Biological Trace Element Research

https://doi.org/10.1007/s12011-018-1286-1

Developmental Neurotoxicity of Arsenic: Involvement of Oxidative

Stress and Mitochondrial Functions
Lalit P. Chandravanshi 1,2 & Richa Gupta 2 & Rajendra K. Shukla 3

Received: 2 January 2018 / Accepted: 23 February 2018

# Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract
Over the last decade, there has been an increased concern about the health risks from exposure to arsenic at low doses, because of
their neurotoxic effects on the developing brain. The exact mechanism underlying arsenic-induced neurotoxicity during sensitive
periods of brain development remains unclear, although enhanced oxidative stresses, leading to mitochondrial dysfunctions
might be involved. Here, we highlight the generation of reactive oxygen species (ROS) and oxidative stress which leads to
mitochondrial dysfunctions and apoptosis in arsenic-induced developmental neurotoxicity. Here, the administration of sodium
arsenite at doses of 2 or 4 mg/kg body weight in female rats from gestational to lactational (GD6-PD21) resulted to increased
ROS, led to oxidative stress, and increased the apoptosis in the frontal cortex, hippocampus, and corpus striatum of developing
rats on PD22, compared to controls. Enhanced levels of ROS were associated with decreased mitochondrial membrane potential
and the activity of mitochondrial complexes, and hampered antioxidant levels. Further, neuronal apoptosis, as measured by
changes in the expression of pro-apoptotic (Bax, Caspase-3), anti-apoptotic (Bcl2), and stress marker proteins (p-p38, pJNK) in
arsenic-exposed rats, was discussed. The severities of changes were found to more persist in the corpus striatum than in other
brain regions of arsenic-exposed rats even after the withdrawal of exposure on PD45 as compared to controls. Therefore, our
results indicate that perinatal arsenic exposure leads to abrupt changes in ROS, oxidative stress, and mitochondrial functions and
that apoptotic factor in different brain regions of rats might contribute to this arsenic-induced developmental neurotoxicity.

Keywords Arsenic . Developmental neurotoxicity . Oxidative stress . Mitochondria . Apoptosis

Introduction various diseases, including cancerous and noncancerous dis-

Arsenic, a naturally occurring neurotoxicant, is one of the risk
factors for cognitive and neurological dysfunctions in humans
[37, 50]. Even though chronic exposure to arsenic at high
levels [3] has been very well recognized as causing different
adverse health outcomes, including neurological diseases, ex-
posure to low and moderate levels [36] of arsenic has only
been more recently correlated with an increased risk for
* Lalit P. Chandravanshi
chandravanshi04@gmail.com
1 lasting effects and may not recover.
Department of Zoology, Institute of Science, Banaras Hindu
University, Varanasi 221005, India
2 neurological dysfunctions in children, particularly at low
Developmental Toxicology Division, CSIR—Indian Institute of
Toxicology Research, Post Box No. 80, MG Marg, Lucknow 226
001, India
3
Department of Biochemistry, All India Institute of Medical Sciences,
Bhopal, India
eases, such as cardiovascular, hypertension, and cognitive im-
pairment in human [5, 41, 65]. However, little is known about
the developmental neurotoxicity of low levels of arsenic dur-
ing crucial periods of brain development. In utero or early life
developments of the child represent a critical window of sus-
ceptibility to the toxicant. Arsenic insult at any step during
development may lead to developing chronic diseases [34,
63]. Arsenic may be a serious threat to the development of
the fetus and infant health because of their combination of
physiological immaturity and exposure during pregnancy
and early life. At times, the changes may not be visible imme-
diately but reflected at later stages of life. The adverse effects
during the developmental periods are likely to have long-
Arsenic may induce oxidative stress, which causes many
levels. Gong and O’Bryant [20], suggested a possible relation-
ship between arsenic exposure and Alzheimer’s disease.
However, the neurotoxic effects and underlying mechanisms
of arsenic-induced developmental neurotoxicity are mostly

