Mol Neurobiol

DOI 10.1007/s12035-014-8928-x

Lead Intoxication Synergies of the Ethanol-Induced Toxic

Responses in Neuronal CellsPC12
V. Kumar & V. K. Tripathi & S. Jahan & M. Agrawal &
A. Pandey & V. K. Khanna & A. B. Pant

Received: 7 August 2014 / Accepted: 7 October 2014

# Springer Science+Business Media New York 2014

Abstract Lead (Pb)-induced neurodegeneration and its link

with widespread neurobehavioral changes are well documented. Experimental evidences suggest that ethanol could enhance the absorption of metals in the body, and alcohol
consumption may increase the susceptibility to metal intoxication in the brain. However, the underlying mechanism of
ethanol action in affecting metal toxicity in brain cells is
poorly understood. Thus, an attempt was made to investigate
the modulatory effect of ethanol on Pb intoxication in PC12
cells, a rat pheochromocytoma. Cells were co-exposed to
biological safe doses of Pb (10 M) and ethanol (200 mM),
and data were compared to the response of cells which received independent exposure to these chemicals at similar
doses. Ethanol (200 mM) exposure significantly aggravated
the Pb-induced alterations in the end points associated with
oxidative stress and apoptosis. The finding confirms the involvement of reactive oxygen species (ROS)-mediated oxidative stress, and impairment of mitochondrial membrane potential, which subsequently facilitate the translocation of triggering proteins between cytoplasm and mitochondria. We
further confirmed the apoptotic changes due to induction of
mitochondria-mediated caspase cascade. These cellular
changes were found to recover significantly, if the cells are
exposed to N-acetyl cysteine (NAC), a known antioxidant.
Our data suggest that ethanol may potentiate Pb-induced
V. Kumar : V. K. Tripathi : S. Jahan : M. Agrawal : A. Pandey :
V. K. Khanna : A. B. Pant (*)
In Vitro Toxicology Laboratory, Indian Institute of Toxicology
Research, P.O. Box: 80, MG Marg, Lucknow 226001, UP, India
e-mail: abpant@rediffmail.com
A. B. Pant
e-mail: abpant@yahoo.com
V. Kumar : V. K. Tripathi : S. Jahan : M. Agrawal : A. Pandey :
V. K. Khanna : A. B. Pant
Council of Scientific & Industrial Research, New Delhi, India

cellular damage in brain cells, but such damaging effects

could be recovered by inhibition of ROS generation. These
results open up further possibilities for the design of new
therapeutics based on antioxidants to prevent neurodegeneration and associated health problems.
Keywords Neurotoxicity . Pb . Ethanol . PC12 cells

Introduction
The toxicological consequences due to heavy metal exposure
from anthropogenic and environmental sources have been
recognized as a major health concern globally [1, 2]. Among
the group of heavy metals with proven toxicity in human, lead
(Pb), a ubiquitous pollutant in the ecosystem, is known to be
highly neurotoxic [3] and developmental neurotoxic [4]. The
major Pb-induced neurological disorders reported in humans
and experimental animals are behavioral abnormalities, learning impairment, decreased hearing, impaired cognitive function, etc. [5]. Despite several in vitro [6, 7], in vivo [8, 9] and
epidemiological [10] studies, the exact mechanism(s) of Pbinduced neurotoxicity in both fetus/children and adults have
still not been elucidated and are also not well understood.
However, among the several proposed mechanisms of Pbinduced neurotoxicity, the oxidative stress-mediated apoptosis
is the most prominent [1113]. Pb exposure increases the
levels of reactive oxygen species (ROS) and intracellular
Ca2+, which in turn disturbs the mitochondrial membrane
potential, leading to release of mitochondrial cytochrome-c
in the cytoplasm by activating caspase cascade-mediated apoptotic cell death [1416]. It also disturbs the antioxidant
defense system via thiol binding [17, 18] and by its calcium
mimicking ability [15]. Previous reports showed significant
altered manifestations of Pb intoxication due to individuals

Mol Neurobiol

habits such as intake of total food, amount and type of fat,

calcium and iron, etc. [19].
Alcohol consumption is one of the major lifestyle factors.
After intake, alcohol is distributed throughout the body tissues
and rapidly crosses the blood-brain barrier and damages the
nervous system [2023]. Teratogenic and developmental neurotoxic effects of ethanol are also well documented [24, 25].
Chronic exposure to ethanol has been reported to cause neuronal degeneration [26], cognitive impairment [27] with permanent structural damages in the brain [28, 29]. At cellular
level, ethanol mediates neurodegeneration by altering the
expression of markers (messenger (m)RNA and proteins)
associated with myelin degeneration [30], insulin responsiveness [31], ROS-mediated oxidative stress [32] and apoptosis
[33], and deregulated acetylcholine homeostasis [34], etc.
There are numerous studies demonstrating high vulnerability of human subjects towards Pb exposure, especially those
who regularly consume alcohol [35]. Reports have shown the
presence of high Pb levels in the blood of alcoholics when
compared with non-alcoholics of same occupation and residing in the same vicinity [36]. Enhanced Pb intoxication in
alcoholics has been suggested in part due to increased permeability of membranes and eventually increased burden of Pb
and depletion of essential metals [37, 38]. Both Pb and alcohol
are known neurotoxicants, and alcohol is known to cross the
blood-brain barrier. As a consequence, this can result in increasing the neurotoxicity level further by facilitating the entry
of Pb in the brain [39].
In this regard, the in vivo reports are available suggesting
the additive/synergic toxic responses of co-exposure to ethanol and Pb [40]. However, detailed studies on the exact
mechanism of ethanol-Pb co-exposure inducing neuronal impairment, oxidative stress, and subsequent apoptosis in controlled in vitro conditions using neuronal cells have not been
reported so far. Thus, the present investigations were carried
out to study the modulatory effect of ethanol concentrations
on Pb-induced oxidative stress, apoptosis, and subsequent
neurodegeneration in PC12 cells, one of the most widely
studied cell line for neurobiology/neurotoxicity studies.

Materials and Methods

Reagents and Consumables All the specified chemicals, reagents, diagnostic kits were purchased from Sigma Chemical
Company. (St. Louis, MO, USA). Nutrient mixture F-12
Hams culture medium, antibiotics, fetal bovine serum (FBS),
and horse serum (HS) were purchased from Gibco BRL,
USA. Culture wares and other plastic consumables used in
the study were purchased from Nunc, Denmark. All other
chemicals were of analytical grade of purity and were procured locally.

