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Int. J. Devl Neuroscience 34 (2014) 60–75

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International Journal of Developmental Neuroscience

journal homepage: www.elsevier.com/locate/ijdevneu

Reversibility of changes in brain cholinergic receptors and

acetylcholinesterase activity in rats following early life arsenic
exposure
Lalit P. Chandravanshi a , Rajesh S. Yadav a,c , Rajendra K. Shukla a , Anshuman Singh a ,
Sarwat Sultana b , Aditya B. Pant a , Devendra Parmar a , Vinay K. Khanna a,∗
a
CSIR-Indian Institute of Toxicology Research, Post Box 80, MG Marg, Lucknow, 226 001, India
b
Neurotoxicology Laboratory, Department of Medical Elementology and Toxicology, Jamia Hamdard, New Delhi, 110 062, India
c
Department of Criminology and Forensic Science, Harisingh Gour University, Sagar,- 470003, India

a r t i c l e i n f o a b s t r a c t

Article history: In view of the increasing incidences of arsenic induced health effects and the vulnerability of the devel-
Received 10 October 2013 oping brain to its toxic effects, studies have been carried out to investigate the mechanism of arsenic
Received in revised form 25 January 2014 induced cholinergic alterations and understand if such changes are persistent or transient on with-
Accepted 31 January 2014
drawal of arsenic exposure. Male rats were exposed to arsenic (2 mg/kg or 4 mg/kg body weight, p.o)
from post-lactational day (PD)22 to PD59, and the effect on selected behavioral and neurochemical end
Keywords:
points associated with cholinergic functions was assessed on PD60 and PD90. Decrease in the binding
Arsenic
of muscarinic-cholinergic receptors in frontal cortex (26%, 43%) and hippocampus (21%, 34%) associated
Learning and memory
Cholinergic receptors
with reduced CHRM2 mRNA levels, acetylcholinesterase activity and expression of ChAT and PKC ␤-1
Oxidative stress was observed in arsenic exposed rats on PD60 as compared to controls. Spatial learning and memory and
Apoptosis muscle strength were affected following arsenic exposure in rats on PD60 and associated with arsenic
induced cholinergic alterations. Enhanced oxidative stress associated with increased expression of pro-
apoptotic proteins and decreased expression of anti-apoptotic proteins was distinct in both frontal cortex
and hippocampus following arsenic exposure in rats on PD60. The cholinergic alterations and other neu-
rochemical modifications were found to be linked with increased arsenic levels in frontal cortex (1.39,
3.90-fold) and hippocampus (3.23, 5.48-fold) on PD60. Although a trend of recovery was observed both in
behavioral and neurochemical endpoints on withdrawal of arsenic exposure on PD90, the results indicate
that continuous arsenic exposure may have detrimental effects.
© 2014 ISDN. Published by Elsevier Ltd. All rights reserved.

1. Introduction
Arsenic (As), a metalloid is present in the environment in three
oxidation states (elemental-0, trivalent-3+ , pentavalent As-5+ ) both
in inorganic and organic forms (Miteva et al., 2005; Kapaj et al.,
2006; Vahidnia et al., 2007). Due to natural occurrence of arsenic in
the earth’s crust and moreover, its ubiquitous presence in rocks and
soil, high levels of arsenic are present in drinking water as a con-
taminant in Indo-Gangetic plains, regions of South East Asia and
many parts of South America (Argos et al., 2010; Lindberg et al.,
2010; Steinmaus et al., 2010; Smith et al., 2011). As per an estimate,
over 140 million people are exposed to arsenic by drinking water
∗ Corresponding author at: Developmental Toxicology Division, CSIR-Indian Insti-
tute of Toxicology Research, M.G. Marg, P.O. Box No. 80, Lucknow – 226 001 U.P.,
India. Tel.: +91 522 2627586; fax: +91 522 2628227.
E-mail address: vkkhanna1@gmail.com (V.K. Khanna).
throughout the globe (Kris, 2009) which has resulted in increased
health problems (Chen et al., 2011; Hughes et al., 2011). Identi-
fied as a human carcinogen, arsenic is figured number one in the
category of hazardous chemicals by the US Agency for Toxic Sub-
stances and Disease Registry (ATSDR). Risk of developing cancers
of kidney, bladder, skin, lungs, breast and liver has been found to
increase following arsenic exposure (Heck et al., 2009; Smith et al.,
2009, 2012; Yuan et al., 2010; Yorifuji et al., 2011; Du et al., 2012).
Hyper-pigmentation and skin lesions are frequently reported in
individuals exposed to arsenic at high levels (Ling et al., 2013). Con-
sumption of arsenic contaminated water has been found to affect
early human development, and is therefore, a cause of concern
among the health scientists and exposed population (Hamadani
et al., 2010, 2011; Parvez et al., 2011; Roy et al., 2011; Parajuli et al.,
2013). Arsenic in drinking water has been found to be an etiological
factor for type 2 diabetes in the population of Taiwan, Bangladesh
and Serbia (Navas-Acien et al., 2006; Jovanović et al., 2012). In view
of deleterious health effects associated with arsenic exposure, the

