylenediamine in deionized water. Subsequently, the

Griess reagent was added directly to the cell super-

natant and incubated under reduced light at room

temperature for 10 min. Samples were analyzed at

550 nm on a Cambridge UV microplate spectrophot-

ometer. Controls and blanks were run in parallel, and

subtracted from the final value to eliminate interfer-

ence. A standard curve was generated from dilutions of

sodium nitrite (1–100 mM) prepared in plating med-

ium. Protein content was assessed by the Lowry

method and data were expressed as either nM

NO2�/mg protein or as the percentage of the control.

Western Blot—iNOS protein

Western blot analysis was performed using the

procedure described by Chandler et al. (1995) with

minor modifications (Mazzio et al., 2003). After

experiments, the supernatant for each sample was

removed and discarded. The monolayer was washed

with PBS and placed in a lysing buffer. The lysing

buffer consisted of 5% glycerol, 1 mM sucrose,

200 mM phenylmethylsulfonyl fluoride, 10 mM [Tris

(hydroxymethyl) aminomethane hydrochloride] (Tris),

5 mg/ml pepstatin A, 1 mM EDTA, 10 mg/ml aprotinin,

10 mg/ml leupeptin, 2 mM DL-dithiothreitol (DTT),

3 mM urea prepared in 18 MV water. The samples

were stored at �80 8C for 24 h and lysed by freeze

thaw. The cell lysate was placed in Laemmli sample


buffer containing 5% mercaptoethanol. Samples were

boiled for 5 min and centrifuged at 13,000 � g for

5 min. The supernatant was removed for western blot.

The proteins were separated on a 10% SDS—poly-

acrylamide gel and transferred to nitrocellulose at

125 V for 1 h in Towbin-SDS transfer buffer contain-

ing Tris (25 mM), glycine (192 mM) and 20% metha-

nol. After the transfer, the blot was washed twice with

PBS containing 0.05% Tween 20 (TTBS). For the

determination of iNOS, a rapid 2 h protocol for immu-

nostaining developed by R&D Systems (Minneapolis,

MN, USA) was utilized. Briefly, the primary antibody

used was specific for iNOS, macNOS and was devel-

oped in rabbit, Sigma Chemical Co., (St. Louis, MO,

USA). The primary antibody was diluted to 1:2000 in

1% BSA (Fraction V) in TTBS containing 0.2%

sodium azide. The primary antibody was added to

the nitrocellulose membrane and incubated on a rocker

at room temperature for 1 h. The nitrocellulose mem-

brane was washed three times, and the secondary

antibody (anti-rabbit IgG, whole molecule, peroxidase

conjugate) 1:2000 was added and incubated on a rocker

at room temperature for 1 h. After a final wash, per-

oxidase was detected with SIGMA FASTTM DAB

(3,30-diaminobenzidine tetrahydrochloride) with a

metal enhancer COCl2. Controls were established with

monoclonal anti-b-actin mouse IgG1/anti-mouse igG

(Fc Specific), with antibody at (1:2000)/(1:2000) dilu-

tions, respectively. The standard was established with

b-actin derived from bovine muscle.

Cell Viability Measurements

Almar blue (AB) indicator dye was used to assess

cell viability (Evans et al., 2001; Mazzio and Soliman,

2003a). AB was prepared in phosphate-buffered saline

(0.5 mg/ml). The dye solution was added (15% v/v) to

the samples, and samples were returned to the incu-

bator for 6 h. Quantitative analysis of dye conversion

was measured on a microplate fluorometer—Model

7620—version 5.02, (Cambridge Technologies Inc.)

set at [550/580], [excitation/emissions] wavelengths.

The data were expressed as percentage control.