Cell Culture PC12, a rat pheochromocytoma cell line, was

originally procured from National Centre for Cell Science,
Pune, India and cultured in 82.5 % Nutrient Mixture (F-12
Hams), supplemented with 2.5 % heat-inactivated FBS, 15 %
HS, 0.2 % sodium bicarbonate (NaHCO3), and antibiotic and
anti-mycotic solution (100X, Invitrogen, Life Technologies,
USA). Cells were maintained in a humidified atmosphere of
95 % air and 5 % CO2 at 37 C in an incubator. The culture
medium was replaced twice a week, and cell cultures were
passaged at a ratio of 1:6 in a week. Prior to use the cells in the
experiments, they were screened for their viability using
trypan blue dye exclusion assay and batches showing more
than 95 % viability were used in the study.
3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide) Assay Prior to use in the experiments, cytotoxic effect
of Pb, ethanol, and their combination on PC12 cells were
carried out using 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide) (MTT) assay as described earlier [41]. Briefly, 1104 cells were allowed to adhere to polyL-lysine-coated 96-well culture plates for 24 h under humid
conditions in 5 % CO2 and 95 % atmospheric air at 37 C. The
medium was aspirated, and cells were incubated with medium
containing graded series of Pb (0.1200 M) and ethanol (50
600 mM) for 24 h. To address the synergistic cytotoxic effect,
PC12 cells were incubated with combined doses of Pb
(10 M) and ethanol (200 mM) for 24 h. After incubation,
the cells were washed twice with phosphate-buffered saline
(PBS), and 200 l of culture medium containing 5 mg/ml of
MTT dye was added to each well and incubated further for
4 h. The reaction mixture was carefully taken out and 200 l
of culture grade dimethylsulphoxide (DMSO) was added to
each well and mixed by pipetting up and down several times
until the content was homogenized. After 10 min, the color
was read at 550 nm using multi-well microplate reader (Synergy HT, Bio-Tek, USA). The unexposed cells which served
as control were also run simultaneously under identical
conditions.
Measurement of Intracellular Calcium [Ca2+]i The calcium
levels have been demonstrated to play a key role in signal
transduction and in various metabolic and physiological processes in the cells. Thus, even a small change in the level of
intracellular calcium [Ca2+]i may induce major alterations in
the cellular activities [14]. [Ca2+]i was measured by fluorometric analysis using molecular probes fura-2
acetoxymethyl ester pentapotassium (fura-2-AM; Sigma-Aldrich, USA). PC12 cells were loaded with 3 mol/l fura-2AM in loading solution containing 125 mM NaCl, 5 mM KCl,
1.2 mM MgSO4, 1.2 mM KH2PO4, 2 mM CaCl2, 6 mM
glucose, 25 mM Heps-NaOH (pH 7.4) buffer and incubated
for 30 min at 37 C in 5 % CO2 and 95 % atmospheric air.
Unbound fura-2-AM was removed by rinsing the cells twice

Mol Neurobiol

with titration solution and saline A containing NaCl, 8.182 g/l;

KCl, 0.372 g/l; NaHCO3 0.336 g/l; and glucose, 0.9 g/l. The
cells (1106 ml1) receiving Pb (10 M) and/or ethanol
(200 mM) or the unexposed control groups were represented
in titration solution or saline A. Ca2+ fluorescence was measured by BMG FLUOstar Omega at an emission wave length
of 510 nM using a pair of excitation length at 355 and 380 nM,
and (Fo) excitation was obtained at the ratio of that generated
by 355/380. Fmax was obtained after cells were lysed with
0.1 % Triton X-100. Fmin was obtained after the addition of
EGTA (5 mM/l final concentration). Ca2+ fluorescence was
examined simultaneously at time points 5, 15, 30, and 45 min
and 1 h. The results shown are representative of three independent experiments carried out under identical experimental
conditions. The concentration of intracellular calcium [Ca2+]i
was calculated using the following formula:



Ca2 i K d  FoFmin=FmaxFo

where Kd is the dissociation constant of fura-2-AM calcium

complex and its value is 224 nmol/l. The results are expressed
in terms of nanomoles of calcium.
ROS Generation Pb- and/or ethanol-induced ROS generation
in PC12 cells which was assessed using 2,7dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma-Aldrich, USA) dye as the fluorescence agent [42]. Briefly, cells
(5104/well) were seeded in poly-L-lysine-pre-coated tissue
culture slides flasks and allowed to adhere. Cells were then
exposed to either of Pb (10 M), ethanol (200 mM), Pb
(10 M) + ethanol (200 mM), NAC (10 M), or Pb
(10 M)+ethanol (200 mM)+(10 M) for 24 h. After exposure, cells were washed twice with PBS and incubated for
30 min at 37 C in the dark in incomplete culture medium
containing H2DCFH-DA (20 M). Slides were washed twice
again with PBS and mounted for microscopic analysis. Intracellular fluorescence was measured using an upright fluorescence microscope by grabbing the images (Nikon Eclipse 80i
equipped with Nikon DS-Ri1 12.7 megapixel camera). Quantification of fluorescence was done using the image analysis
software Leica Qwin 500, and data were expressed as the fold
change of unexposed control cells. Cells exposed to H2O2
(100 M) for 6 h under identical conditions were used as a
positive control.
Glutathione Content Measurement Intracellular glutathione
content was estimated following the protocol of Akhtar et al.
2010 [43]. The exposure of cells to the test chemical and other
conditions were identical to that of ROS generation. After
exposure, cells were washed twice in cold PBS and lysed by
four cycles of freeze thaw in distilled water containing
deoxycholic acid plus sucrose and centrifuged at 10,000g
for 10 min at 4 C. The protein estimation was done in

