Article

Human and Experimental Toxicology

2018, Vol. 37(3) 321–328
Effects of cadmium on Bcl-2/Bax ª The Author(s) 2017
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expression ratio in rat cortex DOI: 10.1177/0960327117703687
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brain and hippocampus

S Mahdavi1, P Khodarahmi1 and NH Roodbari2

Abstract
To investigate the underlying mechanism of neurotoxicity of cadmium, we examined the effects of intraper-
itoneal injection of cadmium on messenger RNA (mRNA) expression of Bcl-2 (B-cell lymphoma 2) and Bax
(Bcl2-associated x) genes and caspase-3/7 activation in rat hippocampus and frontal cortex. Twenty-eight male
Wistar rats weighing 200–250 g were randomly divided into four groups. Control group received saline and
three other groups received cadmium at doses of 1, 2 and 4 mg/kg (body weight) for 15 successive days. One
day after the last injection, the hippocampus and frontal cortex were dissected and removed and then the
expression of Bcl-2 and Bax genes was evaluated using real-time polymerase chain reaction and apoptotic
studies was done using caspase-3/7 activation assay. Cadmium reduced the mRNA level of Bcl-2 in the control
group at doses of 1 (p < 0.01), 2 and 4 mg/kg (p < 0.001) in rat hippocampus and cortex cells. The mRNA level
of Bax increased significantly compared to the control group at the doses of 1 (p < 0.05), 2 and 4 mg/kg (p <
0.001) in rat hippocampus. The mRNA level of Bax was increased significantly compared to the control group
at the doses of 2 and 4 mg/kg (p < 0.001) in rat cortex cells. Cadmium increased caspase-3/7 activity at doses of
1, 2 and 4 mg/kg in rat hippocampus. Caspase-3/7 activity was increased significantly at dose of 4 mg/kg in rat
cortex. This decreased Bcl-2/Bax mRNA ratio induces cell apoptosis. Apoptotic effect of cadmium may be
through the mitochondrial pathway by the activation of caspase-3/7.

