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
fax: +1 8
E-mail ad
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%
E.A. Mazzio et al. / NeuroToxicology 26 (2005) 49–6252
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
E.A. Mazzio et al. / NeuroToxicology 26 (2005) 49–62 53
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
E.A. Mazzio et al. / NeuroToxicology 26 (2005) 49–6254
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
E.A. Mazzio et al. / NeuroToxicology 26 (2005) 49–62 55
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
E.A. Mazzio et al. / NeuroToxicology 26 (2005) 49–6256
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
E.A. Mazzio et al. / NeuroToxicology 26 (2005) 49–62 57
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.
E.A. Mazzio et al. / NeuroToxicology 26 (2005) 49–6258
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/
E.A. Mazzio et al. / NeuroToxicology 26 (2005) 49–62 59
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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