supernatant. The supernatant protein was then precipitated

with 1 % perchloric acid and centrifuged again at 10,000g
for 5 min at 4 C. The precipitated protein (20 l) was mixed
with 160 l of 0.1 M phosphate, 5 mM EDTA buffer (pH 8.3),
and 20 l o-phthalaldehyde (OPT, 1 mg/ml in methanol) in a
black bottom 96-well plate. The plates were incubated for 3 h
at room temperature in the dark, and fluorescence was measured at an emission/excitation wavelengths of 460/355 nm
respectively, along with similarly prepared standards of glutathione (GSH) in 1 % perchloric acid. The results are
expressed as nanomole of GSH per milligram of cellular
protein. Cells exposed to H2O2 (100 M) for 6 h under
identical conditions were used as a positive control.
Lipid Peroxidation Lipid peroxidation was evaluated by the
thiobarbituric acid-reactive substance (TBARS) assay, which
identifies mainly malondialdehyde (MDA), an end product of
lipid peroxidation [44]. MDA was measured by following the
protocol of Akhtar et al. 2010 [43]. Briefly, after exposure to
Pb (10 M), ethanol (200 mM), Pb (10 M) + ethanol
(200 mM), NAC (10 M), and Pb (10 M) + ethanol
(200 mM)+NAC (10 M) for 24 h, cells were washed twice
by isotonic trace element-free Tris-HCl buffer (400 mM, pH
7.3). A 200-l aliquot of cell suspension was subsequently
mixed with 800 l of lipid peroxidation (LPO) assay cocktail
containing 0.4 % (w/v) thiobarbituric acid, 0.5 % (w/v) sodium
dodecyl sulfate (SDS), and 5 % (v/v) acetic acid (pH 3.5) and
incubated for 60 min at 95 C. The sample was cooled using
tap water and centrifuged at 2500g for 5 min. The absorbance of the supernatant was read at 532 nm against a standard
curve prepared using the MDA standard (10 mM) standard
(10 mM 1,1,3,3-tetramethoxypropane in 20 mM Tris-HCl, pH
7.4). The results are calculated as nanomoles of MDA per
milligram of protein. Cells exposed to H2O2 (100 M) for 6 h
under identical conditions were used as a positive control.
Mitochondrial Membrane Potential Mitochondrial membrane potential is one of the earliest biomarkers to identify
the induction of the oxidative stress and apoptosis. Flow
cytometric analysis of apoptotic cell population was done by
Mitolight Apoptosis Detection Kit (APT142, Chemicon,
USA). Mitolight (5,5,6,6-tetrachloro-1,1,3,3-tetraethyl
benzimidazolyl carbocyanine chloride) is a mitochondrial
dye that stains the mitochondria of living cells only. Briefly,
cells after exposure to Pb (10 M), ethanol (200 mM), Pb
(10 M) + ethanol (200 mM), NAC (10 M), and Pb
(10 M)+ethanol (200 mM)+NAC (10 M) for 24 h were
re-suspended in 1 ml of pre-diluted Mitolight solution for
15 min at 37 C. Cells were then immediately analyzed by
flow cytometer (BD FACSCanto ) equipped with the
FACSDiva Version 6.0.0. software. Cells exposed to H2O2
(100 M) for 6 h under identical conditions were used as a
positive control.

Mol Neurobiol

TUNEL Assay Deoxynucleotide transferase dUTP nick end

labeling (TUNEL) is a method for detecting DNA fragments
during the cell cycle by labeling of the deoxynucleotide transferase dUTP nick end. This assay has been carried out using
APO-BrdU TUNEL Assay Kit with Alexa Fluor 488 antiBrdU (Molecular Probes, Invitrogen Detection Technologies,
USA, Catalog No. A23210) by flow cytometer (BD
FACSCanto, USA) equipped with BD FACSDiva, Version
6.1.2, software. Debris was excluded by forward and side-way
light-scattering. Though, the positive controls provided with
kit were used, but at the same time, cells receiving H2O2
(100 M) exposure for 6 h were also used as positive controls.
Transcriptional Changes (Real-Time PCR) Pb (10 M) and/
or ethanol (200 mM) induced alterations in the expression
level of mRNA of marker genes involved in oxidative stress,
and apoptosis-mediated pathways were studied using quantitative real-time PCR (RT-PCR). Total RNA was isolated from
both exposed and unexposed control cells using Trizol reagent
(Invitrogen). Total RNA (1 g) was reverse transcribed into
complementary (c)DNA by superscript 111 first strand cDNA
synthesis kit (Catalog No 1808005, Invitrogen Lifescience
USA). RT-PCR was performed by SYBR Green dye (ABI,
USA) using ABI PRISM 7900HT sequence detection system (Applied Biosystem, USA). Real-time reactions were
performed in triplicate for each sample. The specificity for
each primer (Table 1) was assessed by melting curve analysis
and running no template control (NITCs) for respective
primers. -Actin was used as internal control to normalize
the data. Expressional changes (mRNA) are expressed in
relative quantification (RQ).
Translational Changes (Western Blotting) Alteration in the
expression of marker proteins associated with oxidative stress
(glutathione S-transferase A4-4 (GSTA4-4), HSP70) and apoptosis (CYP-2E1, Bax, Bcl2, PARP, p53, Cytochrome-c, and
activated Caspase-9 and 3) was studied using Western blot
analysis. In brief, after exposure of the cells to Pb (10 M)
and/or ethanol (200 mM), cells were pelleted and lysed using

CelLytic M cell lysis reagent (Sigma) in the presence of

protein inhibitor cocktail (Sigma). After protein estimation
by Bradfords reagent (Fermentas Inc., Glen Burnie, MD),
equal amounts (50 g/well) of denatured proteins were resolved onto TricineSDS/SDS-PAGE gel and blotted onto a
polyvinylidene fluoride membrane (Millipore). After
blocking, the membranes were incubated overnight at 4 C
with primary antibodies of p53, CYP-2E1 (1:1000, Millipore,
USA), activated caspase-9 and 3 (1:200, Millipore, USA),
GSTA4-4 (1:3000, Abcam, USA), PARP, Cytochrome-c
(1:1000, CST, USA), Bax, Bcl2 (1:1000, Santa Cruz, USA),
and -actin (1:500, Millipore, USA). The membranes were
washed three times with PBS and then re-incubated with
horseradish peroxidase-conjugated secondary antibodies
(Calbiochem, USA). The blots were developed using luminol
(Catalog No. 34080, Thermo Scientific, USA), and densitometry for protein-specific bands was done in Gel Documentation System (Alpha Innotech, USA) using AlphaEase FC
StandAlone V. 4.0.0 software. -actin was used as internal
control to normalize the data. The data are expressed in terms
of fold change comparing the control.
Translocation Studies Translocation of Bax protein from cytosol to mitochondria and cytochrome-c from mitochondria to
cytosol due to exposure-induced mitochondrial membrane permeabilization was studied by Western blotting and immunocytochemical analysis using specific anti-Bax (Santa Cruz, USA)
and anti-Cytochrome-c primary antibodies (CST, USA). After
exposure of cells to Pb (10 M), ethanol (200 mM), Pb
(10 M) + ethanol (200 mM), Pb (10 M) + ethanol
(200 mM)+NAC (10 M), and NAC (10 M) for 24 h, cells
were harvested and processed for isolation of mitochondrial and
cytosolic fraction using the protocols of Ghosh et al. 2002 [45]
and Waterhouse et al. 2004 [46]. The cross-contamination of
cytosolic protein in the mitochondrial fraction and vice versa was
also assessed by running separate blots using antibodies specific
to mitochondria and cytosolic proteins. Translocation proteins
between cytosol and mitochondria were also confirmed by immunocytochemistry using anti-Bax- and anti-Cytochrome-c-

Table 1 Real-time PCR: the sequences of primers for real-time PCR were
Gene name

Forward primer (5-3)

Reverse primer (5-3)