Data Analyses

Statistical analysis was performed using Graphpad

Prism—version 3.0: Graphpad Software Inc. (San

Diego, CA). Each data point was expressed as the

mean � S.E.M. for each group. Significance of dif-

ference 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

Cytoprotection of CS Against Neurotoxins

The validity of almar blue detection of cell viability

was determined by quantifying fluorescence intensity

with variation in N2A cell density and time. Saturation

kinetic analysis indicated that dye conversion was

linear with cell density [1.0 � 10�3 to 1.0 �10�6 cells/ml] and reached maximum dye conversion

at approximately 4 h, with stability maintained through

12 h (data not shown). The potential cytoprotective

effects of cigarette smoke condensates CSCs were

examined against the experimental toxins—6-OHDA

(100 mM), H2O2 or MPP+ (500 mM) in N2A cells at

24 h (Fig. 1). The results demonstrate that both MAR–

CSC and 2R4F–CSC are not cytoprotective against 6-

OHDA or H2O2. Moreover, they are both toxic at high

concentrations, and exert only mild protective effects

against MPP+. Controls were established to ensure that

the level of toxicity for each neurotoxin was not

extensive to a degree to prohibit observation of cyto-

protective effects. Controls revealed that reduced glu-

tathione [5 mM] protected against the toxicity of 6-

OHDA and H2O2 from [7.0 � 3% to 67 � 2%], [1.0 �0.4 to 81.0 � 2%,], respectively and glucose [10 mM]

protected against the toxicity of MPP+ from [32 � 2 to

79 � 4%] (data not shown). GSH is a potent antiox-

idant, and has been established as protective against the

in vitro toxic effects of 6-OHDA via H2O2 scavenging

(Blum et al., 2000; Blum et al., 2001; Mazzio et al.,

2004). Glucose is protective against MPP+ toxicity

through propelling anaerobic glycolysis, during sus-

tained impairment of the mitochondria (Mazzio and

Soliman, 2003a; Chalmers-Redman et al., 1999).

These findings suggest that CSCs do not substantially

protect against mitochondrial toxins or oxidative stress

in neuroblastoma.

Nitrite Composition of CS

It was evident from visual observation of the blank

controls, that CSCs contained significant quantities of

NO2� (Fig. 2). Furthermore, NO2

� is the product

formed by iNOS enzyme activity in cytokine activated

glioma. Therefore, NO2� was quantified in CSCs

blanks relative to NO2� produced under the glioma

cellular model of inflammation using LPS/IFN-g at 24


Fig. 1. The neuroprotective effects of MAR–CSC and 2R4F–CSC were evaluated in N2A treated with CSCs � MPP+ (500 mM), H2O2 (500 mM) or 100 mM 6-

OHDA for 24 h at 37 8C. The data represent viability as % live control and are expressed as the mean� S.E.M., n = 4. Significance of difference from the control

group was determined by a one-way ANOVA, followed by a Tukey mean comparison post-hoc test, *P < 0.001.

and 48 h. Increasing the concentration of LPS/IFN-g

elevated iNOS to significant levels at 24 and 48 h in

glioma cells. However, the inherent NO2� concentra-

tion of CSCs was larger than endogenous production in

cytokine activated glioma.

iNOS Activity

In order to evaluate if CSCs alter endogenous NO2�

generated by LPS/IFN-g glioma iNOS, critical experi-

mental controls were established (Fig. 3—top panel).

Due to the inherent high levels of NO2� in CSCs

diluents, several volume-adjusted controls were run

in parallel to subtract for interference. The NO2� in C6

cells were calculated by subtracting absorbance values

of the reagent blank control and those of untreated

cells. A two-way ANOVA, revealed that NO2� content

in the reagent blank was not statistically different from

values in untreated cell controls, indicating that CSCs

do not augment NO2� of iNOS in resting C6 cells.

These data suggest that both CSCs inhibit NO2� by

attenuating iNOS in LPS/IFN-g stimulated glioma.

Western blot analysis revealed inhibitory effects of

CSCs on iNOS protein expression in the presence of

LPS/IFN-g (Fig. 3- bottom panel). These data clearly

indicate potent inhibitory effects on iNOS enzyme

induction. In order to ensure that inhibitory effects

on iNOS protein levels, were not due to the loss of cell

viability, a control was established for viability in C6

Cells (Fig. 4). At 2 and 10% solution, both CSCs had

minimal toxic effects on C6 cells, corroborating that

iNOS induction was attenuated by CSCs. A control

was also established to determine the effects of the

ethanol solvent used to prepare CSCs on NO2� pro-

duced in C6 cells (data not shown). The data indicate

that the concentration of ethanol used in this study [up

to 1.9% solution], had no effect on cell viability, but a

slight potentiating effect on NO2�

. This slight inter-

ference was subtracted for evaluation of the data in

Fig. 3.