Keywords
Cadmium, Bcl-2, Bax, caspase-3/7, cortex, hippocampus

Introduction the brain’s microvasculature and leads to BBB dys-

Cadmium is one of the most toxic heavy metals that is
distributed in our environment.1 The main sources of
cadmium intake are food, water and smoking cigar-
ette. Cadmium can reach the brain by the olfactory
route.2–5 Cadmium has been linked to health problems
including osteoporosis, damage to liver and kidneys,
increased mortality and cardiovascular system dys-
function,1,6 and it is classified as a group I human
carcinogen by the International Agency for Research
on Cancer.7
Several studies have shown cadmium toxicity in
humans and animals. Furthermore, the different tis-
sues increased the antioxidant molecules to prevent
the oxidative damage caused by cadmium.8 Blood–
brain barrier (BBB) has high levels of antioxidant
enzymes that protect from oxidative stress. Exposure
to cadmium has been linked to antioxidant defenses in
function, resulting in more metal entering the brain.9
Cadmium can directly damage the choroids plexus
and increase BBB permeability in rats,10 leading to
brain intracellular accumulation, cellular dysfunction
and cerebral oedema.11 Khan and Parvez showed that
the highly deleterious capacity of cadmium to cross
the BBB induces neurotoxicity.12
1
Department of Biology, Parand Branch, Islamic Azad University,
Parand, Iran
2
Department of Biology, Sciences and Research Branch, Islamic
Azad University, Tehran, Iran
Corresponding author:
P Khodarahmi, Department of Biology, Parand Branch, Islamic
Azad University Parand Branch, Parand 37613-96361, Iran.
Email: khodarahmiparvin@yahoo.com
322
Previous studies have shown that the brain is a
target for cadmium,13,14 wherein cadmium can induce
neurotoxicity,15 changes in the neurochemistry of the
brain16 and genotoxicity in endothelial cells.17 Amara
et al. showed that due to cadmium exposure the anti-
oxidant enzyme activity reduced in rat hippocampus
and cortex.18
The mechanism of toxic and neurotoxic effects of
cadmium could be due to oxidative stress,19 free radi-
cals generation, mitochondria membrane depolariza-
tion 20 and of reactive oxygen species (ROS)
generation,9 altering genes expression and eventually
inducing apoptosis.21 ROS is a regulator of cell death.
ROS can activate phosphatases and protein kinases,
activate or inactivate transcription factors, altering
gene expression in cells and apoptotic cell death.22
However, the mechanism of action of cadmium-
induced apoptosis is still unclear.
Apoptotic cell death occurs in response to environ-
mental stimuli. Regulation of apoptosis is important
for treatment of cancer, normal growth and also for
development and embryogenesis.23 The studies of
human brain tissue and experimental animal models
have been shown that the Bcl-2 (B-cell lymphoma 2)
family regulates cell death of apoptosis in the nervous
system. Bcl-2 operates as an anti-apoptotic in contrast
to Bax (Bcl2-associated x) that has pro-apoptotic
properties. The Bcl-2 gene is important in regulating
apoptosis that encodes various proteins that play key
roles in regulation of cell apoptosis. Bax gene is
expressed in brain and identified as a pro-apoptotic
that is homologous to Bcl-2. Interactions between Bcl-
2 family members both in the cytosol and in mito-
chondria determine survival or death.24 It has been
known that the ratio of Bax/Bcl-2 determines the
death or survival fate of the cell following an apopto-
tic stimulus.25
Memory dysfunction and learning ability destruc-
tion occur in the hippocampus and cortex due to
neuronal apoptosis.26 In particular, stress induced
neuronal death, possibly through apoptosis,27 and
apoptosis-associated molecules can be involved in
neurodegenerative diseases of the central nervous sys-
tem such as Alzheimer’s disease and Parkinson’s dis-
ease.28 An acute intraperitoneal injection of cadmium
causes behavioural, biochemical and neurochemical
dysfunctions in a dose-dependent manner.29
Thus, in this study, the exposure to cadmium was
examined to assess messenger RNA (mRNA) expres-
sion of Bcl-2 and Bax genes and caspase-3/7 activa-
tion capability in the hippocampus and cortex of rats.
Human and Experimental Toxicology 37(3)
Real-time polymerase chain reaction (PCR), one of
the most sensitive and reliable methods for gene
expression analyses, was implemented to detect
changes in gene expression induced by cadmium in
the hippocampus and cortex of rat. The caspase-3/7
assay system was carried out to assess apoptosis in the
hippocampus and cortex of rat.
Materials and methods
Animals and experimental groups
Experiments were conducted on 28 male Wistar rats
weighing 200–250 g at 8 weeks of age, and the rats
were procured from Veterinary Medicine of Tehran
University (Iran). Animals were housed at 22 C + 3 C
and 12-h/12-h light/dark cycle in the animal house of
Parand Islamic Azad University and fed rodent chow
and water. After 2 weeks of adaptation to the new
environment, they were randomly allocated into four
groups of seven each: a control and three experimental
groups. All experiments conformed to guidelines of the
ethical committee of Parand Islamic Azad University.
Cadmium nitrate administration
Cd(NO3)2 (cadmium nitrate) solution was purchased
from Kimia Pars, Inc. (Merck, Germany). The dose
of cadmium was chosen according to previous
research.30–33 Injections were performed intraperito-
neally with a final volume of 1 cc for each dose. The
control group received saline (vehicle of cadmium)
and the experimental groups were administrated cad-
mium concentrations of 1, 2 and 4 mg/kg body weight
for 15 consecutive days. One day after the last injec-
tion, the rats were deeply anaesthetized with chloro-
form and rapidly decapitalized. The brains were
dissected and placed on an ice-cold cutting board.
After removal of the meninges, hippocampus and
frontal cortex were extracted, snap frozen in liquid
nitrogen and stored at 70 C until further tests.
RNA extraction and complementary DNA
synthesis
Total RNA of hippocampus and cortex tissue were
isolated using the RNX-TM plus (CinnaGen Inc.,
Tehran, Iran). The quantity and purity of the extracted
RNA was determined using a spectrophotometer (Nano-
Drop ND-2000, NanoDrop Technologies; Wilmington,
Delaware, US), and only the extracted RNAs with an
A260/A280 ratio ranging from 1.8 to 2.0 were used for
complementary DNA (cDNA) synthesis. Real-time
Mahdavi et al. 323

Table 1. Primer sequences used for real-time PCR.