-actin
CYP-2E1

ACT ATC GGC AAT GAG CGG TTC

AAA GCG TGT GTG TGT TGG AGAA

AGA GCC ACC AAT CCA CAC AGA

AGA GAC TTC AGG TTA AAA TGC TGC A

GSTA4-4
p53
Bax
Bcl2
HSP70
Caspase-9
Caspase-3

GAA GTT CTA GTG ACA GCG TGC TTT A

CCC CAC CGC CTG TAA GAT T
GGG TGG TTG CCC TTT TC TAC T
CTG TAC GGC CCC AGC ATG CG
ACC AGG ACA CTG TTG AGT TC
AGC CAG ATG CTG TCC CAT AC
CAG AGC TGG ACT GCG GTA TTG A

TGT AGC TGC TGC TGT GAT TGG

ATG GGT CCG GAG GAT ACA GAT
CCC GGA GGA AGT CCA GTG TC
GCT TTG TTT CAT GGT ACA TC
ACT CAT CTC CGA GTT CAC AC
CAG GAG ACA AAA CCT GGG AA
CAG GAG ACA AAA CCT GGG AA

Mol Neurobiol

specific monoclonal antibodies. For immunocytochemical analysis studies, cells were seeded in PLL-pre-coated eight-well
chamber slides at a density of 1104 cells/well. After the exposures, the cells were washed with PBS (100 l/well) and stained
with Mitotracker dye (100 nM) for 1 h at 37 C, then fixed in 4 %
paraformaldehyde. Cells were then washed twice with PBS and
incubated for 15 min in PBS containing 0.02 % Triton100 and
0.1 % BSA to block the nonspecific binding sites. The cells were
then incubated with primary antibodies of Cytochrome-c and
Bax for 24 h. The antibodies were diluted in PBS containing
0.02 % Triton100 and 0.1 % BSA. The cells were then washed
three times with PBS and re-incubated for 2 h at room temperature with secondary anti-primary immunoglobulin G (IgG)
conjugated with rhodamine fluorescence dye (Calbiochem,
USA). Cells were visualized under upright microscope (Nikon
Eclipse 80i equipped with Nikon DS-Ri1 12.7 megapixel camera, Japan) and quantified by measuring the change in percent
area of protein expression with the help of Leica Qwin 500
Image Analysis Software (Leica, Germany).
Caspase-9/3 Activity Caspase-9/3 activity was assessed as per
the protocols of the manufacturer (Biovision, Catalog no. K119
and K106, USA). After exposure to Pb (10 M), ethanol
(200 mM), Pb (10 M)+ethanol (200 mM), NAC (10 M),
and Pb (10 M)+ethanol (200 mM)+NAC (10 M) for 24 h,
cells were pelleted, re-suspended in pre-chilled extraction buffer (50 l), and incubated on ice for 10 min. The samples were
then centrifuged for 5 min at 500g, and supernatant (50 l/
well) was transferred to 96-well culture plates. Assay buffer
(50 l) and substrate conjugate (5 l) of caspase-9 and caspase3 were added, mixed well, and incubated for 1 h at 37 C in
dark. Upon completion of incubation period, cells were mixed
thoroughly and read absorbance was read at 405 nm for both
caspases. The values of exposed groups were compared with
unexposed control sets, and the data were expressed as a
percentage of control. Cells exposed to H2O2 (100 M) for
6 h under identical conditions were used as a positive control.
Statistical Analysis Results were expressed as meanstandard error of mean (SEM) for the values obtained from at
least three independent experiments. Statistical analysis was
performed using one-way analysis of variance (ANOVA) and
Dunnetts multiple comparison test using GraphPad Prism
(Version 5.0) software. The valves */#p<0.05 were considered
as significant, ** /##p < 0.01 more significant, and *** /
###
p<0.001 highly significant.

found to be non-cytotoxic. The higher concentrations of Pb,

i.e., 50, 100, and 200 M, have shown statistically significant
reduction in percent cell viability (Fig. 1a). Exposure to ethanol up to 100 mM did not elicit any cytotoxic response.
Thereafter, there was a statistically significant dosedependent decrease in the percent cell viability (Fig. 1b).
Based on the cytotoxicity data of individual toxicants, further
experiments were carried out to evaluate the effect of coexposure to Pb (10 M) and ethanol (200 mM) in PC12 cells.
The co-exposure to Pb and ethanol showed an additive effect
on the loss of cell viability in comparison to individual exposure to Pb and ethanol at the same concentration (Fig. 1c).
Intracellular Calcium Levels [Ca2+]i Both Pb and ethanol
induce a significant increase in the levels of [Ca2+]i at each
time point studied, with maximum levels at 30 and 45 min for
Pb and ethanol exposure, respectively. There was an additive
effect in cells co-exposed to Pb and ethanol, and this additive
effect increased gradually till 45 min. At 60 min, the trend was
similar but the magnitude of increase in the levels of [Ca2+]i
was lower than that at 45 min. In general, the level of increase
was statistically significant in all the cases, i.e., Pb (p<0.05),
ethanol (p < 0.05), and co-exposure to Pb and ethanol
(p<0.001) (Fig. 2).
ROS Generation Results of Pb- and/or ethanol-induced ROS
generation in PC12 cells are depicted in Fig. 3. Fluorescence
microscopy studies showed that PC12 cells receiving coexposures of Pb 10 M+ethanol 200 mM have a significantly
high (p<0.05) ROS production in comparison to the individual
exposures to Pb (10 M) and ethanol (200 mM). Similarly, the
positive control (H2O2 100 M) also induced significant
(p<0.01) ROS generation as compared to control. However,
NAC (10 M) (a known antioxidant) exposure showed a significant (p<0.05) restoration in ROS levels, when compared with
Pb 10 M+ethanol 200 mM exposure group (Fig. 3a, b).
Glutathione Levels A significant reduction in the levels of
GSH was observed in all groups receiving individual and
co-exposures to Pb and ethanol when compared with unexposed control sets. Ethanol 200 mM showed significant
(p<0.01) additive effect in the Pb (10 M)-induced alterations
in GSH levels, while NAC treatment showed significant
(p<0.05) restoration in GSH levels when compared with Pb
10 M+ethanol 200 mM exposure group. As anticipated, a
highly significant (p<0.01) reduction in GSH levels was
recorded in cells exposed to the positive control (H2O2
100 M) group (Fig. 4a).