Monoamine Oxidase

Experimental controls were established for MAO

activity in C6 cell lysates with variation in time (0, 3, 6,

12 h) and substrate concentration (10–1000 mM). DA

was initially compared to tyramine as an MAO sub-

strate, however DA rapidly degraded in aqueous bio-

logical buffer to produce H2O2, the end product for

MAO, yielding a false positive. On the other hand,

tyramine was a stable substrate, and did not react

with the chromogen. The validation controls indicated

linearity of product formation with increase of time

and substrate concentration, only in the presence of

glioma cell lysate. In order to ensure that the signal

was specifically due to MAO activity, product was


Fig. 2. Nitrite concentration in MAR–CSC and 2R4F–CSC solvent blanks

were compared to that produced endogenously in C6 cells exposed to LPS

(6 mg/ml)/IFN-g (100 Units/ml) at 24 and 48 h. The data represent NO2�

[nM/mg protein or volume equivalent] and are expressed as the mean �S.E.M., n = 4. Significance of difference from the treated control was

determined by a one-way ANOVA, followed by a Tukey mean comparison

post-hoc test, *P < 0.001.

monitored in the presence of MAO inhibitors, clorgy-

line, deprenyl and pargyline at 3 h (Fig. 5A–C) (Maz-

zio et al., 2003). Data derived from velocity and

concentration � inhibitors were used to establish a

Michaelis–Menten plot. Both pargyline and deprenyl,

are non-competitive inhibitors, where clorgyline

was competitive with the substrate. The effects of

CSCs were also evaluated on glioma MAO activity

(Fig. 6A, B). Both MAR–CSC and 2R4F–CSCs were

effective in reducing MAO enzyme activity. The MAO

data derived from velocity and concentration � CSCs

were used to establish a Michaelis–Menten plot. The

CSCs displayed a kinetic profile of competitive enzyme

inhibitors, however the MAR–CSC appeared to exert

both competitive and non-competitive inhibition. All

MAO inhibitors and CSCs were tested for cross reac-

tivity with the MAO product, which is H2O2 (Table 1).

There were no interferences detected. This control test

exerts the following functions: (1) the data evaluate the

ability of each compound to scavenge H2O2 in solution

(antioxidant capability) and (2) the data eliminate a

false positive result pertaining to MAO enzyme activ-

ity, based on inhibitor product reactivity. Unlike other

antioxidants, such as GSH and, that can rapidly sca-

venge 500 mM H2O2 in solution, all MAO inhibitors

tested did not. The results indicate a lack of antioxidant

properties exhibited by MAO inhibitors or CSCs, and

therefore effects on MAO activity were validated.

DISCUSSION

The results from this study indicate that cigarette

smoke condensates, derived from either regular or low

tar cigarettes do not contain antioxidant properties and

are not protective against 6-OHDA or H2O2 toxicity.

This was somewhat anticipated, as the toxicity 6-

OHDA in vitro, is known to occur as a result of the

oxidative stress incurred by H2O2 produced through

autoxidation (Blum et al., 2000). Therefore, antioxi-

dants [N-acetyl-L-cysteine and glutathione] and

enzymes, such as catalase, are quite effective in redu-

cing toxicity against these neurotoxins, both in vitro

and in vivo (Blum et al., 2000; Soto-Otero et al., 2000).

In contrast, CSCs are not antioxidants and contain

significant concentration of pro-oxidant radicals,

which should theoretically exacerbate neurotoxicity.

Previous reports corroborate that a number of reactive

oxygen species, such as peroxynitrite (ONOO–), nitric

oxide (NO) and H2O2 are present in CS and formed

readily during combustion (Pryor and Stone, 1993).