unknown. Emerging studies have proved that accumulation of
arsenic and free radicals in the central nervous system will
induce oxidative stress and that antioxidant defenses are relat-
ed to the pathogenesis of neurodegenerative diseases [59].
Oxidative stress induced by arsenic can lead to mitochondrial
dysfunctions and neuronal apoptosis. Arsenic generates reac-
tive oxygen species (ROS) and disrupts the antioxidant de-
fense mechanism [54]. Antioxidant enzymes play a very im-
portant role in protecting the cell molecules from free radical-
generated oxidative stress. A lot of animal studies have report-
ed enhanced oxidative stress accompanied by the reduction in
antioxidant enzyme activities in rats exposed to arsenic [46,
52]. Neurodegeneration in pathological condition can occur as
a result of endogenous oxidative stress and reactive oxygen
species (ROS) generation in the central nervous system (CNS)
[12, 15]. The pathogenesis resulting from neuronal cell death
is believed to involve in the imbalance of oxidative and anti-
oxidant system and increase in ROS production with a con-
comitant impairment of the mitochondrial functions [14, 25].
Oxidative state plays an important function in the regulation
and control of neuronal cell death in the brain through its
interaction with cellular molecules and cell signaling path-
ways. Oxidative stress has been considered as an important
mechanism in the neurotoxicity [6, 10]. In addition, mitochon-
dria have been implicated in the cellular damage and induction
of apoptotic cell signaling pathways during this neurotoxicity
[47, 57], and the fact that mitochondrial dysfunctions may
lead to the severe pathological condition in neurodegenerative
diseases. Being the major source of energy in the cell, mito-
chondria may be an important factor contributing to the cell
death and redox signaling [27]. Generation of ROS in the
electron transport chain may cause mitochondrial respiratory
chain dysfunction. Generation of ROS in the mitochondrial
respiratory chain has been linked to neurotoxicity-associated
changes in the CNS through the impairment in mitochondrial
functions and neuronal homeostasis. In order to better under-
stand the mechanisms underlying arsenic toxicity, the present
study evaluated the redox homeostasis and energy metabolism
in the frontal cortex, hippocampus, and corpus striatum of
arsenic-exposed rats. Regarding bioenergetics, we investigat-
ed the activities of respiratory chain complexes and mitochon-
drial membrane potential (ΔΨm). Impairment in the functions
of mitochondria plays a major role in apoptosis.
Although neurotoxicity of arsenic in adults is well charac-
terized, information regarding the developmental neurotoxic-
ity is limited. The presence of arsenic in groundwater and
extensive industrial uses of arsenic have raised concerns about
its adverse effects on public health. Therefore, the rationale of
the present study was to elucidate the mechanism of arsenic-
induced neurotoxicity in the developing brain. However, no
report currently exhibits the effect of arsenic on the oxidative
stress, mitochondrial membrane potential, mitochondrial
complexes of the electron transport chain, and the apoptosis
Chandravanshi et al.
during the perinatal period. Further, we also tried to investi-
gate the persistent effect of arsenic 23 days after the withdraw-
al of arsenic exposure.
Material and Methods
Animal and Treatment
Perinatal exposure of rats to sodium arsenite: Adult female
rats of proven fertility were kept for mating with the males
in the ratio of 2:1. The day of appearance of the vaginal plug
was considered as day one (GD-1) of gestation, and the preg-
nant rats were separated into individual cages. Pregnant rats
were divided into three groups (6 pregnant rats in each group)
and treated with sodium arsenite (SIGMA, USA) on day six of
gestation (GD-6) until postnatal day (PD) 21 as follows:
Group I- Pregnant rats were treated with normal saline (p.o.)
identically as in groups II and III and served as
controls.
Group II- Pregnant rats were treated with sodium arsenite
dissolved in distilled water (2 mg/kg body weight,
p.o.) once daily from GD6 to PD21.
Group III- Pregnant rats were treated with sodium arsenite
(4 mg/kg body weight, p.o.) dissolved in distilled
water once daily from GD6 to PD21.
The pregnant rats were allowed to deliver naturally and to
nurse their pups until PD21. Martínez et al. [40] reported that
200 to 250 g rats consumed 36 ppm of arsenic, a concentration
that results in ingestion of 3–4 mg/kg/day of arsenic. This
parts per million range (3100 μg/L) of arsenic has also been
reported in drinking water in the United States of America
(USA) and Bangladesh [60]. Exposure to arsenic at doses
0.3–0.4 mg/kg/day (an exposure level that has already been
reported in human population) has not been found toxic ef-
fects. However, structural alterations in myelinated tracts in
brain regions have been found following exposure to arsenic
at doses 3–4 mg/kg body weight/day [40, 48, 66]. The doses
of arsenic (2 mg/kg or 4 mg/kg body weight/day) were select-
ed in the present study based on these reports.
All the litters were culled on PD2; 8–10 pups with an equal
allocation of males and females. The animals were sacrificed,
and the brain was taken out quickly, washed in ice-cold saline,
and dissected into specific regions—frontal cortex, corpus
striatum, and hippocampus following the standard procedure
as described by Glowinski and Iversen [19]. The dissected
brain regions were stored at −80 °C for neurochemical assays.
To understand whether arsenic-induced changes are transient
or persistent, another set of experiment was done on the same
conditions as mentioned above. In this set of experiment, male
pups were separated from mothers on PD22 in each group and
Developmental Neurotoxicity of Arsenic: Involvement of Oxidative Stress and Mitochondrial Functions
left as such until PD45. Effects on neurochemical endpoints
were studied on PD45. Unfortunately, we did not assess the
effect of arsenic on female pups.
The study was approved by the institutional animal ethics
committee of CSIR-IITR, Lucknow (Ref No. ITRC/
IAEC/35/11-41/12, dated September 4, 2012), and all exper-
iments were carried out in accordance with the guidelines
approved by the Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA), Ministry
of Environment and Forests (Government of India), New
Delhi, India.
Assessment of Oxidative Stress and ROS Generation
Assessment of Oxidative Stress
Assay of Lipid Peroxidation, Protein Carbonyl, and Reduced
Glutathione (GSH) Levels Malondialdehyde (MDA) as an end
product of lipid peroxidation levels was measured in frontal
cortex, hippocampus, and corpus striatum by the method of
Ohkawa et al. [42]. The end products were read at 535 nm, and
the values are expressed as μmol MDA formed/h/mg protein.
Protein carbonyl content was measured in the selected
brain regions by the method of Levine et al. [35] using 2,4-
dinitrophenylhydrazine (DNPH) as the substrate. Amount of
protein carbonyl was calculated using a molar extinction co-
efficient (€) 22 mM−1 cm−1 for aliphatic hydrazine, and values
are expressed as nmol/mg protein.
GSH was assayed in selected brain regions by the method
of Hasan and Haider [22] using 5′5′-dithiobis 2-nitrobenzoic
acid (DTNB) as the color reagent. Further, GSH (ranging from
2 to 10 μg) as a standard was run in parallel to plot the stan-
dard curve. The values are expressed as μg GSH/g tissue
weight.
Assay of Superoxide Dismutase (SOD), Catalase (CAT), and
Glutathione Peroxidase Activity SOD activity in brain regions
was measured following the method of Kakkar et al. [31]
using NADH as a substrate in the post-mitochondrial fraction.
The activity of SOD has been expressed as unit/min/mg
protein.
CAT activity was determined by the method of Aebi
[1] in the post-mitochondrial fraction of selected brain
regions using hydrogen peroxide (H2O2) as the substrate.
The decrease in optical density was measured at 240 nm
for 150 s using the spectrophotometer. The activity of the
enzyme was calculated using the molar extinction coef-
ficient 43.6 M cm −1 , and values are expressed as
μmol/min/mg protein.
The activity of glutathione peroxidase in brain regions was
assayed by the method of Flohe and Gunzler [16]. The values
are expressed as nmol GSH oxidized/min/mg protein.
Estimation of ROS Generation Amount of ROS generation
was assessed fluorometrically by measuring the conversion
of DCFH-DA dye into highly fluorescent DCF product in
the presence of H2O2 [13]. Briefly, the mitochondrial fraction
was incubated with DCFH-DA (10 mM, 5 μl) in the water
bath (30 min, 37 °C) in a total reaction mixture of 1 ml. After
incubation, the mitochondria were pelleted by centrifugation
(1200 g, 10 min) and re-suspended in fresh HEPES buffer.
ROS generation was assessed in triplicate for each sample,
and the fluorescence was recorded at excitation/emission—
488/525 nm. The ROS generation has been expressed as
fluorescence/μg protein [51].
Assessment of Mitochondrial Membrane Potential (ΔΨm)
MMP was assessed using JC-1 dye following the procedure
as described by Kapoor et al. [32] with minor modifications.
Briefly, the dissected brain regions—frontal cortex, hippo-
campus, and corpus striatum—were kept in Hank’s balanced
salt solution (HBSS) containing antibiotic and antimycotic.
Mincing of tissues was carried out, followed by trypsinization
at 37 °C for 5–7 min with trypsin (0.5 mg/ml) in HBSS con-
taining BSA (0.5%). Following incubation, trypsin was aspi-
rated, and tissues were incubated (37 °C, 15 min) with DMEM
containing FBS (12%). Cells were dissociated from the tissues
by passing the tissue pieces 20–30 times through 1-ml pipette
tip, and then by passing it through the 200-μl pipette tip. The
resulting cell suspension was filtered through a 50-μm diam-
eter nylon mesh and pelleted by centrifugation (800 ×g for
6 min). Cells were washed with PBS, centrifuged (800 ×g
for 6 min), and re-suspended in PBS. The prepared cell sus-
pension was then incubated with JC-1 (10 μM, 15 min,
37 °C), washed with PBS, and re-suspended in PBS at a con-
centration of 106 cells/ml and analyzed through the flow
cytometer. Values are expressed as the number of depolarized
cells in the single-cell suspension.
Assay of Electron Complex Activity
Isolation of Rat Brain Mitochondria Mitochondria from frontal
cortex, hippocampus, and corpus striatum were isolated fol-
lowing the method of Bagh et al. [4]. Briefly, the tissues were
homogenized in 10 ml of buffer (pH 7.4) containing mannitol
(225 mM), sucrose (75 mM), HEPES (5 mM), EGTA (1 mM),
and BSA (1 mg/ml) and centrifuged (2000 ×g for 3 min) at
4 °C. The supernatant was centrifuged (20,000 ×g for 10 min)
at 4 °C to obtain the crude pellet of synaptosomes and mito-
chondria, which was further dissolved in homogenizing buffer
containing digitonin (0.02%) to lyse the synaptosomes and to
release the intra-synaptosomal mitochondria followed by cen-
trifugation (12,000 ×g for 10 min). The pellet obtained in the
process was washed twice in same buffer devoid of EGTA,
BSA, and digitonin. Finally, the pellet was suspended in iso-
tonic phosphate buffer (50 mM, pH 7.4).