Product
Gene Primers size (bp) Annealing ( C)
GAPDH (glyceraldehyde 3-phosphate Forward: 50 - TGC CAC TCA GAA GAC TGT GG -30 119 53
dehydrogenase) Reverse: 50 - GGA TGC AGG GAT GAT GTT CT -30
Bcl-2 (B-cell lymphoma 2) Forward: 50 - GAG TAC CTG AAC CGG CAT CT -30 90 53
Reverse: 50 - GAA ATC AAA CAG AGG TCG CA -30
Bax (Bcl2-associated x) Forward: 50 - TTG CTA CAG GGT TTC ATC CA -30 112 53
Reverse: 50 - GAG TAC CTG AAC CGG CAT CT -30
GAPDH: glyceraldehyde 3-phosphate dehydrogenase; PCR: polymerase chain reaction.

transcription was performed with 1 mg of RNA and a
first strand cDNA synthesis kit (Fermentas; Thermo Sci-
entific, Waltham, MA, USA), according to the manu-
facturer’s instructions.
Real-time quantitative PCR using SYBER Green
Real-time PCR was used to evaluate the quantitative
expression of mRNA for Bcl-2, Bax and glyceralde-
hyde 3-phosphate dehydrogenase (GAPDH) as the
control. The relative quantification was performed by
measuring increased fluorescence light as a result of
SYBR Green bonding using an Illumina real-time PCR
system Illumina, Inc., (San Diego, California, USA).
Amplification was performed in a final volume of 25
ml, which included 1 ml of cDNA, 12.5 ml of SYBR
Green Master Mix (Master mix Green-No Rox, Ampli-
qon, Denmark), 5 mmol of each complementary primer
of volume 0.5 ml and 10.5 ml of deionized water. The
selected primers were designed and underwent an
extensive search using BLAST tool (NCBI,
www.ncbi.nlm.nih.gov/blast). Sequences of Bcl-2, Bax
and GAPDH primers and annealing temperature used
for real-time PCR are shown in Table 1. The amplifi-
cation conditions were optimized as follows: pre-
denaturation 94 C for 5 min followed by 35 cycles
of denaturation at 94 C for 1 min, annealing at 53 C
for 1 min and extension at 72 C for 5 min. Quantitative
gene expression was analyzed by comparative CT
(

CT) method,34 using GAPDH as an internal con-
trol. The relative fold increase (RFI) was calculated
using the following equation: RFI ¼ 2–

CT.
Caspase-3/7 activity assay
The apoptosis assessments in hippocampal and cortex
cells were carried out using the caspase-Glo 3/7 lumi-
nescent assay system (Promega). The cell lysates
were prepared by cytosolic fractionation method.35
Briefly, Caspase-Glo1 3/7 reagent (5 mL) was added
to equal total protein concentrations of control group
cells and experimental groups’ cells of hippocampal
and cortex, followed by gentle mixing for 30 s. Incu-
bation was followed for 30 min. The luminescence
was measured by a luminometer for each sample
(Berthold Detection System, Germany).35
Data analysis
The data collected from the experiment was recorded
and analyzed using SPSS 22 statistical software pack-
age. The results are presented as the mean + standard
deviation. Statistical significance of differences
throughout this study was assessed using one-way
analysis of variance (Tukey’s test). A p value of less
than 0.05 was considered statistically significant.
Results
Melting curve analysis for real-time PCR products
obtained with the specific primer pairs for Bcl-2, Bax
and GAPDH genes in hippocampus and frontal cortex
(Figure 1).
Figure 2 shows the effect of cadmium on the
expression of Bcl-2 gene in the rat hippocampus
and cortex cells. In hippocampus cells, cadmium
decreased mRNA level in Bcl-2 at doses of 1, 2 and
4 mg/kg (body weight) by 0.2, 0.15 and 0.02 times,
respectively, compared with control cells. In cortex
cells, cadmium decreased the mRNA level in Bcl-2 at
doses of 1, 2 and 4 mg/kg (body weight) by 0.27, 0.21
and 0.05 times, respectively, compared with control
cells. As illustrated, cadmium causes a statistically sig-
nificant decrease in Bcl-2 mRNA levels at doses of 1, 2
(p < 0.01) and 4 mg/kg (p < 0.001) compared to the
control group in cortex and hippocampus cells.
Figure 3 shows the effect of cadmium on the
expression of Bax gene in the rat hippocampus and
cortex. In hippocampus cells, cadmium increased the
mRNA level in Bax at doses of 1, 2 and 4 mg/kg
324 Human and Experimental Toxicology 37(3)