Moreover, smoking has been linked to increased inci-

dence of diseases that involve oxidative stress, evident

by low blood circulating antioxidants, such as ascor-

bate and b-carotene, elevated serum lipid peroxidation,

and increased risk for artherosclerosis and coronary

heart disease (Alberg, 2002). Meanwhile, CS contains

aldehyde compounds that can deplete glutathione and

thiol containing proteins (Reddy et al., 2002) leading to

reduced GSH/GSSG plasma ratio in smokers, relative

to non-smoking controls (Moriarty et al., 2003). These

findings indicate that smoking should exacerbate sys-

temic oxidative stress, in line with previous research,

however in the brain, smoking has a neuroprotective

role against oxidative diseases.

Dopaminergic nigral degeneration associated with

progressive PD, involves oxidative stress itself, evident

by DA oxidation, elevated levels of free iron, reduced

concentration of glutathione, lowered GSH/GSSG

and reduced catalase activity in the basal ganglia

(Blum et al., 2001). PD also involves mitochondrial


Fig. 3. Endogenous NO2� was quantified in C6 cells treated with LPS (6 mg/ml)/IFN-gamma (100 Units/ml) � variation in CSCs at 24 h. Due to the inherent

NO2� concentration in the CSCs, the data was quantified by subtracting the NO2

� content of CSCs in reagent controls or basal cells not treated with LPS/IFN-g.

All experiments were maintained at equal sample volume. The data represent NO2� as % control and are expressed as the mean � S.E.M., n = 4. Significance of

difference from the treated control was determined by a one-way ANOVA, followed by a Tukey mean comparison post-hoc test, *P < 0.001. Patterns of iNOS

protein expression were evaluated in C6 cells treated with LPS (6 mg/ml)/IFN-gamma (100 Units/ml) � variation in CSCs at 24 h acquired with SDS-PAGE

protein separation using western blot.

aberrations in the substantia nigra. MPP+ is a potent

experimental mitochondrial toxin that inhibits NADH

oxidoreductase in complex I of the electron transport

chain, and can espouse pathological lesions similar to

those inherent to PD (Ebadi et al., 2001). The data in

this study, demonstrate that cigarette smoke does not

directly protect against these oxidative processes and

similar to the findings in this study, nicotine only

mildly protects against MPP+ (Jeyarasasingam et al.,

2002). Therefore, indirect effects of CS may alter

vulnerabilities to these neurotoxins within the brain.

One potential indirect effect may involve the effect

of smoking on physiological glucose metabolism,

which could significantly alter toxic vulnerability to

mitochondrial toxins (Chalmers-Redman et al., 1999;

Mazzio and Soliman, 2003a). Cigarette smoking is

correlated with increased risk for developing type 2

diabetes (Persson et al., 2000), insulin resistance


Fig. 4. The effects of CSCs were evaluated for toxicity in C6 cells in the

presence of LPS (6 mg/ml)/IFN-gamma (100 Units/ml) at 24 h. The data

represent cell viability as % control and are expressed as the mean �S.E.M., n = 4. Significance of difference from the treated control was

determined by a one-way ANOVA, followed by a Tukey mean comparison

post-hoc test, *P < 0.001.

(Skurnik and Shoenfeld, 1998; Nakanishi et al., 2000),

and elevated glucose transport into skeletal muscle

tissue (Rincon et al., 1999). The potentiating effects

of cigarette smoke on systemic glucose metabolism

Fig. 5. TOP PANEL: Glioma MAO activity was characterized in the presence

concentration constant (1 mM tyramine). The data represent nM product formed/m

difference from the control for each group was determined by a one-way ANOVA,

PANEL: A Michaelis–Menten plot was generated from data on product formed vs

deprenyl (500 mM), clorgyline (2.5 mM) and pargyline (5 mM). Significance of dif

ANOVA. All inhibitor groups were statistically significant from the control, P <

may be due, in part, due to the nicotine content of

cigarettes, which may mediate central effects through

glucocorticoid release (Bornemisza and Suciu, 1980).

Although speculative, future research will be required

to elucidate if smoking alters brain glucose metabo-

lism, and contributes indirectly to the protection

against mitochondrial insults.

A second plausible indirect effect of CS, may

involve known effects on downregulation of MAO.