Assay of activity of mitochondrial complexes I, II–III, and
IV: The activity of NADH (Complex I) was measured through
ferricyanide as the electron acceptor [23]. Briefly, the assay
was carried out at 30 °C in a reaction system containing
NADH (0.17 mM), potassium ferricyanide (0.6 mM),
Triton-X 100 (0.1%) in phosphate buffer (50 mM, pH 7.4).
The reaction was initiated after the addition of mitochondrial
suspension (10~30 μg of protein) to the sample cuvette. The
rate of oxidation of NADH was monitored by the decrease in
absorbance at 340 nm. The activity of complex I has been
expressed as nmol NADH/oxidized/min/protein (mg).
The activity of succinate-cytochrome c reductase (com-
plexes II–III) was measured by reduction of ferricytochrome
c to ferrocytochrome c under influence of succinate at 550 nm.
The reaction cocktail in cuvette consisted of succinate
(2 mM), KCN (1 mM), EDTA (0.3 mM), and cytochrome c
(1.2 mg/ml) in phosphate buffer (100 mM, pH 7.4) in a total
volume (1 ml). The reaction was initiated by adding mito-
chondrial suspension (10~30 μg protein). The activity of com-
plexes II–III has been expressed as nmol oxidized cytochrome
c reduced/min/protein (mg) [11].
The activity of cytochrome oxidase (complex IV) was mea-
sured by oxidation of reduced cytochrome c (ferrocytochrome
c) at 550 nm. The reaction cocktail consisted of phosphate
buffer (10 mM, pH 7.4) at room temperature and ferricyanide
(1 mM) which was added to oxidize ferrocytochrome c. The
reaction was initiated by adding mitochondrial suspension
(10~30 μg protein) [62]. The activity of complex IV has been
expressed as nmol reduced cytochrome c oxidized/min/pro-
tein (mg).
Western Blotting Expression of proteins linked with apo-
ptosis and stress (Caspase-3, Bax, Bcl2, P-38, p-JNK, Ap-
1) in the frontal cortex, hippocampus, and corpus striatum
was assayed following the method of Yadav et al. [64].
The prepared samples (30 μg protein/lane) were fractioned
by 12% SDS-PAGE. Proteins were electrically transferred
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-Bax, anti-Bcl2, cas-
pase-3, anti-P-38, anti-p-JNK, and anti-Ap-1 (rabbit) (Cell
Signaling Technology, USA, 1:1000) for 24 h at 4 °C.
Anti-β actin was used as a loading control. Blots were
subsequently incubated with horseradish peroxidase-linked
secondary antibody (anti-rabbit IgG 1:2000) for 60 min at
room temperature. After the incubation, blots were washed
and developed using an Immobilon western chemilumines-
cent HRP substrate (Millipore, USA) following the recom-
mended procedure. The densitometry for protein-specific
bands was carried out using Gel Documentation System
(Alpha Innotech) with the help of Alpha Ease FC Stand
Alone V.4.0 software.
Chandravanshi et al.
Protein Estimation Protein content in the samples was mea-
sured following the procedure as described by Lowry et al.
[38] using bovine serum albumin as a reference standard.
Statistical Analysis Results of the experiments were expressed
as mean ± standard error, using a statistical software package
(GraphPad Prism). The differences between the mean values
were evaluated by one-way ANOVA followed by Newman–
Keuls test for post-hoc comparisons. p values less than 0.05
have been considered significant.
Results
Effect on the Extent of Oxidative Stress and ROS
Generation
Effect on Lipid Peroxidation, Protein Carbonyl, and GSH
Levels in Brain Regions Arsenic-induced oxidative stress was
evident by the increased thiobarbituric acid reactive sub-
stances (TBARS) and protein carbonyl contents measured in
the frontal cortex, hippocampus, and corpus striatum of devel-
oping rats. Perinatal exposure to arsenic (2.0 or 4.0 mg/kg
body weight) significantly increased the levels of TBARS in
the frontal cortex (43%, p < 0.05; 74%, p < 0.01), hippocam-
pus (34%, p < 0.05; 66%, p < 0.05), and corpus striatum
(36%, p < 0.01; 62%, p < 0.001) of arsenic-exposed rats on
PD22 as compared to controls (Fig. 1a). The TBARS level
remained significantly increased in the frontal cortex (20%,
p > 0.05; 34%, p < 0.05), hippocampus (19%, p > 0.05; 22%,
p < 0.05), and corpus striatum (18%, p > 0.05; 38%, p < 0.01)
of rats exposed to arsenic at a higher dose even on PD45 as
compared to respective controls; a general trend of recovery
was however seen (Fig. 1a).
An increase in protein carbonyl levels in the frontal cortex
(30%, p < 0.05; 63%, p < 0.001), hippocampus (31%,
p < 0.05; 54%, p < 0.01), and corpus striatum (54%,
p < 0.01; 77%, p < 0.01) was observed in rats perinatally ex-
posed to arsenic (2.0 or 4.0 mg/kg body weight) on PD22 as
compared to controls (Fig. 1b). Protein carbonyl levels
remained significantly increased in the frontal cortex (18%,
p > 0.05; 38%, p < 0.05) and hippocampus (17%, p > 0.05;
25%, p > 0.05) in rats exposed to arsenic at a higher dose
(4.0 mg/kg body weight) on PD45. However, protein carbonyl
levels remained significantly higher in the corpus striatum
(21%, p < 0.05; 34%, p < 0.01) of arsenic-exposed rats at both
doses, even on PD45 as compared to respective controls (Fig.
1b). In the corpus striatum, recovery of protein carbonyl con-
tent was seen less than other regions, after the withdrawal of
arsenic exposure.
A decrease in the GSH levels in the frontal cortex (33%,
p < 0.01; 55%, p < 0.001), corpus striatum (28%, p < 0.01;
44%, p < 0.001), and hippocampus (27%, p < 0.01; 39%,
Developmental Neurotoxicity of Arsenic: Involvement of Oxidative Stress and Mitochondrial Functions