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L.P. Chandravanshi et al. / Int. J. Devl Neuroscience 34 (2014) 60–75
maximum limit of arsenic in drinking water has been reduced to
10 ppb by the World Health Organization and US Environmental
Protection Agency (WHO 2001; US EPA, 2001).
While chronic arsenic poisoning affects the functioning of
respiratory, cardiovascular, gastro-intestinal, genito-urinary and
endocrine systems in humans, the nervous system is more vulner-
able to its toxic effects (Borenstein et al., 2006; Dauphine et al.,
2011; Abhyankar et al., 2012). It has been found that arsenic may
affect both the central and peripheral nervous systems causing sub-
clinical to clinical effects (Navas-Acien et al., 2006; Kim et al., 2013).
Epidemiological studies have revealed an association between
arsenic in drinking water and the risk of cognitive impairment
including disturbed visual perception and visuomotor integration,
psychomotor speed, attention, speech and memory (Calderon et al.,
2001; Rodriguez et al., 2003; Tsai et al., 2003). Decrease in the IQ
of children exposed to ground water arsenic has frequently been
reported (Wang et al., 2007). Cognitive deficits and slower nerve
conduction associated with cumulative developmental exposure
to arsenic have been reported in Taiwanese adolescents (Tsai et al.,
2003; Tseng, 2009). Urinary arsenic levels and its metabolites have
been found to be good indicators to assess the risk of exposure
of the metal in children and exposed workers (George et al., 2013).
An inverse association between urinary arsenic levels and cognitive
functions in children has been shown by many investigators, sug-
gesting that arsenic exposure during development can affect the
functioning of the central nervous system (Calderon et al., 1999,
2001; Tsai et al., 2003; Hamadani et al., 2011). A number of exper-
imental studies on rats and mice have been carried out to assess
the impact of arsenic on neurobehavioral performance (Luo et al.,
2009; Bardullas et al., 2009; Rodriguez et al., 2010; Yadav et al.,
2011). It was found that arsenic exposure leads to neurochemi-
cal alterations associated with impaired motor activity, learning
and memory functions (Itoh et al., 1990; Nagaraja and Desiraju,
1994; Rodriguez et al., 2001, 2003). Enhanced oxidative stress asso-
ciated with increased apoptosis was found to be linked to arsenic
induced neurotoxicity (Estus et al., 1994; Namgung and Xia, 2001;
Ahmed et al., 2011, 2012). Arsenic has been found to cross the
placental barrier and cause developmental abnormalities includ-
ing malformations, decreased growth rate, mortality and neural
tube defects (Golub, 1994; Tabocova et al., 1996; Stump et al.,
1999). In view of increasing risk of exposure to arsenic and asso-
ciated vulnerabilities of the developing brain, studies have been
carried out to assess the impact of arsenic exposure during pre-
natal and early post-natal periods (Anderson et al., 2000; Xi et al.,
2010a; Lu et al., 2012; Herrera et al., 2013). In a study to assess
the developmental toxicity of arsenic acid, Nemec et al. (1998)
found that pentavalent arsenic was equally toxic to mothers and
developing offspring both in mice and rabbits. Further, it is well
reported that the risk of spontaneous abortions, still births and
increased risk of infant mortality is enhanced in pregnant women
consuming drinking water contaminated with high arsenic lev-
els (Vahter et al., 2006; Von Ehrenstein et al., 2006; Huyck et al.,
2007; Rahman et al., 2007, 2010). In a hospital based study on
Nepalese, Parajuli et al. (2013) found an inverse relation with cord
blood arsenic levels and neurodevelopment indicators in neonates,
suggesting that in utero exposure to arsenic could be detrimen-
tal. Tranplacental and early life exposure to arsenic was found to
impair learning and memory and sensory reflexes (Xi et al., 2009).
While studies to assess the impact of arsenic on neurobehavioral
performance following prenatal and early postnatal life have been
carried out, direct exposure to arsenic could also occur in devel-
oping children during post-lactational period. Exposure to arsenic
during early postnatal life may also affect the neurobehavioral per-
formance (Martínez et al., 2011; Luo et al., 2013). Prenatal and
lactational periods in rats are critical and crucial for brain develop-
ment as structural and functional maturation takes place primarily
61
during this time. The period PD22–PD60 is pre-adolescent stage
in rats during which synapse and receptor overproduction occurs
and maturation of cholinergic neurotransmitter system takes place
(Krinke, 2000). However, impact of arsenic exposure during this
period of brain development on the neurobehavioral outcome is
not understood. Also, it is not known whether changes following
arsenic exposure during this period of brain development are tran-
sient or persistent. The study has, therefore, been carried out to
assess the impact of arsenic exposure from post-lactational day
(PD)22 to PD59 on selected behavioral and neurochemical end-
points and investigate the changes immediately after exposure
on PD60 and 30 days after withdrawal of arsenic exposure on
PD90.
2. Materials and methods
2.1. Animals and treatment
Male rats (PD21) of Wistar strain obtained from the animal breeding colony
of CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Lucknow, India were
housed in polypropylene cages. The animals were housed in a temperature con-
trolled room (22 ± 2 ◦ C) under standard animal house conditions in a 12-h light–dark
cycle and fed pellet diet and water ad libitum. Rats were divided in to three groups.
On PD22, rats in groups I and II were administered sodium arsenite (either at 2 mg/kg
or 4 mg/kg body weight, p.o.) till PD59. Rats in the group III served as controls and
were given normal saline identically as in rats of groups I and II. Effect on behav-
ioral parameters – grip strength, learning and continuous alternation was studied
24 h after the last dose of arsenic treatment on PD60. A separate set of rats in each
group was sacrificed on PD90 and brain was taken out quickly and washed in ice
cold saline. Brain was dissected into specific regions – frontal cortex and hippocam-
pus following the standard procedure as described by Glowinski and Iversen (1966)
and kept frozen at −80 ◦ C for neurochemical assays and estimation of arsenic lev-
els. To understand whether neurobehavioral changes following arsenic exposure
are transient or persistent, a set of rats was left as such till PD89 and monitored
daily. Effect on selected behavioral and neurochemical endpoints was studied on
PD90.
3. Behavioral studies
3.1. Grip strength
Forelimb grip strength was measured in the control and arsenic
exposed rats using a computerized grip strength meter (TSE,
Germany) following the standard procedure as described by Terry
et al. (2003). Rats were gently held by the nape of the neck and base
of the tail. The forelimbs of the rats were placed on the tension bar.
Followed by this, the rat was pulled back gently until it released the
bar and the reading was recorded automatically on the computer.
Five successive pulls for each animal in the study group were tried
by a person unaware of their treatment status. The mean of all the
values was taken and processed for statistical analysis.
3.2. Learning ability – active avoidance test
The learning ability of rats was measured by assessing con-
ditioned avoidance behavior using a shuttle box (Techno, India)
following the standard protocol as described by Yadav et al. (2009).
Briefly, rats from the control and arsenic treated groups were placed
individually in one chamber of the shuttle box and habituated for
5 min. A conditioned stimulus in the form of a buzzer for 10 s fol-
lowed by a buzzer and foot shock of 0.5 mA for 10 s was given to
each rat. The avoidance was considered positive if the rats jumped
to a shock-free chamber to avoid the foot shock. All rats in the treat-
ment and control groups were subjected to 20 trials per day for 3
days. The inter-trial interval was 1 min. Of the 20 trials per day,
the total number of avoidances and the number of trials for the
first avoidance were recorded for each rat. The data of the final day
are presented in the results. The percentage of conditioned avoid-
ance response has been recorded as a measure of learning ability
in treated groups and compared with controls.
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62 L.P. Chandravanshi et al. / Int. J. Devl Neuroscience 34 (2014) 60–75
3.3. Spatial memory – continuous alternation test
Continuous alternation in a single session was measured using a
computerized Y-maze (TSE, Germany), to assess short term spatial
memory (Maurice et al., 1994; Roghani et al., 2006). The equip-
ment consisting of three arms – 40 cm long, 15 cm wide and 30 cm
high converged in an equilateral triangular central area with 15 cm
at its longest axis. Rat in each case was placed at the end of one
arm and allowed to move freely through the maze for 5 min. The
sequence of entry in to each arm was recorded automatically on
the computer. Measure of spatial memory has been defined as the
number and the sequence of entries of rat in to the arm recorded
during 5 min. All the arms look alike and no reinforcer, motivation
or punishment was used in this set of experiment. The percentage
of alternation has been calculated as the number of alterna-
tions (entries into three different arms consecutively) divided by
the total possible alternations (i.e. the number of arms entered
minus 2).
4. Neurochemical studies
4.1. Assay of brain muscarinic-cholinergic receptors
Assay of muscarinic-cholinergic receptors in frontal cortex and
hippocampus was carried out by radioligand receptor binding fol-
lowing the procedure as described in detail by Yadav et al. (2011).
Briefly, crude synaptic membrane was prepared by homogenizing
the frontal cortex and hippocampus individually in 19 volumes of
Tris–HCl buffer (5 mM, pH 7.4). The homogenate was centrifuged at
40,000 × g for 15 min at 4 ◦ C and the sedimented pellet was washed
twice by resuspending in homogenization buffer. The suspended
pellet in buffer was recentrifuged at the same speed for 15 min at
4 ◦ C and finally suspended in Tris–HCl buffer (40 mM, pH 7.4) and
stored at −20 ◦ C till binding assays.
Binding incubations in triplicate were carried out in a final vol-
ume of 1.0 ml. For the assay of muscarinic-cholinergic receptors,
3 H-QNB (42 Ci/mmol, Perkin Elmer, USA) was used as a radioli-
gand and atropine sulfate (1 × 10−6 M, Sigma, USA) was used as
a competitor to assess the extent of non-specific binding. The reac-
tion mixture containing Tris–HCl buffer (40 mM, pH 7.4) together
with membrane protein (300–400 ␮g) and 3 H-QNB (1 × 10−9 M)
was incubated for 15 min at 37 ◦ C in the presence or absence of
competitor to assess the non-specific and total binding respec-
tively. At the end of incubation, contents of the binding tubes
were immediately filtered on glass fiber discs (25 mm diameter,
0.3 ␮m pore size, Whatman GF/B) and washed twice rapidly with
5 ml chilled Tris–HCl buffer (40 mM, pH 7.4). Filters were dried and
transferred into vials and scintillation cocktail containing the mix-
ture (2,5-diphenyl oxazole; 1,4-bis-5, phenylozazolyl-benzene;
naphthalene; toluene; methanol and 1,4 dioxane) added. The
radioactivity was counted using ␤-scintillation counter (Packard,
USA) at an efficiency of 30–40% for 3 H to determine membrane
bound radioactivity. Specific binding has been determined by
subtracting the non-specific binding from the total binding and
expressed as pmoles ligand bound/g protein. Scatchard analysis has
been carried out at varying concentrations of 3 H-QNB (ranging from
0.25 nM to 4 nM) to ascertain whether change in the binding is due
to alteration in the affinity (Kd) or number of receptor binding sites
(Bmax).
4.2. mRNA expression of brain muscarinic-cholinergic receptor
(CHRM2) gene
CHRM2 receptor gene expression in frontal cortex and hip-
pocampus of arsenic exposed rats has been determined by
RT-PCR (ABI, USA) using the glyceraldehydes 3-phosphated
dehydrogenase (GAPDH) for normalization. The cDNA was
amplified by RT-PCR using ABI SYBER green qPCR Kit (ABI,
USA) on the 7900HT Fast Real-Time PCR System running at
40 cycles following the protocol: one cycle of denaturation
(95 ◦ C, 10 s), followed by 40 cycles of denaturation (95 ◦ C,
10 s) annealing (60 ◦ C, 10 s) and extension (72 ◦ C, 45 s). A final
extension (72 ◦ C, 5 min) was performed. The primer sequences
used for CHRM2 and GAPDH described earlier (Borges et al.,
2001) are mentioned below, CHRM2, FP5 -GGCAAGCAAGAGTAG
AATAAA-3 and CHRM2, RP 5 -GCCAACAGGATAGCCAAGATT-3
GAPDH, FP 5 -CGGGAAGCTTGTGATCAATGG-3 and GAPDH, RP 5 -
GGCAGTGATGCCATGGACT G-3 .
4.3. Assay of brain acetylcholinesterase activity
Activity of acetylcholinesterase was assayed following the pro-
cedure as described by Ellman et al. (1961) using acetylthiocholine
iodide as a substrate and 5,5 -dithiobis-2 nitrobenzoic acid (DTNB)
as the coloring agent. Briefly, the reaction mixture in a final
volume of 1.0 ml contained phosphate buffer (0.1 M, pH 7.4),
post-mitochondrial fraction of frontal cortex or hippocampus (con-
taining around 15 ∼ 20 ␮g protein), acetylthiocholine iodide (ACTI),
and DTNB (5 mM). The degradation of acetylthiocholine iodide was
measured at 412 nm and the results are expressed as ␮mol ACTI
hydrolysed/min/mg protein.
4.4. Assessment of brain oxidative stress:
4.4.1. Assay of lipid peroxidation, protein carbonyl and reduced
glutathione levels
As a measure of lipid peroxidation, malonaldehyde (MDA) for-
mation was estimated in brain regions following the method of
Ohkawa et al. (1979) and the intensity of pink color formed dur-
ing the reaction was read at 532 nm.The values are expressed
as ␮mol/hour/mg protein Protein carbonyl content in brain
regions was measured following the procedure of Levine et al.
(1990) using 2,4-dinitrophenylhydrazine (DNPH) as a substrate.
Amount of protein carbonyl was calculated using a molar extinc-
tion coefficient (D) 22 mm−1 cm−1 for aliphatic hydrazine and