Figure 1. Melting curve analysis of real-time PCR for Bcl-2 (B-cell lymphoma 2), Bax (Bcl2-associated x) and GAPDH genes. (a)
Hippocampus and (b) frontal cortex. PCR: polymerase chain reaction; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Bcl-2 gene Hippocapus Cortex

1.5 Bax gene Hippocampus Cortex
RFI of Bcl-2 gene

120
*** ***
RFI of Bax gene

1 100
80
0.5 ** *** 60
** *** ***
*** *** 40
***
0 20 *
Control 1 mg/kg 2 mg/kg 4 mg/kg 0
Dose of cadmium Control 1 mg/kg 2 mg/kg 4 mg/kg
Dose of cadmium

Figure 2. Effect of cadmium at doses 1, 2 and 4 mg/kg

(body weight) on expression of Bcl-2 gene in the rat cortex
and hippocampus. GAPDH was amplified as a housekeeping
gene and showed no changes during the experiment. Data
are expressed as mean + SD of ratio of the treated rats to
sham controls (n ¼ 7 per group). There was significant
decrease in Bcl-2 (B-cell lymphoma 2) mRNA levels at doses
1, 2 (cortex and hippocampus, **p < 0.01) and 4 mg/kg
(cortex and hippocampus, ***p < 0.001) versus control
(Tukey’s post hoc test). GAPDH: glyceraldehyde 3-
phosphate dehydrogenase; mRNA: messenger RNA.
Figure 3. Effect of cadmium at doses 1, 2 and 4 mg/kg on
expression of Bax gene in the rat cortex and hippocampus.
GAPDH was amplified as a housekeeping gene and showed
no changes during the experiment. Data are expressed as
mean + SD of ratio of treated rats to sham controls (n ¼ 7
per group). There was significant increase in Bax (Bcl2-
associated x) mRNA levels at doses of 1 (hippocampus, *p <
0.05), 2 and 4 mg/kg (cortex and hippocampus, ***p < 0.001)
versus control group. GAPDH: glyceraldehyde 3-phosphate
dehydrogenase; mRNA: messenger RNA.
Mahdavi et al.
Table 2. The ratio of Bcl-2/Bax mRNA in hippocampus
and cortex.
Concentration of
cadmium (body Bcl-2/Bax ratio in
weight, mg/kg) hippocampus
1 0.02668
2 0.006b
4 0.0005b
mRNA: messenger RNA.
a
p < 0.05.
b
p < 0.001.
Caspase-3/7 activity
(Relative luminescence units)
2000
***
1500
RLU/ sec
1000
500
0
Control 1 mg/kg
Dose of cadmium
Figure 4. Cadmium-induced activation of caspase-3/7 in the
rat hippocampus and cortex cells. Apoptosis was assessed by
measuring caspase-3/7 activity using the Promega caspase-3/
7 assay kit. Data are expressed as mean + SD of ratio of
treated rats to sham controls (n ¼ 3 per group). There was
significant increase in caspase-3/7 activity at doses of 1, 2 and
4 mg/kg (in hippocampus, ***p < 0.001), and 4 mg/kg (cortex,
*p < 0.05) versus control group.
(body weight) by 7.45, 24.68 and 37.65 times, respec-
tively, compared with control cells. In cortex cells,
cadmium increased the mRNA level of Bax at doses
of 1, 2 and 4 mg/kg (body weight) by 8.78, 93.14 and
95.72 times, respectively, compared with control
cells. As illustrated, in hippocampus cells, cadmium
causes a statistically significant increase in Bax
mRNA levels at doses 1 (p < 0.05), 2 and 4 mg/kg
p < 0.001) compared to the control group. In cortex
cells, cadmium causes a statistically significant
increase in Bax mRNA levels at doses 2 and 4 mg/
kg (p < 0.001) compared to the control group.
As Table 2 shows, in hippocampus, Bcl-2/Bax
mRNA ratio was calculated and shows that it was
significantly decreased in doses of 2 and 4 mg/kg
cadmium body weight (p < 0.001). In cortex, Bcl-2/
Bax mRNA ratio was significantly decreased in doses
of 1 (p < 0.05), 2 and 4 mg/kg cadmium body weight
(p < 0.001).
325
In apoptosis, the critical caspases are caspase-3/7,
the activities of which have been detected in differ-