Fig. 1 Effects of perinatal A

12 CONT As I As II
exposure (GD6 to PD21) to ***

μ MDA/hour/mg protein
arsenic (2 or 4 mg/kg body 10 **
**
weight/day) on MDA (a), protein * **
8 * *
carbonyl contents (b), and the *
levels of GSH (c) were assessed 6 *
in the frontal cortex,
4
hippocampus, and corpus
striatum of rats on PD22 and 2
23 days after withdrawal of
0
exposure on PD45. Data are PD22 PD45 PD22 PD45 PD22 PD45
reported as means ± SEM for five Frontal Cortex Hippocampus Corpus Striatum
animals in each group. B
80
Statistically significant **

Unit/min/mg protein
differences from control group, **
60
*p < 0.05, **p < 0.01, and
***p < 0.001, were determined **
*
by one-way-ANOVA with 40 *** **
* * *
Newman–Keuls test
20

0
PD22 PD45 PD22 PD45 PD22 PD45
Frontal Cortex Hippocampus Corpus Striatum
C
25
n mole GSH oxidized/

20
min/mg protein

*
15 ** *
* * * * ***
** *
10 **

0
PD22 PD45 PD22 PD45 PD22 PD45
Frontal Cortex Hippocampus Corpus Striatum

p < 0.001) was evident in rats perinatally (GD6-PD21) exposed
to arsenic (2.0 or 4.0 mg/kg body weight) on PD22 as com-
pared to controls (Fig. 1c). A trend of recovery in decreased
levels of GSH was observed in the frontal cortex and hippo-
campus of rats exposed to arsenic at a low dose as compared to
those exposed at a higher dose on withdrawal of exposure on
PD45 (Fig. 1c). While a trend of recovery was also found in the
decreased levels of GSH in the corpus striatum (28%, p < 0.05;
44%, p < 0.05) of arsenic-exposed rats, changes remained in
rats exposed to arsenic at both doses of arsenic, on PD45 in
comparison to respective controls (Fig. 1c).
Effect on the Activity of SOD, CAT, and Glutathione
Peroxidase in Brain Regions Activity of SOD was found to
be decreased significantly in the frontal cortex (35%, p < 0.01;
58%, p < 0.001), hippocampus (24%, p < 0.05; 48%,
p < 0.001), and corpus striatum (31%, p < 0.01; 53%,
p < 0.001) of rats perinatally exposed to arsenic on PD22 as
compared to controls (Fig. 2a). The activity of SOD in the
frontal cortex and hippocampus of arsenic-exposed rats exhib-
ited a trend of recovery on withdrawal of arsenic exposure on
PD45 as compared to controls (Fig. 2a). The decrease in the
activity of SOD was found to persist in the corpus striatum
(21%, p < 0.05; 35%, p < 0.01) in arsenic-exposed rats on
PD45 as compared to controls (Fig. 2a).
Perinatal exposure to arsenic (2.0 or 4.0 mg/kg body
weight) from GD6 to PD21 resulted to a decrease in the ac-
tivity of CAT in the frontal cortex (29%, p < 0.01; 46%,
p < 0.001), hippocampus (23%, p < 0.05; 46%, p < 0.01),
and corpus striatum (25%, p < 0.01; 51%, p < 0.001) on
PD22 as compared to controls (Fig. 2b). The CAT activity in
the selected brain regions was remained significantly de-
creased at a higher dose of arsenic (4.0 mg/kg body weight)
on PD45 as compared to respective controls (Fig. 2b).
Perinatal exposure to arsenic (2.0 or 4.0 mg/kg body
weight) resulted in decreased activity of glutathione peroxi-
dase in the frontal cortex (24%, p < 0.05; 36%, p < 0.01), hip-
pocampus (24%, p < 0.05; 39%, p < 0.01), and corpus stria-
tum (31%, p < 0.01; 42%, p < 0.001) of rats on PD22 as com-
pared to controls (Fig. 2c). The decrease in the activity of
glutathione peroxidase was found to persist in the frontal cor-
tex (19%, p < 0.05; 26%, p < 0.05) and corpus striatum (23%,
p < 0.05; 33%, p < 0.05) of rats exposed to arsenic at both the
doses on withdrawal of arsenic exposure as compared to re-
spective controls on PD45 (Fig. 2c). No significant change in
the activity of glutathione peroxidase in the hippocampus
 Chandravanshi et al.