values are expressed as n moles/mg protein. Reduced glu-
tathione (GSH) levels both in frontal cortex and hippocampus
were measured spectrophotometrically using 5,5 -dithiobis (2-
nitrobenzoic acid) (DTNB) as the color reagent following the
method of Hasan and Haider (1989). A range (2–10 ␮g) of
glutathione as standard was also run in parallel to plot the stan-
dard curve and the values are expressed as ␮g GSH/g tissue
weight.
4.4.2. Assay of superoxide dismutase, catalase and glutathione
peroxidase activity
Activity of superoxide dismutase (SOD) in frontal cortex and hip-
pocampus was assayed following the method of Kakkar et al. (1984)
using NADH as a substrate in the post-mitochondrial fraction. The
SOD activity has been expressed as units/min/mg protein. Activity
of catalase in both the brain regions was assayed spectrophotomet-
rically following the method of Aebi (1984) in post-mitochondrial
fraction using hydrogen peroxide (H2 O2 ) as substrate. The activity
of the enzyme was calculated using the molar extinction coefficient
(D) 43.6 M cm−1 and has been expressed in ␮mole/min/mg protein.
Activity of glutathione peroxidase (GPx) both in frontal cortex and
hippocampus was measured following the procedure of Flohe and
Gunzler (1984). The activity of the enzyme has been expressed as
nmol GSH oxidized/min/mg protein.
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5. Immunoexpression of proteins by Western blotting
5.1. Expression of ChAT, PKC ˇ-1 and selected proteins associated
with apoptosis
Expression of choline acetyltransferase, PKC ␤-1 and proteins
linked with apoptosis and stress (Caspase-3, Cleaved Caspase-
3, Bax, Bcl2, phospho-p38, phospho-JNK, Ap-1) in frontal cortex
and hippocampus was assayed following the method of Jamal
et al. (2007). Briefly, brain regions were homogenized in RIPA
buffer containing Tris–HCl (50 mM, pH 6.8), NaCl (150 mM), sodium
deoxycholate (0.5%), SDS (0.1%), protease inhibitor and Triton-
100X (1%) and centrifuged at 12,000 × g (15 min, at 4 ◦ C) to remove
the insoluble material. The pellet was discarded and the super-
natant further mixed with loading buffer containing Tris–HCl
(60 mM, pH 6.8), SDS (2%), glycerol (10%), ␤-mercaptoethanol
(5%), bromophenol blue (0.01%) and boiled for 5 – 7 min. The
prepared samples (30 ␮g protein/lane) were electrophoresed on
12% SDS–PAGE, electroblotted on to nitrocellulose membranes
(Millipore, USA) and blocked with blocking buffer (Western
blocker solution TM Sigma, USA). After subsequent washing, the
blots were incubated with primary antibody (Anti-TH, Sigma,
USA, 1:2000 and Anti-ChAT, Sigma, USA, 1:2000) for 24 h at
4 ◦ C followed by incubation with horseradish peroxidase linked
secondary antibody (anti-mouse IgG, 1:4000) at room temper-
ature for 60 min. After the incubation, blots were washed and
developed using an immobilon western chemiluminescent HRP
substrate (Millipore, USA) following the recommended procedure.
␤-actin was probed as an internal control and used to confirm
that an equal amount of protein was loaded in each lane. A
digital gel image analysis system (VersaDoc, Model 1000, Bio
Rad,Quantity 1) was used for semi-quantification of immunoreac-
tivity.
5.2. Estimation of arsenic levels in brain regions
Arsenic levels in both brain regions were estimated using the
hydride system 60 atomic absorption spectrophotometer (HSAAS,
ZEEnit 700) following the method of Ballentine and Burford (1957).
Briefly, concentrated nitric acid (1 ml) and perchloric acid (1 ml)
were added in the flask containing tissue (100 mg approximately).
The sample was then digested over a sand bath until the solu-
tion becomes yellow in color. If the color of the digest was
brown, more nitric acid and perchloric acid were added and the
process of oxidation was repeated. The digest was made up to
known volume with deionized water and aliquots were used
to estimate arsenic using atomic absorption spectrophotometer.
A calibration curve was constructed by adding known amounts
of arsenic standard (Sigma) to calculate arsenic levels in brain
regions.
5.3. Protein estimation
Protein content in samples was measured following the method
of Lowry et al. (1951) using bovine serum albumin as a reference
standard.
5.4. Statistical analysis
Graph Pad prism software has been used to analyze data
of neurobehavioral, neurochemical and immunoblotting end-
points. The data have been analyzed by one-way analysis
of variance (ANOVA) followed by Newman–Keuls test for
posthoc comparisons. Values up to p < 0.05 have been considered
significant.
63
Table 1
Early life exposure (PD22–PD59) to sodium arsenite and effect on body and brain
weights of rats on PD60 and PD90.
Age in postnatal day
Treatment groups PD60 PD90
Body weight (gm)
Control 120.7 ± 6.50 138.3 ± 4.13
ARS I (2 mg/kg) 112.7 ± 4.50 131.0 ± 3.97
ARS II (4 mg/kg) 101.3 ± 5.9* 126.0 ± 3.62
Brain weight (gm)
Control 1.51 ± 0.01 1.55 ± 0.02
ARS I (2 mg/kg) 1.45 ± 0.02 1.51 ± 0.02
1.47 ± 0.01 1.49 ± 0.02
ARS II (4 mg/kg)
Rats were exposed to sodium arsenite (ARS I – 2.0 or ARS II – 4.0 mg/kg body
weight/day, p.o.) from PD22 to PD59. Effect on body and brain weight parameters
studied on PD60 and 30 days after withdrawal of sodium arsenite exposure on PD90.
Values are mean ± SEM of 15 animals in each group for body weight and five ani-
mals in each group for brain weight. Data have been analyzed by one-way analysis
of variance followed by Newman–Keuls test.
*
Significantly differs from controls (p < 0.05).
6. Results
To assess if changes in the behavioral and neurochemical param-
eters are transient or persistent, the effect of arsenic on body and
brain weights and selected behavioral and neurochemical end-
points was studied in rats following early life exposure from PD22
to PD59 on PD60. The effect was further monitored, 30 days after
withdrawal of arsenic exposure on PD90.
6.1. Effect of arsenic on body and brain weight of rats
Early life exposure to arsenic (2.0 mg/kg or 4.0 mg/kg body
weight) caused a decrease in the body weight (F(2,42) = 3.101, 6%,
p > 0.05; 15%, p < 0.05) of rats with significant changes in those
exposed at high dose of arsenic on PD60 as compared to con-
trols exhibiting impairment in the growth of developing rats
(Table 1). However, a trend of recovery was observed in the body
weight (F(2,42) = 2.511, 5%, p > 0.05; 8%, p > 0.05) of rats exposed to
arsenic at both the doses, 30 days after withdrawal of exposure on
PD90.
No significant change in the brain weight was observed in rats
exposed to arsenic (2.0 mg/kg or 4.0 mg/kg body weight) during
early life on PD60 and 30 days after withdrawal of exposure on
PD90 as compared to respective controls (Table 1).
6.2. Behavioral studies
6.2.1. Effect on grip strength
To assess the effect on muscle weakness if any, fore limb grip
strength was monitored following early life exposure of rats from
PD22 to PD59 to arsenic (2.0 mg/kg or 4.0 mg/kg body weight). A
significant decrease in the forelimb grip strength (F(2,12) = 13.81,
18%, p < 0.01; 30%, p < 0.001) was observed in rats exposed to arsenic
at both the doses (2.0 mg/kg or 4.0 mg/kg body weight) respectively
on PD60 in comparison to controls (Fig. 1). Interestingly, a trend
of recovery in the grip strength was observed (F(2,12) = 1.951, 3%,
p > 0.05; 8%, p > 0.05) in arsenic exposed rats 30 days after with-
drawal of exposure on PD90 in comparison to respective controls
(Fig. 1).
6.2.2. Effect on learning ability – active avoidance test
Effect of arsenic exposure on learning and memory was assessed
by monitoring the active avoidance response using shuttle box and
the results are presented in Fig. 2A. Impairment in the learning and
memory (F(2,12) = 31.94, 22%, p < 0.01; 50%, p < 0.001) was observed
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64 L.P. Chandravanshi et al. / Int. J. Devl Neuroscience 34 (2014) 60–75
Fig. 1. Early life exposure (PD22–PD59) to sodium arsenite and effect on grip
strength of rats. Rats were exposed to sodium arsenite (ARS I – 2.0 or ARS II –
4.0 mg/kg body weight/day, p.o.) from PD22 to PD59. Effect on grip strength assessed
immediately after exposure to sodium arsenite on PD60 and 30 days after with-
drawal of sodium arsenite exposure on PD90. Values are mean ± SEM of five animals
in each group. Data have been analyzed by one-way analysis of variance followed
by Newman–Keuls test. *Significantly differs from controls (p < 0.01); **significantly
differs from controls (p < 0.001).
in rats exposed to arsenic (2.0 mg/kg or 4.0 mg/kg body weight) dur-
ing early life as compared to controls (Fig. 2A). The decrease in the
learning and memory was more marked in rats treated with arsenic
at the higher dose. It was interesting to observe a trend of recovery
in learning and memory (F(2,13) = 3.25, 6%, p > 0.05; 10%, p > 0.05)
in arsenic exposed rats 30 days after withdrawal of exposure on
PD90 as compared to respective controls (Fig. 2A).
Fig. 2. Early life exposure (PD22–PD59) to sodium arsenite and effect on learning
and memory assessed by active avoidance response (A) and continuous alternation
test (B) of rats. Rats were exposed to sodium arsenite (ARS I – 2.0 or ARS II – 4.0 mg/kg
body weight/day, p.o.) from PD22 to PD59. Effect on active avoidance response (A)
and continious alternation test (B) assessed immediately after exposure to sodium
arsenite on PD60 and 30 days after withdrawal of sodium arsenite exposure on
PD90. Values are mean ± SEM of four to five animals in each group in the data pre-
sented for active avoidance response (A) and five to six animals in each group in
the data presented for continuous alternation test (B). Data have been analyzed by
one-way analysis of variance followed by Newman–Keuls test. *Significantly differs
from controls (p < 0.01); **significantly differs from controls (p < 0.001).
6.2.3. Effect on spatial memory – continuous alternation test
Effect on the spatial memory was assessed by monitoring the
continuous alternation test using Y-Maze in rats exposed to arsenic
during early life and the results are presented in Fig. 2B. A signifi-
cant decrease in the spatial memory (F(2,14) = 19.23, 35%, p < 0.01;
59.6%, p < 0.001) was observed in rats exposed to arsenic dur-
ing early life from PD22 to PD59 at both the doses (2.0 mg/kg or
4.0 mg/kg body weight) on PD60 as compared to rats in the control
group (Fig. 2B). Although a trend of recovery in the spatial mem-
ory was observed (F(2,14) = 2.364, 15%, p > 0.05; 23%, p < 0.05) in
arsenic exposed rats 30 days after withdrawal of exposure on PD90,
the decrease was found to persist in rats exposed to arsenic at the
higher dose (Fig. 2B).
7. Neurochemical studies
Effect on muscarinic-cholinergic receptors, expression of
CHRM2 receptor gene and acetylcholinesterase activity associated
with the integrity of brain cholinergic system was assessed follow-
ing early life exposure from PD22–PD59 to arsenic (2.0 mg/kg or
4.0 mg/kg body weight) on PD60 and 30 days after withdrawal of
arsenic exposure on PD90 and the results are described below:
7.1. Effect on brain muscarinic-cholinergic receptors
Fig. 3A illustrates effect of early life arsenic exposure of rats
on brain muscarinic-cholinergic receptors. A significant decrease
in the binding of 3 H-QNB to frontocortical (F(2,10) = 15.74, 26%,
p < 0.01; 43%, p < 0.01), and hippocampal (F(2,9) = 7.792, 21%,
p < 0.05; 34%, p < 0.01) membranes, known to label muscarinic-
cholinergic receptors was observed in arsenic treated rats on PD60
as compared to controls (Fig. 3A). Scatchard analysis revealed that
decrease in the binding of 3 H-QNB to frontocortical and hippocam-
pal membranes was due to decreased number of binding sites
(Table 2). A trend of recovery was observed in the binding of
3 H-QNB to frontocortical and hippocampal membranes in arsenic
treated rats on PD90 as compared to respective controls (Fig. 3A).
7.2. Effect on mRNA expression CHRM2 receptor gene
Early life exposure of rats to arsenic (2.0 mg/kg or 4.0 mg/kg
body weight) caused a significant decrease in the expression
of CHRM2 receptor genes in frontal cortex (F(2,6) = 39.46, 29%,
p < 0.01; 48%, p < 0.001) and hippocampus (F(2,6) = 32.00, 23%,
p < 0.05; 43%, p < 0.001) on PD60 as compared to controls. These
changes exhibited a trend of recovery 30 days after withdrawal of
arsenic exposure as compared to respective controls (Fig. 3B).
7.3. Effect on brain acetylcholinesterase activity
Early life exposure of rats from PD22 to PD59 to arsenic
(2.0 mg/kg or 4.0 mg/kg body weight) caused a significant decrease
in the activity of acetylcholinesterase (AChE) in frontal cor-
tex (F(2,9) = 7.519, 24%, p < 0.05; 39%, p < 0.05) and hippocampus
(F(2,12) = 22.35, 26%, p < 0.001; 42%, p < 0.001) on PD60 as com-
pared to controls (Fig. 4). A trend of recovery in the activity of AChE
both in frontal cortex (F(2,10) = 1.040, 6%, p > 0.05; 14%, p > 0.05)
and hippocampus (F(2,10) = 1.457, 10%, p > 0.05; 14%, p > 0.05) was
observed 30 days after withdrawal of exposure on PD90 in com-
parison to respective controls (Fig. 4).
7.4. Assessment of oxidative stress
To assess the extent of oxidative stress following early life expo-
sure to arsenic (2.0 mg/kg or 4.0 mg/kg body weight) in rats, effect
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Fig. 3. Early life exposure (PD22–PD59) to sodium arsenite and effect on muscarinic-cholinergic receptors (A) and CHRM2 mRNA levels (B) in frontal cortex and hippocampus
of rats. Rats were exposed to sodium arsenite (ARS I – 2.0 or ARS II – 4.0 mg/kg body weight/day, p.o.) from PD22 to PD59. Effect on muscarinic-cholinergic receptors (A)
and CHRM2 mRNA levels (B) assessed immediately after exposure to sodium arsenite on PD60 and 30 days after withdrawal of sodium arsenite exposure on PD90. Values
are mean ± SEM of four to five animals in each group in the data presented for muscarinic-cholinergic receptors (A) and three animals in each group in the data presented
for CHRM2 mRNA levels (B). Data have been analyzed by one-way analysis of variance followed by Newman–Keuls test. *Significantly differs from controls (p < 0.01);
**significantly differs from controls (p < 0.001).