Results
Cytotoxicity Studies A concentration-dependent decrease in
the percent cell viability was observed in PC12 cells exposed
to Pb (10200 M) for 24 h. Pb concentration at 10 M was

Lipid Peroxidation The results indicate that co-exposure to Pb

(10 M)+ethanol (200 mM) significantly adds to the individual exposure to Pb (10 M) or ethanol (200 mM) induced
increase in LPO. But, the level of LPO was reduced following

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Fig. 1

Percent cell viability assessed by MTT assay in PC 12 cells a

Cells exposed to Pb (0.1 to 200 M) for 24 h. b cells exposed to ethanol
(50600 mM) for 24 h. *p<0.05, **p<0.01, ***p<0.001. c Cells
exposed to Pb (10 M) and/or ethanol (200 mM) for 24 h. */#p<0.05,
**/## p < 0.01, *** /### p < 0.001;*p < 0.05, **p < 0.01, *** p < 0.001
(unexposed control vs. experimental group); #p < 0.05, ##p < 0.01,
###
p<0.001 (compared between treated groups)

the exposure to Pb (10 M)+ethanol (200 mM) with antioxidant (NAC 10 M) in comparison to LPO induction due to
Pb (10 M)+ethanol (200 mM) exposure. Moreover, cells
exposed to H2O2 (10 M) for 6 h have also shown highly
significant increase in the levels of LPO (Fig. 4b).
Mitochondrial Membrane Potential Flow cytometric analysis
clearly showed that co-exposure to Pb (10 M) and ethanol
(200 mM) induced maximum reduction (19.3 %) in mitochondrial membrane potential (MMP) compared with individual
exposures to Pb 10 M (15.7 %) and ethanol 200 mM (7.4 %),
respectively. On the other hand, NAC (10 M) reduced the
adverse effect of Pb (10 M)+ethanol (200 mM) by 15.8 %
when given together. Cells exposed to H2O2 (100 M) for 6 h
have also shown a significant reduction (17.0 %) in MMP
(Fig. 5)
TUNEL Assay Pb- and/or ethanol-induced apoptosis was also
assessed using the TUNEL assay with the help of FACS
analysis (Fig. 6). Exposure to Pb and ethanol separately was
found to induce apoptosis of 7.9 and 3.9 %. These changes
were synergistic in case of co-exposure to Pb and ethanol, i.e.,
12 %. Pb and ethanol induced apoptotic changes, which could
be reverted back towards the basal level (6.8 %) in cells
simultaneously exposed to NAC (10 M) (Fig. 6)
Transcriptional Changes Pb and/or ethanol exposureinduced alterations in the mRNA expression of marker genes
associated with oxidative stress and apoptosis and are presented in Fig. 7. Though, the individual exposures to Pb and
Fig. 2 Measurement of
intracellular Ca2+ levels (nmol/l)
in PC 12 cells. Cells exposed to
Pb (10 M) and/or ethanol
(200 mM) at various time periods
from 5 to 60 min. *p<0.05,
**p<0.01, ***p<0.001
(unexposed control vs.
experimental group)

ethanol have shown significant upregulation of oxidative

stress and pro-apoptotic genes and downregulation of
anti-apoptotic genes, the magnitude of these alterations
was significantly increased in the cells co-exposed to Pb
(10 M)+ethanol (200 mM), i.e., p53 4.14-, Bax 3.33-,
caspase-9 9.69-, caspase-3 4.15-, CYP-2E1 7.95-,
GSTA4-4 10.84-, Hsp70 3.75-, and Bcl2 0.376-fold of
control, respectively.
Translational Changes The results of Western blot analysis to
evaluate the altered expressions of marker proteins of oxidative stress and apoptosis are presented in Fig. 8. Co-exposure
to Pb and ethanol effectively upregulated the protein expression of p53 (2.78-fold), cytochrome-c (2.26-fold), Bax (1.87fold), activated caspase-9 (1.68-fold,) activated caspase-3
(1.67-fold), CYP-2E1 (1.93-fold), GSTA4-4 (1.97-fold), and
HSP-70 (1.81-fold) and downregulated Bcl2 (0.51-fold) of
respective controls. Protein expression for poly (ADPribose) polymerase (PARP) showed the fragments of cleavage
of 116 and 89 kDa proteins exposed to toxic insult, which was
higher in co-exposure to Pb and ethanol in comparison to its
individual groups. There was linearity in the data at both
transcriptional and translational levels in co-exposure as well
as in individual exposure groups.
Translocation of Bax and Cytochrome-c The involvement of
mitochondria-mediated apoptotic pathways in PC12 cells coexposed to Pb and ethanol was confirmed by the translocation
of Bax protein from cytosol to mitochondria and cytochromec protein from mitochondria to cytoplasm using the Western
blot analysis and immunocytochemical localization. There
was a significantly higher translocation of proteins between
mitochondria and cytoplasm in cells co-exposed to Pb and
ethanol, when compared with magnitude of translocation
following individual exposure to Pb or ethanol. The NAC
treatment was found to be significantly effective in reducing
the Pb and ethanol co-exposure-mediated translocation of Bax

Mol Neurobiol
Fig. 3 ROS generation
measurement by fluorescence
microscope using 2,7dichlorodihydrofluorescein
diacetate (DCFH-DA) dye. a
Cells (1106) received exposure
of Pb (10 M), ethanol
(200 mM), Pb (10 M)+ethanol
(200 mM), NAC (10 M), and Pb
(10 M)+ethanol (200 mM)+
NAC (10 M) for 24 h.
Following the exposure, cells
were incubated with DCFH-DA
(20 M) for 30 min at 37 C, and
then, microphotographs were
taken by Nikon phase contrast
cum fluorescence microscope
(model 80i), and for fluorescence,
quantification was done using
Leica Q win500 image analysis
Software. b Relative
quantification of fold induction in
ROS generation by intracellular
image analysis, receiving the
exposure of Pb (10 M), ethanol
(200 mM), Pb (10 M)+ethanol
(200 mM), NAC (10 M), and Pb
(10 M)+ethanol (200 mM)+
NAC (10 M) at 24 h. Cells
exposed to H2O2 (100 M) for
6 h under identical experimental
conditions and served as positive
control. */#p<0.05, **/##p<0.01,
***/###p<0.001,*=p<0.05;
**p<0.01, ***p<0.001
(unexposed control vs.
experimental group); #p<0.05,
##
p<0.01, ###p<0.001 (compared
between treated groups)

and cytochrome-c between mitochondria and cytoplasm. The

high purity of both cytosolic and mitochondrial fractions
could be ascertained as there was no cross-contamination of
the cytosol- and mitochondria-specific proteins. The observations recorded through Western blot analysis and immunocytochemistry were found to have similar trends and linearity
(Fig. 9a, b).
Activity of Caspase-9/3 Highlights of Pb- and/or ethanolinduced alterations in the activity of caspase-9/3 and ameliorative responses of NAC are shown in Fig. 10a, b. The activity
of both caspase-9 and caspase-3 was found to be increased
significantly in all the exposure groups when compared with
the control group. The magnitude of induction was significantly higher (p<0.01) in cells co-exposed to Pb and ethanol.
Similarly, the positive control (H2O2 100 M) also induced

the activity of caspase-9 and caspase-3 significantly (p<0.01)

in comparison to control. NAC was found to be effective in
ameliorating the activity of caspase activity significantly
(p<0.05), as NAC (10 M) significantly brought down the
caspase-9 and caspase-3 activity in cells co-exposed to Pb and
ethanol, which was comparable to cells receiving individual
exposure of either of Pb or ethanol (Fig. 10a, b).