Fig. 2 Effects of perinatal CONT As I As II

exposure (GD6 to PD21) to A
2.5
arsenic (2 or 4 mg/kg body

Unit/min/mg protein
weight/day) on activities of SOD 2
(a), CAT (b), and glutathione *
*
peroxidase (c) were assessed in 1.5 ** *
the frontal cortex, hippocampus, ***
1 ***
and corpus striatum of rats on **
PD22 and 23 days after ***
withdrawal of exposure on PD45. 0.5
Data are reported as means ±
0
SEM for five animals in each PD22 PD45 PD22 PD45 PD22 PD45
group. Statistically significant Frontal Cortex Hippocampus Corpus Striatum
differences from control group, B 9
*p < 0.05, **p < 0.01, and 8

μmole/min/mg protein
***p < 0.001 were determined by 7
one-way ANOVA with Newman– *
6
Keuls test
5 * ** *
**
4 ** *
***
3 ***
2
1
0
PD22 PD45 PD22 PD45 PD22 PD45
Frontal Cortex Hippocampus Corpus Striatum

C
350
GSH μgm/gm tissue weight

300
250
*
200 * ** * *
** **
***
150 *** ***

100
50
0
PD22 PD45 PD22 PD45 PD22 PD45
Frontal Cortex Hippocampus Corpus Striatum

(12%, p > 0.05; 21%, p < 0.05) was observed in rats exposed
to arsenic at a low dose while the activity of glutathione per-
oxidase remained significantly decreased in those exposed to
arsenic at a higher dose as compared to respective controls on
PD45 (Fig. 2c).
Effect on the ROS Generation in Brain Regions ROS genera-
tion was found to be markedly affected by both doses of arse-
nic on PD22. ROS generation was increased by the arsenic
exposure in the frontal cortex (48%, p < 0.05; 86%,
p < 0.001), hippocampus (48%, p < 0.05; 68%, p < 0.01),
and corpus striatum (54%, p < 0.05; 90%, p < 0.01) of devel-
oping rats on PD22 as compared to controls. The increase in
the ROS generation was more intense in corpus striatum than
in frontal cortex and hippocampus. Although a trend of recov-
ery in the ROS generation was observed on withdrawal of
arsenic exposure, the increase in the ROS generation was
found to persist in the frontal cortex (30%, p < 0.05; 36%,
p < 0.05), hippocampus (19%, p < 0.05; 29%, p < 0.05), and
corpus striatum (32%, p < 0.05; 43%, p < 0.05) on PD45 as
compared to controls (Fig. 3a).
Effect on the Mitochondrial Membrane Potential (ΔΨm) in
Brain Regions Exposure to arsenic for perinatal period
(GD6–PD21) had a marked effect on MMP in selected brain
regions. Arsenic dose dependently increased the number of
depolarized cells in the frontal cortex (7.9%, p < 0.05;
12.1%, p < 0.001), hippocampus (8.66%, p < 0.01; 11.53%,
p < 0.001), and corpus striatum (11.57%, p < 0.01; 16.27%,
p < 0.001), indicating a decrease in the MMP on PD22 as
compared to controls. Percentage of depolarize cells was
found to persist in the frontal cortex (7.2%, p < 0.05; 7.8%,
p < 0.01), hippocampus (5.7%, p < 0.05; 6.83%, p < 0.01),
and corpus striatum (9.36%, p < 0.05; 11.67%, p < 0.001) of
rats exposed to arsenic as compared to the respective controls
after the 23 days after cessation of exposure (Fig. 3b).
Effect on the Activity of Mitochondrial Complexes in Brain
Regions As there was a loss of MMP following arsenic
administration in rats, it was of curiosity, interest to ex-
plore if this would have an impact on the electron trans-
port chain. Activities of mitochondrial complexes were
found to be altered by arsenic during the perinatal period
Developmental Neurotoxicity of Arsenic: Involvement of Oxidative Stress and Mitochondrial Functions

Fig. 3 Effects of perinatal A

exposure (GD6 to PD21) to 8 CONT As I As II

DCF fluorescence/μg protein

**
arsenic (2 or 4 mg/kg body 7 ***
weight/day) on ROS levels (a) *
6 **
and MMP (b) were assessed in
* *
the frontal cortex, hippocampus, 5 *
*
*
and corpus striatum of rats on * *
PD22 and 23 days after 4
withdrawal of exposure on PD45. 3
In the upper panel figure of b,
representative dot plot of an 2
independent experiment in each 1
group is presented. Depolarize
0
cells (%) in each group are PD22 PD45 PD22 PD45 PD22 PD45
calculated and presented in the Frontal Cortex Hippocampus Corpus Striatum
lower panel of b. Data are
reported as means ± SEM for five B Frontal Cortex Hippocampus Corpus Striatum
animals in each group.
Statistically significant CONT
5 2.9 4.8 3.3
differences from the control 5.2 3.2

group, *p < 0.05, **p < 0.01, and

***p < 0.001 were determined by As I 5.6 11.8 7.2
7.3 6.7 8.4
one-way ANOVA with Newman–
Keuls test
As II 12.4 7 12.8 6.6 18.5 12.2

PD22 PD45 PD22 PD45 PD22 PD45

18 ***

15
Depolarize cells

*** *** **
12 ***
*
9 **
* **
* **
6 *

0
PD22 PD45 PD22 PD45 PD22 PD45
Frontal Cortex Hippocampus Corpus Striatum

in selected brain regions of rats at both doses. The activity
of NADH (complex I) was found to be decreased signif-
icantly in the frontal cortex (18%, p > 0.05; 29%,
p < 0.05), hippocampus (20%, p > 0.05; 31%, p < 0.05),
and corpus striatum (22%, p < 0.05; 35%, p < 0.001) of
rats perinatally arsenic exposed rats to 4 mg/kg arsenic
on PD22 as compared to controls (Fig. 4a). A significant
loss in the activity of succinate-cytochrome c reductase
(complexes II–III) in the frontal cortex (29%, p < 0.05;
45%, p < 0.01), hippocampus (27%, p < 0.05; 49%,
p < 0.01), and corpus striatum (34%, p < 0.05; 66%,
p < 0.001) was also evident in rats perinatally exposed to
arsenic (2.0 mg/kg or 4.0 mg/kg body weight) in compar-
ison to those in the control group (Fig. 4b). Arsenic-
exposed rats also exhibited a decrease in the activity of
cytochrome oxidase (complex IV) in the frontal cortex
(17%, p > 0.05; 32%, p < 0.05), hippocampus (24%,
p < 0.05; 33%, p < 0.05), and corpus striatum (23%,
p < 0.05; 33%, p < 0.01) on PD22 in comparison to con-
trols (Fig. 4c). While a trend of recovery in the activity of
complex I and complex IV was observed both in frontal
cortex and hippocampus, the activity of both complexes
remained decreased in the corpus striatum of rats exposed
to arsenic at a higher dose on withdrawal of exposure.
Decrease in the activity of complexes II–III remained in
the frontal cortex (14%, p > 0.05; 31%, p < 0.05) and hip-
pocampus (16%, p > 0.05; 27%, p < 0.05) of rats exposed
to arsenic at a higher dose on the withdrawal of exposure
on PD45 as compared to controls. The decrease in the
activity of complexes II–III in the corpus striatum (22%,
p < 0.05; 35%, p < 0.05) was found to persist in rats ex-
posed to arsenic at both doses on PD45 in comparison to
controls. Maximum recovery was observed in complexes I
and IV in selected brain regions of arsenic-exposed rats,
while complexes II–III showed less recovery after the
withdrawal of arsenic exposure.
 Chandravanshi et al.