Fig. 4. Early life exposure (PD22–PD59) to sodium arsenite and effect on the acetylcholinestrase activity in frontal cortex and hippocampus of rats. Rats were exposed to
sodium arsenite (ARS I – 2.0 or ARS II – 4.0 mg/kg body weight/day, p.o.) from PD22 to PD59. Effect on acetylcholinestrase activity assessed immediately after exposure to
sodium arsenite on PD60 and 30 days after withdrawal of sodium arsenite exposure on PD90. Values are mean ± SEM of four to five animals in each group. Data have been
analyzed by one-way analysis of variance followed by Newman–Keuls test. *Significantly differs from controls (p < 0.05); **significantly differs from controls (p < 0.001).
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66 L.P. Chandravanshi et al. / Int. J. Devl Neuroscience 34 (2014) 60–75
Table 2
Scatchard analysis of 3 H-QNB to frontocortical and hippocampal membranes in rats exposed to sodium arsenite during early life (PD22–PD59).
Age in postnatal day
Treatment groups PD60
Kd
Frontal cortex
Control 1.56 ± 0.013
ARS I (2 mg/kg) 1.83 ± 0.18
ARS II (4 mg/kg) 2.09 ± 0.18
Hippocampus
Control 1.48 ± 0.09
ARS I (2 mg/kg) 1.74 ± 0.14
ARS II (4 mg/kg) 1.93 ± 0.18
Values are mean ± SEM of three observations in each group. Kd – dissociation constant expressed in nM, Bmax – maximum number of binding sites expressed in pmoles
3
H-QNB bound/g protein. Data have been analyzed by one-way analysis of variance followed by Newman–Keuls test.
*
Significantly differs from controls (p < 0.05).
**
Significantly differs from controls (p < 0.01).
on lipid peroxidation and protein carbonyls implicated as pro-
oxidants and on the activity of superoxide dismutase, catalase and
glutathione peroxidase and glutathione levels associated with anti-
oxidant defense was studied immediately after arsenic exposure on
PD60 and 30 days after withdrawal of arsenic exposure on PD90 and
the results are presented below:
7.4.1. Effect on brain lipid peroxidation, protein carbonyl levels
and reduced glutathione levels
Levels of lipid peroxidation, protein carbonyl and reduced glu-
tathione were measured both in hippocampus and frontal cortex
to assess the extent of oxidative stress. Early life exposure of rats to
arsenic (2.0 mg/kg or 4.0 mg/kg body weight) caused a significant
increase in the TBARS levels associated with enhanced lipid peroxi-
dation in frontal cortex (F(2,9) = 27.23, 40%, p < 0.05; 72%, p < 0.001)
and hippocampus (F(2,9) = 14.33, 41%, p < 0.05; 72%, p < 0.01) on
PD60 as compared to controls (Fig. 5A). Increase in the TBARS
levels was more marked in rats exposed to arsenic at a higher
dose (4.0 mg/kg body weight) suggesting enhanced lipid peroxi-
dation in the brain. Although a trend of recovery in TBARS levels
was observed both in frontal cortex (F(2,9) = 5.176, 28%, p > 0.05;
40%, p < 0.05) and hippocampus (F(2,9) = 4.004, 19%, p > 0.05; 41%,
p < 0.05) 30 days after withdrawal of exposure, levels of TBARS
remained significantly higher both in frontal cortex and hippocam-
pus of rats exposed to arsenic at a higher dose as compared to
respective controls (Fig. 5A).
Exposure to arsenic (2.0 mg/kg or 4.0 mg/kg body weight) from
PD22 to PD59 significantly increased the levels of protein carbonyl
in frontal cortex (F(2,12) = 14.10, 35%, p < 0.01; 68%, p < 0.001) and
hippocampus (F(2,12) = 15.02, 37%, p < 0.05; 78%, p < 0.01) in com-
parison to controls (Fig. 5B). Although a trend of recovery in protein
carbonyl levels was observed both in frontal cortex (F(2,12) = 5.47,
27%, p > 0.05; 48%, p < 0.05) and hippocampus (F(2,12) = 4.883, 19%,
p > 0.05; 46%, p < 0.05) 30 days after withdrawal, protein carbonyl
levels remained significantly higher in rats exposed to arsenic at the
higher dose on PD90 in comparison to respective controls (Fig. 5B).
Decrease in the levels of reduced glutathione in frontal cortex
(F(2,12) = 33.62, 28%, p < 0.001; 40%, p < 0.001), and hippocampus
(F(2,9) = 9.24, 31%, p < 0.05; 49%, p < 0.01) was observed in rats
treated with arsenic (2.0 mg/kg or 4.0 mg/kg body weight) during
early life as compared to controls (Fig. 5C). A trend of recovery in
reduced glutathione was observed in frontal cortex (F(2,10) = 6.79,
9%, p > 0.05; 17%, p < 0.05) and hippocampus (F(2,12) = 3.891, 8%,
p > 0.05; 21%, p < 0.05) of arsenic exposed rats 30 days after with-
drawal of exposure on PD90 in comparison to respective controls
(Fig. 5C).
PD90
Bmax Kd Bmax
737.9 ± 84 1.96 ± 0.12 805 ± 58
592 ± 54* 2.06 ± 0.14 764 ± 46
459 ± 48** 2.11 ± 0.21 749 ± 57
816.6 ± 79 1.67 ± 0.10 993.6 ± 80
646.9 ± 65* 1.75 ± 0.11 884.6 ± 91
465.1 ± 53** 1.82 ± 0.12 841.4 ± 68
7.4.2. Effect on superoxide dismutase, catalase and glutathione
peroxidase activity
A significant decrease in the activity of superoxide dismu-
tase was observed in frontal cortex (F(2,10) = 50.4039%, p < 0.001;
55%, p < 0.001) and hippocampus (F(2,12) = 48.09, 46%, p < 0.001;
65%, p < 0.001) following early life exposure of rats to arsenic
(2.0 mg/kg or 4.0 mg/kg body weight) in comparison to con-
trols (Fig. 6A). A trend of recovery in the activity of superoxide
dismutase was observed 30 days after withdrawal of exposure
on PD90 both in frontal cortex (F(2,12) = 0.9796, 5%, p > 0.05;
12%, p > 0.05) and hippocampus (F(2,9) = 5.670, 12%, p > 0.05; 30%,
p < 0.05) of arsenic exposed rats as compared to respective controls
(Fig. 6A).
Rats treated with arsenic (2.0 mg/kg or 4.0 mg/kg body weight)
exhibited a decrease in the activity of catalase in frontal cor-
tex (F(2,11) = 13.01, 25%, p < 0.01; 43%, p < 0.01) and hippocampus
(F(2,11) = 19.47, 26%, p < 0.05; 57%, p < 0.01) in comparison to con-
trols (Fig. 6B). The activity of catalase remained decreased in arsenic
treated rats even after withdrawal of exposure for 30 days although
the changes were not significant in frontal cortex (F(2,9) = 4.922,
13%, p < 0.05; 29%, p < 0.05) and hippocampus (F(2,9) = 5.260, 15%,
p > 0.05; 22%, p < 0.05) in comparison to respective controls (Fig. 6B).
A significant decrease in the activity of glutathione peroxi-
dase was observed in frontal cortex (F(2,9) = 8.45, 28%, p < 0.05;
40%, p < 0.01) and hippocampus (F(2,11) = 11.26, 32%, p < 0.01; 57%,
p < 0.001) of rats following their exposure to arsenic (2.0 mg/kg or
4.0 mg/kg body weight) from PD22 to PD59 as compared to con-
trols on PD60 (Fig. 6C). The activity of glutathione peroxidase in
arsenic treated rats exhibited a trend of recovery in frontal cor-
tex (F(2,10) = 3.55, 7%, p > 0.05; 15%, p > 0.05) and hippocampus
(F(2,9) = 1.78, 5%, p > 0.05; 14%, p < 0.05) 30 days after withdrawal
of exposure in the arsenic treated rats as compared to respective
controls (Fig. 6C).
8. Immunoexpression of proteins by Western blotting
Effect on the expression of selected proteins associated with
the cholinergic circuitry and those involved in pro-apoptotic, anti-
apoptotic and stress pathways was assessed following early life
exposure from PD22–PD59 to arsenic (2.0 mg/kg or 4.0 mg/kg body
weight) in rats to investigate their involvement in arsenic induced
neurotoxicity and results are presented below:
8.1. Effect on the expression of brain ChAT and PKC ˇ-1 proteins
A decrease in the expression of ChAT protein in frontal cor-
tex (F(2,6) = 59.79, 1.34, 1.44-fold) and hippocampus (F(2,6) = 167.8,
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Fig. 5. Early life exposure (PD22–PD59) to sodium arsenite and effect on lipid peroxidation levels (A), protein carbonyl levels (B) and reduced glutathione levels (C) in frontal
cortex and hippocampus of rats. Rats were exposed to sodium arsenite (ARS I – 2.0 or ARS II – 4.0 mg/kg body weight/day, p.o.) from PD22 to PD59. Effect on these weight
parameters assessed immediately after exposure to sodium arsenite on PD60 and 30 days after withdrawal of sodium arsenite exposure on PD90. Values are mean ± SEM of
four to five animals in each group. Data have been analyzed by one-way analysis of variance followed by Newman–Keuls test. *Significantly differs from controls (p < 0.05);
**significantly differs from controls (p < 0.01); ***significantly differs from controls (p < 0.001).