ent cells. In this study, caspase-3/7 activation capa-
Bcl-2/Bax ratio in bility was evaluated in hippocampus and cortex cells
frontal cortex (Figure 4). In rat hippocampus, cadmium increased
the activity of caspase-3/7 at doses of 1, 2 and 4 mg/kg
0.01a by nearly 3.9, 2.43 and 2.43 times, respectively, com-
0.001b
pared to the control group. In rat cortex, cadmium
1.20b
increased caspase-3/7 activity at doses of 1, 2, and 4
mg/kg by nearly 1.43, 1.02 and 1.86 times, respec-
tively, compared to the control groups. As illustrated,
in hippocampus cells, cadmium causes a statistically
significant increase in caspase-3/7 activity at doses
Hippocampus Cortex of 1, 2 and 4 mg/kg (p < 0.001) compared to the
control group. In cortex cells, cadmium causes a
* statistically significant increase in caspase-3/7 activ-
ity at dose of 4 mg/kg (p < 0.05) compared to the
*** *** control group.
Discussion
2 mg/kg 4 mg/kg Despite several studies investigating the neurotoxic
effects of cadmium, the data on underlying mechan-
ism remain not completely understood. The purpose
of this study was to evaluate the neurotoxic effects of
cadmium on rat hippocampus and cortex through
assessment caspase-3/7 activity and the expression
of genes involved in apoptosis. The present study
showed that cadmium exposure leads to decreases in
mRNA expression of anti-apoptotic Bcl-2, increases
in that of pro-apoptotic Bax genes and decreases in
Bcl-2/Bax ratio in a dose-dependent manner. Further-
more, cadmium increased caspase-3/7 activity in rat
hippocampus and cortex.
We used quantitative real-time PCR to determine
changes in the mRNA levels apoptotic genes of Bcl-2
and Bax in hippocampus and frontal cortex cells of
rats, and apoptotic studies was done using caspase-3/7
activation assay kit.
One of the targets of cadmium is the brain.13,14
Several mechanisms are involved in cadmium-
induced toxicity in the brain. One of the mechanisms
is changing permeability of vascular endothelium in
nerve cells.36 Cadmium crosses the BBC by disrup-
tion in BBC.9,12 Cadmium increases the permeability
of the BBB in rats11,32 and accumulates in the brain of
developing and adult rats.37,38 Moreover, cadmium
decreased enzymatic antioxidants levels in the frontal
cortex tissue.32 In various organs, cadmium induces
heavy metal-binding proteins such as metallothionein
(MT). MT is a cysteine-rich protein and high metal
326
affinity. MT has an important role in the metabolism
of cadmium, which is expressed in the brain. A pro-
tective mechanism against cadmium-induced neuro-
toxicity is MT. Cadmium exposure induces MT
deficiency and Alzheimer’s disease,39 and it also
causes behavioural and neurochemical dysfunctions.31
Based on multiple studies, the possible mechan-
isms of cadmium-related toxicity include change in
gene expression, repress of expression of cell cycle-
regulated protein,40 and alter of the activity of anti-
oxidant enzymes,18 stimulation of oxidative stress and
production of free radical and oxidative damages of
lipids, proteins and DNA in humans and animal.41
Moreover, cadmium changes pro-apoptotic gene
expression and DNA repair-related genes.40
Several studies have shown mechanisms of
cadmium-induced toxicity in production of free radi-
cals and generation of ROS that result in damage of
mitochondrial and apoptosis induction.42 Moreover,
damage of mitochondria can produce ROS and apop-
tosis induction.11 On the other hand, heavy metals