It is important to note that in this study the concentra-

tions of CSC effective in inhibiting MAO were also

toxic to neuroblastoma, potentially questioning a role

for MAO. It is possible that cancer type cells are more

vulnerable to the toxic effects of CS, due to immortal

cells having high requirements for glutathione

(Ferruzzi et al., 2003), and a present vulnerability

through sulfhydryl depletion by CS (Reddy et al.,

2002). Future research will be required to determine

if the toxicity of CS in primary neurons, parallels that

of neuroblastoma. None the less, there are many reports

implicating MAO inhibition as contributing to the

neuroprotective properties of CS. Positron emission

tomography scans with [11C] L-deprenyl and [11C]

clorgyline indicate that in the human brain, cigarette

smoke can effectively inhibit MAO-A and MAO-B

(Fowler et al., 1998; Volkow et al., 1999). In addition,

chemicals within CS, such as norharman and harman

of clorgyline [A], deprenyl [B] or pargyline [C], holding the substrate

g protein/3 h and are expressed as the mean � S.E.M., n = 4. Significance of

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

. time with variation in substrate � inhibitors at a constant concentration of

ference between treatment and control groups was determined by a two-way

0.001.


Fig. 6. Top panel: The effects of 2R4F–CSC [A] and MAR–CSC [B] on MAO activity were assessed, holding the substrate constant (1 mM tyramine). The data

represent nM product formed/mg protein/3 h and are expressed as the mean � S.E.M., n = 4. Significance of difference from the control for each group was

determined by a one-way ANOVA, followed by a Tukey mean comparison post-hoc test, *P < 0.001. Bottom panel: A Michaelis–Menten plot was generated

from data on product formed vs. time with variation in substrate � CSC a constant concentration of 2R4F CSC (20% solution) and MAR–CSC (10% solution).

Significance of difference between treatment and control groups was determined by a two-way ANOVA. All inhibitor groups were statistically significant from

the control, P < 0.001.

are directly responsible for enzyme inhibition

(Rommelspacher et al., 2002). Chronic MAO down-

regulation by cigarette smoking, may prevent the for-

mation of endogenous mitochondrial neurotoxins that

contribute to the etiology of PD (Naoi et al., 2000;

Maruyama, 2001). Oxidation and methylation of TIQs

and THbCs by MAO can lead to the production of

cationic species structurally similar to MPP+ (Soto-

Otero et al., 2001). Furthermore, products of MAO,

such as acetaldehyde, can undergo condensation reac-

tions with DA to form endogenous neurotoxins, such

as isoquinolines, N-methyl-(R)-salsolinol salsolinol,

and tetrahydropapaverine (Maruyama et al., 2000;

Maruyama, 2001). Subsequent methylation, can lead

to formation of N-methyl-R-salsolinol (a potent dopa-

minergic neurotoxin), or oxidative products, such as

isoquinolinium (potent mitochondrial toxin) (Naoi

et al., 1998; Storch et al., 2000). Therefore, the inhibi-

tion of MAO may reduce the vulnerability to age-

related CNS neurological degenerative disorders, in

particular involving central dopaminergic systems

(Zeng et al., 1995; Fowler et al., 1997).

A third plausible indirect effect of CS may involve

anti-inflammatory effects in glial cells of the brain.

During CNS inflammation, the rise in endogenous

cytokine concentration can lead to the induction of

iNOS in astrocytes (Zhao et al., 1998). Meanwhile,

overproduction of NO in the CNS is thought to play a

critical role in the pathology of degenerative diseases,

such as Alzheimer’s disease (Luth et al., 2002) ische-

mia, head trauma (Gahm et al., 2002), multiple sclero-

sis (Broholm et al., 2004) and PD (Hyun et al., 2002).