1.60, 1.84-fold) was clearly evident following early life expo-
sure of rats to arsenic (2.0 mg/kg or 4.0 mg/kg body weight)
from PD22 to PD59 in comparison to controls on PD60 (Fig. 7A).
No significant effect on the expression of ChAT protein both in
frontal cortex and hippocampus was observed in rats 30 days
after withdrawal of exposure as compared to respective controls
(Fig. 7A).
Early life exposure to arsenic (2.0 mg/kg or 4.0 mg/kg body
weight) from PD22 to PD59 in rats caused a significant decrease in
the expression of PKC ␤-1 protein in hippocampus (F(2,6) = 58.30,
1.5-fold, p < 0.001; 2.10-fold, p < 0.001) on PD60 as compared to
controls (Fig. 7B). No significant effect on the expression of PKC
␤-1 protein in hippocampal region was observed in rats 30 days
after withdrawal of arsenic exposure as compared to respective
controls (Fig. 7B).
8.2. Effect on the expression of pro-apoptotic (Bax, Caspase-3,
Cleaved Caspase-3) and anti-apoptotic (Bcl-2) proteins
A significant increase in the Bcl-2/Bax protein ratio in frontal
cortex (F(2,6) = 50.18, 1.47-fold, p < 0.01; 2.19-fold, p < 0.001)
and hippocampus (F(2,6) = 76.42, 1.63-fold, p < 0.001; 2.61-fold,
p < 0.001) was observed in arsenic (2.0 mg/kg or 4.0 mg/kg body
weight) exposed rats on PD60 as compared to controls. In spite of a
trend of recovery observed both in frontal cortex and hippocampus,
ratio of Bcl-2/Bax remained increased in both the brain regions of
rats exposed to arsenic at the higher dose 30 days after withdrawal
of exposure on PD90 in comparison to respective controls (Fig. 8A).
A significant increase in the expression of Caspase-3 pro-
tein in frontal cortex (F(2,6) = 309.8, 1.27-fold, p < 0.001; 1.44-fold,
p < 0.001) and hippocampus (F(2,6) = 167.3, 1.35-fold, p < 0.001;
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68 L.P. Chandravanshi et al. / Int. J. Devl Neuroscience 34 (2014) 60–75