Fig. 4 Effects of perinatal A

exposure (GD6 to PD21) to 500 CONT As I As II

n mole NADH oxidized/

arsenic (2 or 4 mg/kg body
400

min/mg/protein
weight/day) on activity of
mitochondrial complexes *
300 *
(complex I (a), complexes II and * **
*
III (b), and complex IV (c)) were 200
assessed in the frontal cortex,
hippocampus, and corpus 100
striatum of rats on PD22 and
23 days after withdrawal of 0
PD22 PD45 PD22 PD45 PD22 PD45
exposure on PD45. Data are Frontal Cortex Hippocampus Corpus Striatum
reported as means ± SEM for five
animals in each group. B
100

Reduced/ min/mg/protein
Statistically significant

n mole oxidized Cyt C

differences from control group, 80
*p < 0.05, **p < 0.01, and *
60 *
***p < 0.001, were determined * * * *
by one-way ANOVA with ** *
40 ** ***
Newman–Keuls test
20

0
PD22 PD45 PD22 PD45 PD22 PD45
Frontal Cortex Hippocampus Corpus Striatum
C
450
400
350
300
*
250 * ** *
200
150
100 *
*
50
0
PD22 PD45 PD22 PD45 PD22 PD45
Frontal Cortex Hippocampus Corpus Striatum

Effect on the Expression of Key Proteins Associated with
Apoptosis in Brain Regions As shown in Fig. 5a, the ratio
of Bcl-2/Bax was markedly increased in the frontal cortex
(1.90-fold, p < 0.01; 2.86-fold, p < 0.001), hippocampus
(1.52-fold, p < 0.01; 1.99-fold, p < 0.001), and corpus stri-
atum (2.20-fold, p < 0.01; 3.26-fold, p < 0.01) of rats fol-
lowing perinatal exposure to arsenic (2.0 mg/kg or
4.0 mg/kg body weight) compared to the control. A trend
of recovery was, however, seen in the Bcl-2/Bax ratio after
withdrawal of exposure, but a significant increase in ratio
in the frontal cortex, hippocampus, and corpus striatum of
rats or animals exposed to arsenic at both doses (Fig. 5a).
The intensity of change in the ratio of Bcl-2/Bax was more
pronounced in the corpus striatum than in the other re-
gions, even 23 days after withdrawal of exposure.
The results showed that the arsenic administration signifi-
cantly increased the expression of apoptotic hallmark protein
caspase-3 in the frontal cortex (1.45-fold, p < 0.01; 1.61-fold,
p < 0.001), hippocampus (1.48-fold, p < 0.01; 1.82-fold,
p < 0.001), and corpus striatum (1.48-fold, p < 0.01; 1.82-fold,
p < 0.001) of rats perinatally exposed to arsenic (2.0 mg/kg or
4.0 mg/kg body weight) on PD22 in comparison to controls
(Fig. 5b). While a trend of recovery was observed in the ex-
pression of caspase-3 on withdrawal of arsenic exposure.
However, expression of caspase-3 remained increased in the
frontal cortex, hippocampus, and corpus striatum even 23 days
after cessation of exposure in comparison to respective con-
trols (Fig. 5b).
Protein expression of Phospo p-38 was found to be signifi-
cantly increased in the frontal cortex (1.24-fold, p < 0.05; 1.48-
fold, p < 0.001), hippocampus (1.27-fold, p < 0.01; 1.32-fold,
p < 0.01), and corpus striatum (1.98-fold, p < 0.001; 2.41-fold,
p < 0.001) of rats on PD22 in comparison to controls (Fig. 6a).
Expression of Phospo p-38 was found to recover at a low dose
of arsenic in the frontal cortex and hippocampus of rats, but the
change remained persistent in the corpus striatum in arsenic-
exposed rats on withdrawal of exposure on PD45 as compared
to respective controls (Fig. 6a).
Developmental Neurotoxicity of Arsenic: Involvement of Oxidative Stress and Mitochondrial Functions

CONT As I As II
A Hippocampus Corpus Striatum
Frontal Cortex
Bcl2 20 kda
Bax 26 kda
β actin 42 kda
CONT As I As II CONT As I As II CONT As I As II CONT As I As II CONT As I As II CONT As I As II
PD22 PD45 PD22 PD45 PD22 PD45

3.6 **

3 ***

2.4
Fold change

** **
** ***
1.8 ** *
** **
1.2

0.6

0
PD22 PD45 PD22 PD45 PD22 PD45
Frontal Cortex Hippocampus Corpus Striatum
B
Frontal Cortex Hippocampus Corpus Striatum
Caspase-3 35kda
β actin 42 kda
CONT As I As II CONT As I As II CONT As I As II CONT As I As II CONT As I As II CONT As I As II
PD22 PD45 PD22 PD45 PD22 PD45
2
***
*** ***
1.6 *** **
***
** **
*
Fold change

1.2 * *

0.8

0.4

0
PD22 PD45 PD22 PD45 PD22 PD45
Frontal Cortex Hippocampus Corpus Striatum
Fig. 5 Effects of perinatal exposure (GD6 to PD21) to arsenic (2 or densitometric analysis of bands in the control and arsenic-exposed
4 mg/kg body weight/day) on the expression of Bcl2, Bax ratio (a), and groups. Data are reported as means ± SEM for three animals in each
caspase-3 (b) were assessed in frontal cortex, hippocampus, and corpus group. Statistically significant differences from the control group,
striatum of rats on PD22 and 23 days after withdrawal of exposure on *p < 0.05, **p < 0.01, and ***p < 0.001, were determined by one-way
PD45. Each panel shows a representative Western blot (top) and ANOVA with Newman–Keuls test

When compared to control, we found that perinatal expo-
sure to arsenic (2.0 mg/kg or 4.0 mg/kg body weight) on PD22
led to significantly enhanced expression of AP-1 in the frontal
cortex (1.47-fold, p < 0.05; 1.71-fold, p < 0.01), hippocampus
(1.48-fold, p < 0.05; 1.57-fold, p < 0.05), and corpus striatum
(1.55-fold, p < 0.01; 1.61-fold, p < 0.01) (Fig. 6b). A trend of
recovery was recorded 23 days after withdrawal of exposure,
and no significant variations occurred in the frontal cortex and
hippocampus. Interestingly, overexpression of AP-1 was
remained significantly increased in the corpus striatum of rats
exposed to arsenic at both doses after the withdrawal of expo-
sure as compared to controls (Fig. 6b).
Expression of JNK1 and JNK2 was significantly
higher in the corpus striatum of rats exposed to arsenic
 Chandravanshi et al.