Discussion
The working hypothesis for the present investigation was to
study, whether the co-exposure of non-cytotoxic concentrations of Pb and ethanol leads to oxidative stress which further
precedes apoptotic condition in PC12 cells, a most

Mol Neurobiol

Fig. 4 Analysis of oxidative stress marker glutathione and lipid peroxidation. a Levels of glutathione (GSH) expressed in nanomoles per milligram protein following the exposure of Pb (10 M), ethanol (200 mM),
Pb (10 M)+ethanol (200 mM), Pb (10 M)+ethanol (200 mM)+NAC
(10 M), and NAC (10 M) after 24 h. b Levels of lipid peroxidation
(LPO) expressed in nanomoles per 10 mg protein following the exposure
of Pb (10 M), ethanol (200 mM), Pb (10 M)+ethanol (200 mM), Pb
(10 M)+ethanol (200 mM)+NAC (10 M), and NAC (10 M) after
24 h. */#p<0.05, **/##p<0.01, ***/###p<0.001, *p<0.05; **p<0.01,
***p<0.001 (unexposed control vs. experimental group); #p<0.05,
##
p<0.01, ###p<0.001 (compared between treated groups)

representative cell line used in neurotoxicity studies. The

reason for selecting the combination of Pb and ethanol was
due to available reports on the influence of alcohol consumption on Pb intoxication [39] and more precisely to neurotoxicity and developmental neurotoxicity [4]. The association
between Pb exposure and alcohol consumption in industrial
workers has already been demonstrated [47]. It is also reported
that oxidative stress is one of the molecular mechanisms for
Pb toxicity. Pb increases the levels of ROS along with intracellular calcium [Ca2+]i which in turn causes a fall in the
mitochondrial potential leading to apoptosis via cytochromec release [1416]. Chronic alcohol exposure has been reported
to induce cognitive impairment with permanent structural
brain damage by affecting the neuronal cells of both central
and peripheral nervous systems [2729]. Such alcoholmediated neurodegeneration results in loss of neurons and

oligodendroglia by decreasing the expression of genes associated with myelination, insulin-responsiveness and mitochondrial functions, and increased indices of apoptosis, oxidative stress, mitochondrial dysfunction, lipid peroxidation,
DNA damage, and deregulated acetylcholine homeostasis
[3033].
We observed an increase in the production of [Ca2+]i, ROS,
and associated oxidative stress in cells receiving either Pb or
ethanol individually. However, cells co-exposed to Pb and
ethanol demonstrated more pronounced levels of [Ca2+]i,
ROS production, and oxidative stress. Pb is reported to induce
oxidative stress by inhibiting the enzymatic activity by its
binding to the active thiol site, while ethanol-induced oxidative stress results due to conversion of ethanol into acetaldehyde [48]. Based on the results, it may be concluded that Pb
and ethanol synergistically induce the adverse effects. The
synergistic response for oxidative stress under the influence
of co-exposure to Pb and ethanol in experimental animals has
been reported to be associated with increased production of
ROS [49]. ROS are oxygen-containing molecules, such as
hydrogen peroxide (H2O2), superoxide anion (O2), and hydroxyl radical (HO) that are involved in redox signaling. To
confirm the ROS redox signaling under identical experimental
conditions, PC12 cells were exposed to H2O2 (100 M for
6 h), which served as positive control. H2O2-induced ROS
production was similar as in the case of Pb and/or ethanol,
suggesting involvement of similar signal transduction pathways. The significant reduction in ROS in the presence of a
known antioxidant, NAC, with co-exposure to Pb and ethanol
in PC12 cells, further confirms the involvement of ROS in
toxicity. NAC works as a GSH precursor, and is an important
cellular antioxidant and plays a major role in protecting the cells
against oxidative stress through direct scavenging of ROS [50].
NAC treatment provided a sufficiently strong antioxidant defense system, resulting in restoring LPO and GSH of cells
exposed to a combination of Pb and ethanol. A similar kind
of effect had also been noticed in SHSY5Y cell line, which was
treated with the antioxidant NAC against arsenic and dopamine
[51]. Thus, the present study demonstrated that the cytotoxicity induced by the co-exposure to Pb and ethanol could be
attenuated by antioxidant NAC (data not shown), suggesting
that increased ROS generation and reduction in antioxidant
defense system are responsible for the decreased cell viability.
The abundant amount of oxygen supply to the brain and
high lipid contents in the cells make brain cells more susceptible to oxidative stress-mediated toxicity of Pb and ethanol.
The limited regeneration and repair capacities of neuronal
cells also add to that. A dose-dependent loss in cell viability
was observed after independent exposure to Pb and ethanol.
The magnitude of viability loss was significantly increased
upon co-exposure to both Pb and ethanol. This increase in the
loss of cell viability could be correlated well with the increased production of ROS and subsequently associated

Mol Neurobiol

Fig. 5 Apoptotic analysis by Mitolight Apoptosis Detection Kit

(APT142, Chemicon, USA). a Unstained cells. b Experimental positive
control (H2O2 100 M). c Control. d Pb 10 M. e Ethanol 200 mM. f Pb
10 M+Ethanol 200 mM. g. Pb 10 M+Ethanol 200 mM+NAC 10 M.
h NAC 10 M. Briefly, following the exposure of Pb (10 M), ethanol

(200 mM), Pb (10 M)+ethanol (200 mM), Pb (10 M)+ethanol

(200 mM)+NAC (10 M), and NAC (10 M) for 24 h, cells were resuspended in 1 ml of pre-diluted Mitolight solution for 15 min at 37 C.
Cells were then immediately analyzed by flow cytometer (BD
FACSCanto) equipped with the FACSDiva Version 6.0.0. software

events of oxidative stress and apoptotic end points. The thiol

binding affinity of Pb is one among the important underlying
mechanisms responsible for its adverse effect on different
cellular and sub-cellular molecules. GSH, a low-molecularweight non-enzymatic thiol containing antioxidant compound
which is synthesized in the cells in abundance, is known to be
affected by Pb exposure [52]. GSH plays a key role in
protecting cells from oxidative damage and toxicity of xenobiotics and maintaining redox homeostasis. Though, we observed depletion of GSH in all the experimental groups receiving Pb and/or ethanol, but the magnitude of depletion was
statistically greater in cells co-exposed to both Pb and ethanol.
Ethanol is known to deplete GSH via acetaldehyde-mediated

adduct formation. So, the depletion of GSH levels in PC12

cells observed in the present investigation could be attributed
to the increased utilization of GSH-GPx in detoxification of
H2O2 generated by Pb and ethanol.
Though, the brain is capable of handling of increased levels
of ROS by a number of cellular defense systems to lower
down the concentration of free radical species and repair
oxidative cellular damage. But, increased production of ROS
may cause permeabilization of blood-brain barrier (BBB),
which subsequently inhibits the mitochondrial respiration
and known to induce progressive lipid peroxidation [38, 49].
In the present investigations, a significant increase in LPO and
associated reduction in mitochondrial membrane potential