Fig. 6. Early life exposure (PD22–PD59) to sodium arsenite and effect on superoxide dismutase (A), catalase (B) and glutathione peroxidase (C) activity in frontal cortex and
hippocampus of rats. Rats were exposed to sodium arsenite (ARS I – 2.0 or ARS II – 4.0 mg/kg body weight/day, p.o.) from PD22 to PD59. Effect on these parameters assessed
immediately after exposure to sodium arsenite on PD60 and 30 days after withdrawal of sodium arsenite exposure on PD90. Values are mean ± SEM of four to five animals
in each group. Data have been analyzed by one-way analysis of variance followed by Newman–Keuls test. *Significantly differs from controls (p < 0.05); **significantly differs
from controls (p < 0.01); ***significantly differs from controls (p < 0.001).

2.01-fold, p < 0.001) was observed on PD60 in arsenic (2.0 mg/kg
or 4.0 mg/kg body weight) exposed rats in comparison to controls
(Fig. 8A). Exposure to arsenic also resulted to increase the expres-
sion of Cleaved Caspase-3 in frontal cortex (F(2,6) = 79.7, 1.52-fold,
p < 0.05; 2.84-fold, p < 0.001) and hippocampus (F(2,6) = 11.35, 2.33-
fold, p < 0.01; 2.50-fold, p < 0.05) on PD60 as compared to rats in the
control group (Fig. 8B). While a trend of recovery was observed in
the expression of Caspase-3 and Cleaved Caspase-3, 30 days after
withdrawal of exposure, changes were found to persist both in
frontal cortex and hippocampus of rats exposed to arsenic at higher
dose on PD90 in comparison to respective controls (Fig. 8A and B).
8.3. Effect on the expression of stress marker (phospho-p38, AP-1
and phospho-JNK) proteins
Expression of phospho-p38 protein in frontal cortex
(F(2,6) = 69.0, 1.41-fold, p < 0.001; 1.66-fold, p < 0.001) and hip-
pocampus (F(2,6) = 38.2, 1.78-fold, p < 0.01; 2.07-fold, p < 0.001)
was found increased in arsenic (2.0 mg/kg or 4.0 mg/kg body
weight) exposed rats in comparison to controls on PD60 (Fig. 9A).
Change in the expression was more marked in both brain regions
at the higher dose of arsenic. A trend of recovery in the expres-
sion of phospho-p38 both in frontal cortex and hippocampus
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L.P. Chandravanshi et al. / Int. J. Devl Neuroscience 34 (2014) 60–75 69

Fig. 7. Early life exposure (PD22–PD59) to sodium arsenite and effect on the expression of PKC ␤-1 (A) and ChAT (B). Rats were exposed to sodium arsenite (ARS I – 2.0 or
ARS II – 4.0 mg/kg body weight/day, p.o.) from PD22 to PD59. Effect on the expression of these proteins studied immediately after exposure to sodium arsenite on PD60 and
30 days after withdrawal of sodium arsenite exposure on PD90. Values are mean ± SEM of three animals in each group. Data have been analyzed by one-way analysis of
variance followed by Newman–Keuls test. *Significantly differs from controls (p < 0.001).

was observed 30 days after withdrawal of exposure on PD90 in
comparison to respective controls (Fig. 9A).
Exposure to arsenic (2.0 mg/kg or 4.0 mg/kg body weight)
resulted to increase the expression of AP-1 in frontal cortex
(F(2,6) = 73.5, 1.26-fold, p < 0.001; 1.51-fold, p < 0.001) and hip-
pocampus (F(2,6) = 47.1, 1.45-fold, p < 0.001; 1.67-fold, p < 0.001) on
PD60 as compared to controls (Fig. 9A). Increase in the expression
of AP-1 was more marked both in frontal cortex and hippocam-
pus of rats exposed to arsenic at a higher dose. Interestingly, a
trend of recovery in the expression of AP-1 was observed both
in the frontal cortex and hippocampus 30 days after withdrawal
of exposure in rats on PD90 in comparison to respective controls
(Fig. 9A).
No significant change in the expression of phospho-JNK was
observed in rats exposed to arsenic (2.0 mg/kg or 4.0 mg/kg body
weight) during early life from PD22 to PD59 on PD60 and PD90 both
in frontal cortex and hippocampus (Fig. 9B).
9. Effect on arsenic levels
A significant increase in the levels of arsenic in frontal cortex
(F(2,9) = 26.10, 1.39-fold, p < 0.05; 3.90-fold, p < 0.001), and hip-
pocampus (F(2,6) = 65.02, 3.23-fold, p < 0.001; 5.48-fold, p < 0.001)
was observed on PD60 in rats following early life exposure to
arsenic from PD22 to PD59 as compared to controls (Table 3). A
trend of recovery in arsenic levels was observed 30 days after
withdrawal of exposure in arsenic exposed rats in frontal cortex
(F(2,9) = 12.77, 0.45, fold, p > 0.05; 1.00-fold, p < 0.01) and hip-
pocampus (F(2,9) = 7.39, 0.27, fold, p > 0.05; 1.00-fold, p < 0.05) as
compared to respective controls (Table 3).
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70 L.P. Chandravanshi et al. / Int. J. Devl Neuroscience 34 (2014) 60–75

Fig. 8. Early life exposure (PD22–PD59) to sodium arsenite and effect on the expression of selected pro-apoptotic (Caspase-3, Bax (A), Cleaved Caspase-3 (B)) and anti-
apoptotic (Bcl-2 (A)) proteins in frontal cortex and hippocampus of rats. Rats were exposed to sodium arsenite (ARS I – 2.0 or ARS II – 4.0 mg/kg body weight/day, p.o.)
from PD22 to PD59. Effect on the expression of these proteins studied immediately after exposure to sodium arsenite on PD60 and 30 days after withdrawal of sodium
arsenite exposure on PD90. Values are mean ± SEM of three animals in each group. Data have been analyzed by one-way analysis of variance followed by Newman–Keuls
test. *Significantly differs from controls (p < 0.05); **significantly differs from controls (p < 0.01); ***significantly differs from controls (p < 0.001).