A CONT As I As II

Frontal Cortex Hippocampus Corpus Striatum

p-p38 40 kda
β actin 42 kda
CONT As I As II CONT As I As II CONT As I As II CONT As I As II CONT As I As II CONT As I As II
PD22 PD45 PD22 PD45 PD22 PD45
2.8
2.4 ***
Fold change

2 ***

1.6 ***
* ** ** ** ** **
1.2 *

0.8
0.4
0
PD22 PD45 PD22 PD45 PD22 PD45
Frontal Cortex Hippocampus Corpus Striatum

B
Frontal Cortex Hippocampus Corpus Striatum
Ap-1 39 kda
β actin 42 kda
CONT As I As II CONT As I As II CONT As I As II CONT As I As II CONT As I As II CONT As I As II
PD22 PD45 PD22 PD45 PD22 PD45
2
**
1.6 * ** **
* * *
*
1.2
Fold change

0.8

0.4

0
PD22 PD45 PD22 PD45 PD22 PD45
Frontal Cortex Hippocampus Corpus Striatum

Fig. 6 Effects of perinatal exposure (GD6 to PD21) to arsenic (2 or
4 mg/kg body weight/day) on the expression of p-p38 (a) and AP-1 (b)
were assessed in the frontal cortex, hippocampus, and corpus striatum of
rats on PD22 and 23 days after withdrawal of exposure on PD45. Each
panel shows a representative Western blot (top) and densitometric
analysis of bands in the control and arsenic-exposed groups. Data are
reported as means ± SEM for three animals in each group. Statistically
significant differences from the control group, *p < 0.05, **p < 0.01, and
***p < 0.001, were determined by one-way ANOVA with Newman–
Keuls test

on PD22 as compared to controls (Fig. 7). Increase in Discussion

the expression of JNK1 and JNK2 persisted in rats ex-
posed to arsenic at a higher dose (4.0 mg/kg body
weight), while recovered in these exposed at a low dose
(2.0 mg/kg body weight) on withdrawal of exposure on
PD45. No significant changes were observed in the ex-
pression of JNK-1 and JNK-2 proteins in frontal cortex
and hippocampus of arsenic (2.0 mg/kg or 4.0 mg/kg
body weight)-exposed rats as compared to controls on
PD22 and PD45 (Fig. 7).
Developmental neurotoxicity of arsenic in human has been
defined as elevated risks of cognitive dysfunction, learning,
and memory impairment in children [33]. Prenatal arsenic
exposure has been shown to target the different brain regions
of rats [2, 40]. At the molecular level, arsenic is known to
cross the blood brain barrier and interfere with the neuronal
functions in the brain [28]. Previous studies also demonstrated
that early life exposure to arsenic impairs cholinergic
Developmental Neurotoxicity of Arsenic: Involvement of Oxidative Stress and Mitochondrial Functions
CONT
Frontal Cortex Hippocampus
pJNK-2
pJNK-1
β actin
CONT As I As II CONT As I As II CONT As I As II
2.5 PD22 PD45 PD22
2
Fold change
1.5
0.5
0 JNK-2 JNK-1 JNK-2 JNK-1 JNK-2 JNK-1
PD22 PD45 PD22
Frontal Cortex Hippocampus
Fig. 7 Effects of perinatal exposure (GD6 to PD21) to arsenic (2 or
4 mg/kg body weight/day) on the expression of p-JNK was assessed in
the frontal cortex, hippocampus, and corpus striatum of rats on PD22 and
23 days after withdrawal of exposure on PD45. Each panel shows a
representative Western blot (top) and densitometric analysis of bands in
dopaminergic dysfunctions in the frontal cortex, hippocam-
pus, and corpus striatum of rats at low doses of arsenic [8,
9]. Therefore, in order to better understand the developmental
neurotoxicity of arsenic and to confirm the toxicity exerted by
a prenatal exposure to different brain regions of rats, we eval-
uated the effects of arsenic on ROS generation, oxidant, and
antioxidant molecules, and mitochondrial functions in brain
regions of arsenic-exposed rats with normal.
One of the most important mechanisms involved in the neu-
rotoxicity of arsenic is their ability to cause oxidative stress and
mitochondrial dysfunction [46]. Enhanced oxidative stress is
found in cognitive impairment, along with increased and de-
creased levels of oxidant and antioxidant markers, respectively
[17, 18, 29]. High levels of polyunsaturated fatty acids, high
metabolic rate, lower activity of brain antioxidant (CAT, SOD,
and glutathione peroxidase) and reduced capacity of cellular
regeneration make the brain highly susceptible target to dam-
age by reactive oxygen species [43]. Neurodegeneration, one of
the neurotoxic effects of arsenic, was induced by increase in the
generation of ROS in the brain [13, 39]. The present study
indicated that prenatal exposure to 2 or 4 mg/kg arsenic results
in oxidative stress to induce an increment in ROS. Arsenic has
high affinity to glutathione; it acts as a scavenger of reactive
species, which might have implications for the maintenance of
thiol-disulfide balance. Thus, reduced levels of GSH are
As I As II
Corpus Striatum
54 kda
46 kda
42 kda
CONT As I As II CONT As I As II CONT As I As II
PD45 PD22 PD45
***
**
**
*
* *
JNK-2 JNK-1 JNK-2 JNK-1 JNK-2 JNK-1
PD45 PD22 PD45
Corpus Striatum
the control and arsenic-exposed groups. Data are reported as means ±
SEM for three animals in each group. Statistically significant
differences from the control group, *p < 0.05, **p < 0.01, and
***p < 0.001 were determined by one-way ANOVA with Newman–
Keuls test
correlated to enhanced oxidative stress in developing rats after
exposure to 2 or 4 mg/kg arsenic during the perinatal period.
We then evaluated different biomarkers of oxidative stress in
the frontal cortex, hippocampus, and corpus striatum of
arsenic-exposed rats. We observed that GSH levels were de-
creased in the frontal cortex, hippocampus, and corpus striatum
of rats treated with arsenic in comparison with the control
group. In order to further evaluate the activity of antioxidant
enzymes, we examined the activities of SOD, CAT, and gluta-
thione peroxidase. We examined that low levels of arsenic de-
creased the activities of CAT, SOD, and glutathione peroxidase
in selected brain regions of rats. Hence, a reduced level of GSH
combined with a decreased glutathione peroxidase activity may
lead to the accumulation of oxidatively damaged molecules and
enhances the vulnerability of the brain to oxidative insult by
arsenic exposure [30, 54]. A decrease in SOD enzymes may
lead to the generation of superoxide radical and hydrogen per-
oxide in the brain. Hydrogen peroxide is decomposed biolog-
ically by the CAT enzyme whose activity also showed a signif-
icant fall following perinatal arsenic exposure in brain regions.
Accumulation of superoxide and peroxide radicals leads to
peroxidation of lipids in membrane resulting in increased lipid
peroxidation and protein carbonyl contents as observed in the
present study. Based on the observation that accumulation of
TBARS products and protein carbonyl contents was observed