Mol Neurobiol

Fig. 6 DNA damage analysis in PC12 cells using APO-BrdU TUNEL

(deoxynucleotide transferase dUTP nick end labeling) Assay Kit with
Alexa Fluor anti-BrdU (Molecular Probes, Invitrogen detection Technologies, USA, Cat No.# A23210) by a flow cytometer (BD
FACSCanto, USA) equipped with BD FACSDiva, Version 6.1.2,

software. a Positive control (human lymphoma cells) provided with the

kit. b Experimental positive control (H2O2 100 M). c Control. d Pb
10 M. e. Ethanol 200 mM. f Pb 10 M+Ethanol 200 mM. g Pb 10 M+
Ethanol 200 mM+NAC 10 M. h NAC 10 M

was noticed in the cells exposed to Pb and/or ethanol. Similar

to other end points, co-exposure to Pb and ethanol was showing a synergism in the increased lipid peroxidation and reduction in MMP.
The association of oxidative stress and apoptosis induction
in the brain has been reported as indicator of neurotoxic
response to both Pb and alcohol [53]. The results of TUNEL
assay with labeled cells confirming DNA fragmentation also
support our finding of ROS-induced apoptosis. We and others
have already reported the involvement of mitochondrial chain
complexes in ROS-induced apoptotic changes in cytoplasm
[54]; however, such apoptotic changes are known to follow
different pathways [5559]. We observed that exposure to Pb

and/or ethanol significantly upregulated the expression (both

mRNA and protein) of caspase-3, caspase-9, Bax, p53, and
Cytochrome-c and downregulated Bcl2. In general, transcriptional changes were well coordinated with translational changes and with physiological activity in the case of caspase-3 and
caspase-9. Recent literature has shown that upregulation of
cytosolic p53 protein interacts with mitochondria, thereby
promoting mitochondrial membrane permeabilization [60],
and plays a key role in the regulation of apoptosis [61]. This
cytosolic p53 protein has been suggested to induce proapoptotic members of the Bcl2 family such as Bax and its
displacement with anti-apoptotic Bcl2 protein [55]. However,
induction of nuclear p53 protein is involved in preventing

Mol Neurobiol

Fig. 7 mRNA expression profiling of marker genes associated with

oxidative stress DNA damage and apoptosis in PC12 cells following
the exposure of Pb (10 M) and/or ethanol (200 mM) for 24 h. Quantitative real-time PCR (RT-PCRq) was performed in triplicate by SYBR

Green dye using ABI PRISM 7900HT Sequence Detection System

(Applied Biosystems, USA). -Actin was used as internal control to
normalize the data, and changes in mRNA expression are expressed in
relative quantity compared with respective unexposed control groups

Fig. 8 Protein expression profiling of marker genes associated with

oxidative stress, DNA damage, and apoptosis in PC12 cells following
the exposure Pb (10 M) and/or ethanol (200 mM) for 24 h. -Actin was
used as internal control to normalize the data. Lanes A untreated control,
B Pb 10 M, C Ethanol 200 mM, and D Pb 10 M+ethanol 200 mM,
respectively. Molecular weight of protein studied: p53 (53 kDa), Cytochrome-c (12 kDa), Bax (23 kDa), Bcl2 (29 kDa), activated caspase-9
(37 kDa), activated caspase-3 (17 kDa), CYP-2E1 (50 kDa), HSP70
(70 kDa), PARP-Cleavage (116 and 89 kDa), and -actin (42 kDa) for
normalization. Quantification was done in Gel Documentation System
(Alpha Innotech, USA) with the help of AlphaEase FC StandAlone
V.4.0 software

genotoxicity by inducing transcriptional reprogramming,

which finally leads to controlled cell death [62, 63]. Thus,
the alterations in the expression profile of marker genes in this
study indicate that cytosolic p53 triggers the mitochondrial
apoptotic cascade in PC12 cells exposed to Pb and/or ethanol.
Moreover, activation of PARP also suggests the activation of
DNA repair by nuclear p53 in cells exposed to Pb and/or
ethanol.
In the present investigations, a synergistic increase in the
translocation of Bax protein from cytosol to mitochondria and
cytochrome-c protein from mitochondria to cytosol was observed in cells exposed to Pb and ethanol, when compared
with exposures to Pb or ethanol alone. Altogether, these
findings confirm the induction of cytosolic p53 and its role
in triggering the pathway of mitochondrial membrane permeability in PC12 cells as an important phase in the induction of
mitochondria-mediated apoptosis [64]. In pro-apoptotic
phase, embedment of Bax in the mitochondrial membrane
creates permeable channels which permit larger protein molecules to pass through them. This event leads to mitochondrial
leakage from inner membrane followed by greater ROS
generation, uncoupling of oxidation phosphorylation,
and breakdown of mitochondrial membrane potential
[65]. These observations are in agreement with the
earlier reported studies with Pb and alcohol individually,
but the present investigations provide the first experimental evidence of synergistic response of co-exposure
to Pb and ethanol in PC12 cells.
Release of cytochrome-c [66] from mitochondria in the
cytosol activates caspase-9 [67], which further activates caspase-3. Such activated caspase-3 executes the catabolic caspase cascade that eventually results in cell death. Activation of
caspase cascade may occur through two different pathways,
i.e., either by binding of procaspase-9 with Apaf-1 to form the
apoptosome complex after the release of cytochrome-c from