In the CNS, accumulation of singlet NO is relatively

non-toxic, however its combination with superoxide

can yield peroxynitrite/peroxynitrous acid, which con-

tributes to neurodegenerative injury through oxidation/


Table 1

Antioxidant effects of MAO inhibitors and CSCs

mM Clorgyline mM Deprenyl mM Pargyline

mM H2O2 remaining at 30 M (start 250 mM H2O2)

Ctrl 250.0 � 0.9 Ctrl 250.0 � 0.5 Ctrl 250.0 � 1.1

0.50 248.7 � 0.4 100 247.9 � 0.5 1.00 250.6 � 1.3

2.50 248.4 � 0.6 500 250.5 � 0.5 5.00 256.5 � 1.8

5.00 249.5 � 0.7 1000 249.1 � 1.2 10.00 252.7 � 0.9

mM GSH Solution (%) MARLBORO Solution (%) 2R4F

Ctrl 500.0 � 1.4 Ctrl 250.0 � 3.0 Ctrl 250.0 � 1.5

1.00 43.4 � 2.8 2.00 260.8 � 3.0 2.00 255.0 � 3.0

5.00 8.1 � 0.3* 10.00 274.3 � 2.8 10.00 270.5 � 1.4*

10.00 4.5 � 1.3* 20.00 285.8 � 0.9* 20.00 290.0 � 1.9*

The antioxidant capacity of CSCs and MAO inhibitors was evaluated. Experimental compounds were incubated with 250 mM of H2O2 for 30 M. A control was

established for GSH in the presence of 500 mM H2O2. The data represent mM of H2O2 remaining, and are expressed as the mean � S.E.M., n = 4. Significance of

difference from the control was determined by a one-way ANOVA, followed by a Tukey mean comparison post-hoc test.* P < 0.001.

nitrosylation of lipid membrane and protein structures

(Szabo, 2003; Kikugawa et al., 2004). The presence of

ONOO– within or surrounding neurons, can contribute

to toxicity through rendering a loss of anaerobic and

mitochondrial oxidative metabolism and initiation of

apoptosis (Szabo, 2003; Brown and Borutaite, 2002;

Murray et al., 2003; Vieira and Kroemer, 2003).

The findings in this study indicate that chemicals

within CS can reduce expression of glial iNOS in the

presence of cytokines, yet paradoxically CS also con-

tains high concentration of nitrite. It is not known if the

NO2� content of cigarette smoke actually reaches the

brain, however, other studies have corroborated that

cigarette smoke contains high concentration of nitro-

gen oxides, and NO2� (0.34 mg per cigarette) (Toki-

moto and Shinagawa, 2001; Rustemeier et al., 2002).

The concentration of total N-nitroso compounds in

cigarette smoke filter pads is �220 nM per cigarette

(Haorah et al., 2001) and �137–238 ng per cigarette of

the nitrosamine, N0-nitrosonornicotine (Hecht et al.,

1978). While it is known that NO2� in cigarette smoke

can react directly with biological structures, contribut-

ing toward smoking-related illnesses, such as cancer

(Lee et al., 1997; Paik and Dillon, 2000), little is known

about the effects of smoke on NO2� within the brain

and central nervous system. Moreover, reports indicate

that NO donors may provide neuroprotection through

propensity to potentiate glutathione expression, pro-

vide resistance to metal catalyzed oxidative stress

(Canals et al., 2001), reduce free radical formation

in glia, reduce neurological injury associated with

infection, ischemia/reperfusion (Dobrucki et al.,

2000; Mason et al., 2000), lipid peroxidation (Nara

et al., 1999) and intra-neuronal calcium overload

(Hotta et al., 1999). Therefore, it is plausible that

the NO content of cigarette smoke may play a sig-

nificant role in altering vulnerability to neurodegen-

erative diseases of the brain. Future research will be

required to corroborate if the inhibitory effects of CS

on iNOS or NO donor properties of CS, contribute in

part toward reduced neurodegenerative processes in the

brain.

In summary, cigarette smoke contains high concen-

tration of NO2�, yet attenuates expression of cytokine

activated glial iNOS. CS is an inhibitor of MAO, and is

not directly protective against the toxicity of 6-OHDA

or H2O2. Future research will be required to investigate

if the protective effects of CS involve alterations in

central glucose metabolism or antagonizing the expres-

sion of pro-inflammatory proteins in glia.

ACKNOWLEDGMENT

This work was supported by a grant received from

the National Institutes of Health (NCRR 03020). The

authors wish to gratefully acknowledge the use of

Florida A&M University ARCH Core Facility and

the NIEHS grant # ES 11182.

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