in brain regions of exposed rats, it indicates the loss of mem-
brane integrity and function. Increased oxidant and decreased
antioxidant molecules were observed in arsenic-exposed devel-
oping rats as previously shown in other reports [24, 53]. On the
other hand, there is evidence suggesting neurotoxic effects of
arsenic, which accumulate in the frontal cortex, hippocampus,
and corpus striatum of developing rats [8, 9]. In this regard,
arsenic has a high affinity to glutathione that can react with and
induce the production of free radicals [21]. In the present study,
we further studied the role of mitochondrial complexes and
mitochondrial membrane potential in arsenic-induced oxidative
stress and its mechanisms. Accumulation of ROS may trigger
the inhibition of the activity of mitochondrial complexes by
oxidative phosphorylation. In relation to this, we observed that
arsenic decreased the activities of complexes I, II–III, and IV in
the frontal cortex, hippocampus, and corpus striatum of rats.
Our previous study also demonstrated that arsenic impairs the
activities of mitochondrial complexes in the different brain re-
gions of early life exposed rats, supporting the observation that
affected exposed rats present increase levels of ROS [9].
Inhibition of the electron transport complex activity leads
to abnormal accumulation of ROS. Accumulation of ROS is
responsible for the lipid bi-layer damage [52] and caused mi-
tochondrial swelling and mitochondrial membrane potential
(MMP) fall down [58], which suggests that mitochondrial
function was inhibited by the increase level of ROS. In the
present study, fluctuation in the MMP and electron transport
complex activity are considerable to be a distinctive triggers,
underlying the pathogenesis of arsenic-induced mitochondrial
dysfunctions in developing rats.
Further, ample evidence suggested that oxidative stress and
mitochondrial dysfunctions may cause neurodegeneration [7,
44]. Taken together, ROS and the resulting imbalance between
the antioxidant and oxidant molecules can be part of the signal
transduction pathway during apoptosis. It is now established
that various pro-apoptotic factors are released into the cytosol
during oxidative stress and mitochondrial dysfunctions.
Therefore, it seemed likely that arsenic would induce apoptosis
through ROS generation, oxidative stress, and mitochondrial
dysfunctions. Apoptosis has shown to play a major role in the
pathogenesis of various neurological disorders [26]. The bal-
ance between anti-apoptotic and apoptotic proteins determines
the fate of the cells either to undergo apoptosis or to survive in
pathophysiology. Our data presented here reveal that arsenic
enhanced the Bcl2/Bax ratio in different brain regions of rats
in a dose-dependent manner. Increased ratio of Bcl2/Bax and
oxidative stress leads to the change in MMP of brain tissues of
rats; therefore, it could enhance apoptosis. Increased Bcl2/Bax
ratio stimulates apoptotic signaling molecules along with the
variety of caspases and finally caspase-3, which is the main
protein of mitochondrial pathway of apoptosis [45]. We ob-
served that expression of caspase-3 was increased in the frontal
cortex, hippocampus, and corpus striatum following arsenic
Chandravanshi et al.
exposure to different doses. Increased ratio of Bcl2/Bax pro-
teins and increased levels of caspase-3 are reported in various
neurodegenerative conditions [49, 55].
MAPK signaling pathways are activated in response to
stress. Thus, JNK and p38 MAPK signaling pathways are
stimulated by ROS [61]. In this study, we observed that
ROS production seems to induce the activation of JNK or
p38 MAPK pathways. JNK and p38 have classically been
associated with apoptosis. In the present study, exposure to
arsenic at low doses did not affect the expression of p-JNK1
and p-JNK2 both in the frontal cortex and hippocampus of
developing rats. However, expression of p-JNK1 and p-JNK2
was found to increase in corpus striatum of arsenic-exposed
rats as compared to controls. Further, we observed a marked
increased the protein expression of phosphorylated p38
MAPK in different brain regions of arsenic-exposed develop-
ing rats. AP-1 is a nuclear transcriptional factor, which is also
involved in apoptosis. Expression of AP-1 can be modulated
by the MAPK signaling pathway [56]. Increased expression of
AP-1 observed in different brain regions could be easily asso-
ciated with enhanced apoptosis as a result of arsenic exposure.
Up-regulation of p38 and pJNK and AP-1 in developing brain
suggest increased neuronal apoptosis, and these observations
are consistent with our earlier report demonstrating similar
changes in the frontal cortex and hippocampus [8]. These
results suggest that the arsenic-induced activation of AP-1
via a p38 MAPK that includes JNK is involved in the induc-
tion of apoptosis in brain regions of developing rats.
In summary, our study shows that arsenic exposure im-
paired the mitochondrial functions and enhanced oxidative
stress and apoptosis in selected brain regions of perinatal ex-
posed rats. Regarding the response of different brain region
tissues to the neurotoxic effects of arsenic, it is noteworthy
that the corpus striatum was the most vulnerable region to
disruption of redox homeostasis, mitochondrial dysfunctions,
and neuronal apoptosis induced by arsenic. As regards to the
corpus striatum, we cannot explain at present the reasons by
which the corpus striatum presents more susceptibility to ar-
senic in developing rats. In conclusion, arsenic affects the
central nervous system during the perinatal period of brain
development. Consequently, it may be suggested that proper
testing of water for arsenic is important for women of child-
bearing age and pregnant women to avoid arsenic exposure
during critical periods of brain development. Further, the con-
tinuous exposure to arsenic during perinatal periods of brain
development may be an important risk factor for increased
vulnerability to neurodegenerative diseases.
Acknowledgements The authors thank the Director, CSIR—Indian
Institute of Toxicology Research (CSIR-IITR), Lucknow, for his support
and the keen interest in the present study. Financial support by the
University Grants Commission, New Delhi, for carrying out the study
is gratefully acknowledged.
Developmental Neurotoxicity of Arsenic: Involvement of Oxidative Stress and Mitochondrial Functions
Compliance with Ethical Standards
Conflict of Interest None declared.
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