Mol Neurobiol

Fig. 9 a Translocation of cytoplasmic Bax protein to mitochondria and

mitochondrial cytochrome-c protein to cytoplasm in PC12 cells following
the exposure of Pb (10 M), ethanol (200 mM), Pb (10 M)+ethanol
(200 mM), Pb (10 M)+ethanol (200 mM)+NAC (10 M), and NAC
(10 M) for 24 h. A Untreated control. B Pb 10 M. C Ethanol 200 mM.
D Pb 10 M+ethanol 200 m. E Pb 10 M+ethanol 200 mM+NAC
10 M. F NAC 10 M. -Actin and V-DAC were used to assess the
purity of cytosolic and mitochondrial fraction. b Representative microphotographs showing translocation of cytoplasmic Bax protein to mitochondria and mitochondrial cytochrome-c protein to cytoplasm in PC12

cells following the exposure of Pb (10 M), ethanol (200 mM), Pb

(10 M)+ethanol (200 mM), Pb (10 M)+ethanol (200 mM)+NAC
(10 M), and NAC (10 M) for 24 h. A Untreated control. B Pb 10 M. C
Ethanol 200 mM. D Pb 10 M+ethanol 200 mM. E Pb 10 M+ethanol
200 mM+NAC 10 M. F NAC 10 M. The images were snapped by
Nikon DS-Ri1 (12.7 megapixel) camera using upright phase contrast plus
fluorescence microscope (Nikon 80i, Japan) at 10100 oil immersion
magnification. Mitochondria stained with MitoTracker green (Mt-green)
and nuclei stained with DAPI

damaged mitochondria [68] or by activation of caspases

through the OMI and SMACs molecules [69]. The expression
of these changes also correlates with activity of caspase-9 and
caspase-3 obtained in the present investigations which are

indicative of mitochondria-mediated apoptosis that operates

through caspase cascade.
Ethanol is reported to induce impairment in blood-brain
barrier (BBB) [22]; hence, synergistic response of co-

Mol Neurobiol
Fig. 10 Induction in the activity
of caspase-9/3 in PC12 cells
following the exposure of Pb
(10 M), ethanol (200 mM), Pb
(10 M)+ethanol (200 mM),
NAC (10 M), and Pb (10 M)+
ethanol (200 mM)+NAC
(10 M) for 24 h. a Induction in
the activity of caspase-9. b
Induction in the activity of
caspase-3. Cells exposed to H2O2
(100 M) for 6 h under identical
conditions were used as a positive
control. */#p<0.05, **/##p<0.01,
***/###p<0.001,*p<0.05;
**p<0.01, ***p<0.001
(unexposed control vs.
experimental group); #p<0.05,
##
p<0.01, ###p<0.001 (compared
between treated groups)

exposure to Pb and ethanol may be due to accumulation of Pb

in the brain due to impaired BBB. Ethanol and its metabolites
are also known to stimulate inositol 1,4,5-triphosphate receptor [IP(3)R)-operated intracellular calcium (Ca(2+)] release,
which ultimately leads deficiency of calcium [70]. Ethanol is
also reported to alter the distribution and metabolism of Pb
[39]. Though, the mechanisms may be different but both Pb
and ethanol induce oxidative stress and apoptosis in PC12
cells.
The activation of cytochrome P450s and their interaction
with mitochondrial chain complexes have been suggested in
chemical-induced apoptosis [55, 57]. We observed a significant upregulation in the expression of CYP-2E1 after exposure to Pb and/or ethanol, which was found to be up-streamed
to ROS generation in PC12 cells. Such upregulated CYP-2E1
might have played an important role in the production of
reactive oxygenated molecules (ROMs), which are known to
induce ROS generation [71], LPO [72], and GSTs [73] and
finally leads to apoptosis [74]. In the present investigations,

apoptosis induction and oxidative stress were found to be

associated with induction of CYP-2E1. Induced CYP-2E1
was found to cause oxidative stress by reduction of the intracellular GSH levels [75], activation of the p38 MAP kinase
pathway, and induction of the transcription factor Nrf2 [76], in
human hepatoma cell line HepG2. The role of CYP-2E1 has
been suggested in alcohol-induced oxidative DNA damage in
liver of null mice [77]. Pb- and ethanol-induced alterations in
the expression profile of CYP-2E1 could be correlated with an
earlier study showing CYP-2E1 induction by 4hydroxynonenal (4-HNE), a product of lipid peroxidation
[78], and organophosphate pesticide-monocrotophos in
PC12 cells [79]. GSTs, a family of phase II enzymes, participate in detoxification of xenobiotics from cells. 4-HNE, a byproduct of lipid peroxidation, conjugates with GSH, specifically by glutathione S-transferase A4-4 (GSTA4-4), which has
been shown as a major pathway for 4-HNE neutralization
[78]. There are reports demonstrating the protective role of
GSTA4-4 in various cell systems having toxic insults due to a

Mol Neurobiol
Fig. 11 Schematic flow diagram
to represent apoptotic pathway
involved in PC12 cells exposed to
Pb and/or ethanol

variety of environmental chemicals and metabolites [80, 81].

We also observed a similar pattern of elevated levels of
GSTA4-4 in PC12 cells exposed to Pb and/or ethanol. Moreover, upregulation of GSTA4-4 expression may be the defense
mechanism to protect against damage caused by 4-HNE and
other by-products formed via increased lipid peroxidation
induced by Pb and/or ethanol treatment. Heat shock proteins
(HSPs) contain several different families of proteins, and their
expressions are induced in response to wide variety of physiological and environmental insults. One such protein is
HSP70, which is induced under high stress condition.
HSP70 protects cells from stress-induced caspase-dependent
apoptosis and facilitates the survival of cells against toxic
stimuli [82]. Our study shows that Pb or ethanol significantly
increased HSP70 expression both at mRNA and protein levels;
however, combined treatment with both chemicals resulted in
more pronounced HSP70 induction, suggesting greater magnitude of stress in the cells.
Based on the findings, we propose a schematic model on
the synergism in apoptotic effects of co-exposure to Pb and
ethanol to cells (Fig. 11). The co-exposure to Pb and ethanol
first triggers intrinsic generation of [Ca2+]i and ROS, which
subsequently initiates oxidative damage. The increased ROS
levels and genotoxicity upregulate p53 and attenuate Bcl2
protein, leading to a declined ratio of Bcl-2/Bax. The alteration in Bcl2/Bax ratio results in the release of cytochrome-c.
Finally, the release of cytochrome-c activates caspase-9 and
subsequently triggers the caspase-3 cascade leading to apoptosis in PC12 cells. The present investigations conclude
that PC12 cells exposed to both Pb and ethanol show

more pronounced increase in oxidative stress and apoptosis than to the independent exposures to Pb and
ethanol.
Acknowledgments The authors are grateful to the Director, IITR, Lucknow, India, for his keen interest in the study. Financial support from Department of Biotechnology, Ministry of Science & Technology, Government of
India, New Delhi, India [Grant No. 102/IFD/SAN/PR1524/20102011];
Department of Science and Technology, Ministry of Science & Technology,
Government of India, New Delhi, India [Grant No. SR/SO/Z 36/2007/91/10];
and Council of Scientific & Industrial Research, Government of India, New
Delhi, India [Grant No. BSC0111/INDEPTH/ CSIR Network Project] is
acknowledged. Indian Council of Medical Research, (ICMR), New Delhi,
India, is acknowledged for providing the fellowship to Dr. Vivek Kumar. The
funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the article.
Conflict of Interest The authors declare no conflict of interest.

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