10. Discussion doses of arsenic (2 mg/kg or 4 mg/kg body weight/day) selected in

Although a number of experimental studies have been carried
out to investigate the mechanism of arsenic induced neurotoxicity
at different ages and doses with varying period of exposure, the
focus in the present study is to understand the mechanism of brain
cholinergic alterations and associated functions during early life
arsenic exposure at low doses and to assess if such changes are tran-
sient or persistent. Exposure to arsenic at doses 0.3–0.4 mg/kg/day
has been found to cause only morphological alterations in the
myelinated central tracts with no toxic effects. However, structural
alterations in myelinated tracts in brain regions have been observed
following exposure to arsenic at doses 3–4 mg/kg body weight/day
(Rios et al., 2009; Zarazua et al., 2010; Martínez et al., 2011). The
the present study are based on these reports.
Reduction in the body weight following arsenic exposure in
the progeny has been observed both in clinical and experimental
studies (Holson et al., 1999; Schulz et al., 2002; Saha et al., 2008).
Inverse association between environmental arsenic exposure and
growth of children has been reported (Saha et al., 2012; Gardner
et al., 2013). Interestingly, arsenic exposure in early childhood has
a greater effect on body weight and growth than prenatal expo-
sure (Saha et al., 2012). While the prevalence of decreased body
weight and growth was higher in boys, the effect was more marked
in girls (Saha et al., 2012). Adverse effect on body weight due to
arsenic exposure in experimental studies was irrespective of time
and dose in most of the studies (Stump et al., 1999; Ekong et al.,
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L.P. Chandravanshi et al. / Int. J. Devl Neuroscience 34 (2014) 60–75 71

Fig. 9. Early life exposure (PD22–PD59) to sodium arsenite and effect on the expression of selected stress marker proteins (Phospho-p38, AP-1 (A) and Phospho-JNK-1,2 (B))
in frontal cortex and hippocampus of rats. Rats were exposed to sodium arsenite (ARS I – 2.0 or ARS II – 4.0 mg/kg body weight/day, p.o.) from PD22 to PD59. Effect on the
expression of these proteins studied immediately after exposure to sodium arsenite on PD60 and 30 days after withdrawal of sodium arsenite exposure on PD90. Values are
mean ± SEM of three animals in each group. Data have been analyzed by one-way analysis of variance followed by Newman–Keuls test. *Significantly differs from controls
(p < 0.05); **significantly differs from controls (p < 0.01); ***significantly differs from controls (p < 0.001).