such as cadmium affect the mitochondrial function43
and increases ROS production.
In neural tissues, cadmium as a neurotoxic metal
induces histopathological damage.36 In experimental
and clinical conditions, cadmium is demonstrated to
induce oxidative stress which initiates cell damage
and neurodegenerative processes in the brain.44 Cad-
mium has neurotoxic effects on the hippocampus,
parietal cortex, striatum and cerebellum of rats.18,45
In the brain, free radicals can potentially cause dam-
age to neurons.11 On the other hand, oxidative stress
observed especially in the frontal cortex and hippo-
campus of rats exposed to cadmium may result from
the increased production of free radicals.18
Previous studies have shown that cadmium-
induced apoptosis is inhibited by overexpression of
the anti-apoptotic protein Bcl-2 in mammalian
cells.46,47 The Bcl-2 protein family induces pro-
grammed cell death by mitochondrial pathway of
apoptosis which is also known as intrinsic. In
response to various cytotoxic stresses, pro-apoptotic
proteins begin to release apopotogenic factors such as
cytochrome c into the cytosol and promote caspase
activation in the cytosol which are are known in both
initiation and execution of apoptosis.48
It has been known that overexpression of Bcl-2 can
protect cells from apoptosis mediated by ROS.49
However, the mechanism by which Bcl-2 prevents
ROS-induced apoptosis is unknown. Bcl-2 itself does
not possess antioxidant activity, but it may act
Human and Experimental Toxicology 37(3)
indirectly to increase the levels and/or activities of
endogenous antioxidants (e.g. glutathione or superox-
ide dismutase) within cells.50–52 Moreover, Bax can
promote apoptosis by homodimerization or hetrodi-
merization of Bcl-2.53
In this study, we demonstrated that cadmium
decreases the expression of Bcl-2 and increases the
expression of pro-apoptotic Bax genes in rat hippo-
campus and cortex. This is in accordance with prior
studies which semi-quantitatively showed modulation
of the same genes in apoptosis.40,53,54
As we report here, increase in caspase-3/7 activity
and alteration in the ratio of Bcl-2/Bax could be a key
determining factor in the release of cytochrome c, the
activation of caspase-3/7 and the initiation of apoptosis.
A decrease in this ratio may exacerbate apoptosis, and
increasing this ratio may reverse the deleterious effect of
cytotoxic stimuli.53,54 While Bax has been shown to
trigger cell death, the anti-apoptotic Bcl-2 can block
cytochrome c release and caspase activation.54,55
Conclusion
In conclusion, the results of the current study showed
that intraperitoneal administration of cadmium
increases the expression of pro-apoptotic Bax and
decreases the expression of anti-apoptotic Bcl-2 genes
in a dose-dependent manner in rat cortex brain and
hippocampus. Due to a decrement in the Bcl-2/Bax
ratio, it is likely that apoptosis developed by cadmium
in the rat hippocampus and cortex is dependent on
mitochondrial pathway. Furthermore, this result was
also confirmed by increase in caspase-3/7 activity.
Author’s Note
The part of the results presented in this article was based on
a student’s thesis work.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest
with respect to the research, authorship, and/or publication
of this article.
Funding
The author(s) received no financial support for the
research, authorship, and/or publication of this article.
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