2012). Loss of body weight following early life exposure to arsenic at
low dose as observed in the present study indicates growth impair-
ment and exhibit vulnerability of developing rats to the toxic effects
of arsenic and is consistent with earlier experimental reports (Dhar
et al., 2005; Bashir et al., 2006). Recovery of body weight on with-
drawal of arsenic exposure suggests that the effect is transient, and
continuous arsenic exposure may have detrimental health effects.
While arsenic has been found to affect different body organs,
the nervous system is a soft target to its deleterious effects. Increas-
ing incidences of neuropathy and neurological disorders associated
with acute and chronic arsenic exposure further indicate that
arsenic may affect both the peripheral and the central nervous
systems (Vahidnia et al., 2007; Banerjee et al., 2011). A num-
ber of studies have shown that arsenic exposure is closely linked
with increased incidences of neuropsychological disturbances and
cognitive deficits with greater vulnerability in children (Wright
et al., 2006; Rosado et al., 2007). As environmental arsenic expo-
sure has been found to be a potential risk factor for Alzheimer’s
disease (Giasson et al., 2002; Borenstein et al., 2006; Gong and
Bryant, 2010), there is a great need to understand the involve-
ment and the mechanism of cholinergic modifications. An inverse
relationship between arsenic exposure and plasma cholinesterase
activity has been observed in a population based study by Ali et al.
(2010). It was suggested that alteration in plasma cholinesterase
activity could be one of the potential biological mechanisms by
which arsenic induces neurotoxicity in humans. In brain, acetyl-
cholinesterase (AChE), an enzyme largely present in the cholinergic
neurons plays an important role in the metabolism of acetylcholine,
an important neurotransmitter associated with synaptic plastic-
ity (Roger et al., 2006). A number of environmental chemicals
including arsenic have been found to inhibit the activity of brain
acetylcholinesterase in rats (Sankhwar et al., 2012; Yadav et al.,
2011; Ansari et al., 2012). Arsenic exposure has also been found
to inhibit acetylcholinesterase activity in vitro in cultured hip-
pocampal neurons of rats and neuroblastoma cells of mice (Repetto
et al., 1994). Synthesis and generation of acetylcholine in brain
slices has been found to be affected as a result of arsenic expo-
sure (Kobayashi et al., 1987). As most of these studies have been
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72 L.P. Chandravanshi et al. / Int. J. Devl Neuroscience 34 (2014) 60–75
Table 3
Early life exposure (PD22–PD59) to sodium arsenite and effect on arsenic levels in
frontal cortex and hippocampus of rats on PD60 and PD90.
Age in postnatal day
Brain region/Treatment groups PD60 PD90
Frontal cortex
Control 357 ± 19.55 340 ± 39.87
ARS I (2 mg/kg) 853.5 ± 108.5* 495 ± 69.49
ARS II (4 mg/kg) 1751 ± 212.6*** 706 ± 38.9**
Hippocampus
Control 336.5 ± 33.33 296 ± 42.41
ARS I (2 mg/kg) 1517 ± 159.49*** 377 ± 54.24
ARS II (4 mg/kg) 2182 ± 116.6*** 591 ± 68.41*
Rats were exposed to sodium arsenite (ARS I – 2.0 or ARS II – 4.0 mg/kg body
weight/day, p.o.) from PD22 to PD59 and arsenic levels in frontal cortex and hip-
pocampus were analyzed by atomic absorption spectrophotometer immediately
after exposure on PD60 and 30 days after withdrawal of exposure on PD90. Values
are mean ± SEM of four animals in each group. Data have been analyzed by one-way
analysis of variance followed by Newman–Keuls test. Arsenic levels expressed as
ng/gm tissue weight.
*
Significantly differs from controls (p < 0.05).
**
Significantly differs from controls (p < 0.01).
***
Significantly differs from controls (p < 0.001).
carried out at high doses of arsenic and in adult animals, impact of
low doses of arsenic on the integrity of brain acetylcholinesterase
activity in developing animals is not understood. Decrease in
acetylcholinesterase activity both in frontal cortex and hippocam-
pus following arsenic exposure in rats during early life as observed
in the present study is consistent with earlier reports on adult
rats (Patlolla and Tchounwou, 2005; Rodriguez et al., 2005; Flora
et al., 2009). Being the metabolic enzyme, inhibition in the activ-
ity of acetylcholinesterase may be associated with pharmacological
consequences including enhanced accumulation of acetylcholine at
the synaptic cleft and subsequently down regulation of cholinergic
receptors both in frontal cortex and hippocampus. Desensitization
of brain cholinergic receptors due to accumulation of acetylcholine
has been demonstrated earlier (Zheng et al., 2000). Interestingly,
decrease in the mRNA expression of ChRM2 receptor gene both
in the frontal cortex and hippocampus following arsenic expo-
sure observed in the present study is consistent with decreased
binding of cholinergic receptors. Further, decrease in the expres-
sion of choline acetyltransferase (ChAT) both in the frontal cortex
and hippocampus following arsenic exposure at low doses could
be due to auto-feed back regulation as a result of accumulation of
acetylcholine and indicate vulnerability of cholinergic system. The
role of cholinergic system in modulating learning and memory is
well established (Paul, 2003; Havekesa et al., 2011). Decrease in
learning and memory assessed by active avoidance response and
continuous alternation test in arsenic exposed rats could be asso-
ciated with impaired cholinergic system, specially decreased brain
cholinergic-muscarinic receptors in the present study. Moreover,
the role of PKC ␤-1 in modulating long term potentiation and dif-
ferent forms of learning is well accepted (Wu et al., 2007; Paratcha
et al., 2000). Decreased expression of hippocampal PKC ␤-1 there-
fore could also be associated with impaired learning in arsenic
exposed rats as observed in the present study. While studying the
neuroprotective efficacy of curcumin in arsenic induced choliner-
gic dysfunctions, decreased sensitivity of cholinergic-muscarinic
receptors associated with impaired learning was observed by us in
arsenic exposed rats. However, the dose of arsenic (sodium arsen-
ite, 20 mg/kg body weight/day) was very high in these studies.
Cholinergic dysfunctions following early life exposure to arsenic
at low doses as observed in the present study are striking. Another
interesting phenomenon observed in the present study was a trend
of recovery in the cholinergic modifications, PKC ␤-1 expression
and learning and memory in arsenic exposed rats on withdrawal of
exposure. In an interesting study, Liu et al. (2012) found that arsenic
induced effect on hippocampal cell proliferation and neurogenesis
could be recovered in adult mice.
Although there are evidences exhibiting that arsenic may cause
oxidative damage in the brain by creating an imbalance between
pro-oxidant and anti-oxidant system (Yen et al., 2011), the impact
of arsenic during early life exposure on the intensity of brain
oxidative stress is not understood. Increased formation of malon-
aldehyde and protein carbonyls in frontal cortex and hippocampus
of arsenic exposed rats indicate enhanced oxidative damage due
to increased generation of reactive species and the finding is con-
sistent with earlier reports (Bhadauria and Flora, 2007; Flora et al.,
2009; Wang et al., 2012). As arsenic has a high affinity with thiols, it
may affect free thiols and catalytic activity of proteins and enzymes
containing thiols (Shen et al., 2012).
Decrease in reduced glutathione levels both in hippocampus
and frontal cortex as observed in the present study could be due
to arsenic induced toxic manifestations and indicates enhanced
stress. Arsenic exposure has also been found to impair the integrity
of enzymes associated with the antioxidant defense (Kumar et al.,
2013; Sannadia et al., 2013). Decrease in the activity of super-
oxide dismutase following arsenic exposure in frontal cortex and
hippocampus indicates that arsenic may affect the dismutation of
superoxide radicals and thus enhances the vulnerability of both
the brain regions to toxic effects of superoxide radicals. Further,
decreased activity of catalase and glutathione peroxidase, enzymes
associated to reduce hydrogen peroxide into water and oxygen
in both the brain regions of arsenic exposed rats indicate that
enzymatic antioxidant defense in the brain is impaired following
arsenic exposure. The fact that the brain has high utilization of
oxygen associated with impaired antioxidant defense, vulnerabil-
ity of the brain to arsenic induced oxidative stress is likely to be
enhanced as observed in the present study. Oxidative injury in the
brain like other body organs following exposure to environmental
chemicals including arsenic may cause pharmacological, physio-
logical and pathological changes including cellular apoptosis (De
Vizcaya-Ruiz et al., 2009; Khan et al., 2013; Sannadia et al., 2013).
Upregulation in the expression of Caspase-3, cleaved Caspase-3
and Bcl-2/Bax protein ratio both in frontal cortex and hippocampus
indicate enhanced apoptosis and could be associated with arsenic
induced oxidative stress during early life. Increased expression of
AP-1, a stress marker protein in arsenic exposed rats also appears
to be associated with enhanced oxidative stress. While no signif-
icant effect on the expression of phospho-JNK was observed in
the present study, increased expression of phospho-p38 both in
frontal cortex and hippocampus suggest that arsenic induced apo-
ptosis may be modulated by p38 MAPKs protein. The functional
role of MAPKs in modulating proliferation, differentiation, apopto-
sis and other important cellular processes through intracellular
signaling is well accepted (Estus et al., 1994; Ham et al., 1995;
Mesner et al., 1995; Namgung and Zhengui, 2000). As there are
reports exhibiting a close link between increased MAPK signaling
and neurodegenerative disorders associated with enhanced oxida-
tive stress and apoptosis (Miloso et al., 2008; Kim and Choi, 2010),
the increased oxidative stress associated with enhanced apoptosis
following early life arsenic exposure in the present study is convinc-
ing and could be easily linked. It was noticeable that the intensity of
oxidative stress and apoptosis was reduced and exhibited a trend
of recovery both in frontal cortex and hippocampus on withdrawal
of arsenic exposure.
While deciphering the mechanism of recovery in cholinergic
modifications and learning, intensity of oxidative stress and apo-
ptosis in arsenic exposed rats on withdrawal of exposure, the
presence of arsenic in frontal cortex and hippocampus appear to
be an important factor. Arsenic, like many other metals, crosses
the blood brain barrier and accumulates in the brain through active
 Author's personal copy
L.P. Chandravanshi et al. / Int. J. Devl Neuroscience 34 (2014) 60–75
Fig. 10. Schematic representation of sodium arsenite induced cholinergic deficits
associated with oxidative stress and apoptosis in rat brain.
uptake (Xi et al., 2010b; Lu et al., 2012). It has been shown in vitro
that the transporter for arsenic uptake into astrocytes has high Km
value as there was a proportional increase in cellular content of
arsenic in the system (Meyer et al., 2013; Calatayud et al., 2010;
Thomas, 2007). Decreased arsenic levels both in the frontal cortex
and hippocampus on withdrawal of exposure is quite fascinating.
While it is difficult to explain the exact mechanism of low arsenic
levels both in the frontal cortex and the hippocampus on with-
drawal of exposure as observed in the present study, decreased
arsenic levels in the cerebrum of adult mice was observed on
cessation of arsenic exposure by Liu et al. (2012). Arsenic forms
conjugates with cellular GSH and possible role of these conjugates
to eliminate/export arsenic from the cells has been suggested by a
number of investigators (Kala et al., 2000; Liu et al., 2001; Thomas,
2007; Meyer et al., 2013).
The results of the present study exhibit that early life exposure
to arsenic at low doses may cause cholinergic alterations both in
frontal cortex and hippocampus and impair learning and mem-
ory. Increased oxidative stress associated with enhanced apoptosis
appear to be important contributors in arsenic induced cholin-
ergic dysfunctions and exhibit vulnerability of developing rats
to the toxic effects of arsenic. Based on these findings a hypo-
thetical model of arsenic induced neurochemical alterations in
developing rats has been proposed (Fig. 10). A trend of recov-
ery in behavioral and neurochemical changes during early life
arsenic exposure clearly exhibits that these effects are transient
but continuous arsenic exposure may have detrimental effects.
Although it is difficult to comment at present, assay of blood acetyl-
cholinesterase activity could be further explored as a biomarker
to assess the risk of arsenic induced neurotoxicity in the popu-
lation. The findings are interesting and will help the regulatory
agencies and scientists to plan the strategy for biomonitoring on
arsenic exposed population and specially children to re-evaluate
the Tolerable Daily Intake (TDI) of arsenic levels in food or drinking
water.
Acknowledgments
The study is a part of INDEPTH programme of CSIR, New Delhi.
CSIR–IITR, Lucknow Communication no. 3076. The authors thank
73
the Director, CSIR-Indian Institute of Toxicology Research (CSIR-
IITR), Lucknow for his keen interest in the present study. One
of the co-authors, Lalit P Chandravanshi is grateful to the Uni-
versity Grants Commission, New Delhi for providing the research
fellowship. The authors acknowledge the help of Mr. Ritul Kamal,
CSIR-IITR, Lucknow in analyzing the data and Mr. B.S. Pandey
for extending the technical support. The authors also thank Pro-
fessor Madhu Mehrotra, Former Head, Department of European
Languages, University of Lucknow, Lucknow and Mr. B.D. Bhat-
tacharji, CSIR-IITR, Lucknow for going through the manuscript and
making the necessary changes in the language.
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