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Pakistan J. Zool., vol. 46(6), pp. 1719-1730, 2014.
Oxidative Stress and Apoptosis are Markers in Renal Toxicity
Following Egyptian Cobra (Naja haje) Envenomation
Mohamed A. Dkhil,1,2 Saleh Al-Quraishy,1 Abdel Razik H. Farrag,3 Ahmed M. Aref,4 Mohamed S.
Othman5 and Ahmed E. Abdel Moneim2,*
1Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia.
2Department of Zoology and Entomology, Faculty of Science, Helwan University, Cairo, Egypt.
3Pathology Department, Medical Research Division, National Research Centre, Cairo, Egypt.
4Biological Science Department, Faculty of Dentistry, Modern Sciences and Arts University, Giza, Egypt.
5Biochemistry and Molecular Biology Department. Faculty of Biotechnology, Modern Science and Arts, Giza,
Egypt.
Abstract.- Snakebite is a serious and important problem in tropical and subtropical countries including Egypt.
The venom of Egyptian cobra (Naja haje; L.) is complex, and it has been considered as a good source of short
neurotoxins and several cytotoxins. In this study, oxidative stress inductions as well as apoptotic effects of the
Egyptian cobra crude venom at a dose of 0.025mg/kg (intraperitoneal injection; i.p.) has been investigated in kidney of
rats after 4 h. Twelve rats divided into 2 groups, Group I served as control group, Group II received i.p. injection of
0.025mg/kg of crude venom. The venom enhanced lipid peroxidation and nitric oxide productions in the kidney with
concomitant reduction in glutathione content and superoxide dismutase, catalase, glutathione peroxidase, glutathione
reductase and glutathione-S-transferase activities were inhibited. Moreover, the venom induced a renal injury as
indicated by histopathological changes in the kidney tissue with an elevation in serum creatinine and urea. In addition,
the renal ultrastructural changes were in the form of blebbing of visceral epithelial cells, and foot process
disorganization. Also, the glomerular capillaries lined by hypertrophied endothelial cells. These findings were
associated with the pro-apoptotic action in the kidney. The results suggest that Egyptian cobra venom stimulates
oxidative stress to induce apoptosis in renal tissue through inhibition of mitochondrial respiration in male rats.
Keywords: Egyptian cobra venom, renal toxicity, oxidative stress, apoptosis.
INTRODUCTION
Snakebite envenomation is known to man
since antiquity and many references to snakebite are
found in the oldest medical writings. There are more
than 2.5 million venomous snake bites annually,
with greater than 125000 deaths. The risk is highest
in the tropics and West Africa, predominantly
among rural population (Gutierrez et al., 2006).
Snake venoms are a mixture of complex toxins that
may be independent, synergistic or antagonistic (Al-
Quraishy et al., 2014). Broadly, there are two types
of toxins namely neurotoxins, which attack the
central nervous system and hemotoxins which target
the circulatory system (Markland, 1998). It is
important to understand that the actual mixture of
toxins in the venom will vary by individual species
_________________________________
* Corresponding author: aest1977@helwan.edu.eg
0030-9923/2014/0006-1719 $ 8.00/0 Copyright 2014 Zoological Society of Pakistan
and also by age and season (Al-Sadoon et al., 2013).
The venom of Egyptian cobra (Naja haje) is
complex, and it has been a good source of short
neurotoxins and several cytotoxins. From this
venom the purification and the primary structure of
two short neurotoxins and of 14 cytotoxins have
been reported. Besides the toxins, N. haje venom
contains also low-molecular-weight polypeptides,
usually of relative low toxicity and of completely
distinct immunochemical properties (Joubert and
Taljaard, 1978).
Although, nearly all snakes with medical
relevance can induce nephropathy, leading to acute
renal failure (ARF), it is unusual except with bites
by Egyptian cobra. This action was attributed to the
effect of different venom toxins, such as myotoxins,
cytotoxins, phospholipases, and cardiotoxins
(Rahmy, 2001). It was found that myotoxin
probably causes renal damage due to myoglobin
cast nephropathy. Venom phospholipase is known
to be toxic to cells and believed to be responsible for
disturbing the cell membrane permeability
M.A. DKHIL ET AL.
1720
(Mukherjee and Maity, 1998). Phospholipase may
also alter the mitochondrial respiratory functions
and induce a hemolytic activity (Mukherjee et al.,
1998). Cobra cytotoxins and cardiotoxins may also
disturb different cell types (Rahmy, 2001).
Cytotoxin lytic activity in synergy with various
phospholipases of cobra venom was also reported
(Chaim-Matyas et al., 1995).
Nevertheless, the oxidative stress induced by
the venom of N. haje was not sufficiently covered in
the available literature (El Hakim et al., 2011).
Thus, it is so special interest to examine the possible
effects of LD50 of the crude venom in kidney of rats
after 4 h of envenomation.
MATERIALS AND METHODS
Experimental animals
Adult male Wistar albino rats weighing 120–
150 g were obtained from the Holding Company for
Biological Products and Vaccines (Vacsera, Cairo,
Egypt). After an acclimatization period of one week,
the animals were divided into two groups (6 rats per
group) and housed in wire bottomed cages under
standard conditions of illumination with a 12 h
light-dark cycle at 25±1ºC. They were provided
with water and a balanced diet ad libitum. We have
followed the European Community Directive
(86/609/EEC) and national rules on animal care that
was carried out in accordance with the NIH
Guidelines for the Care and Use of Laboratory
Animals 8th edition.
Experimental protocol
Venom was milked from adult snakes
collected from the western Nile delta in Egypt, in
September, dried and reconstituted in saline solution
prior to use. LD50 of venom was determined as
described by Meier and Theakston (1986). To study
the effect LD50 of the venom in kidney of rats after 4
h, rats were divided into two groups, six rats of
each. Group I served as a control and received saline
(0.2 ml saline/ rat) by intraperitoneal (i.p.) injection.
Group II was injected i.p. with LD50 dose of N. haje
venom in saline (0.025mg/kg). The animals of the
two groups were sacrificed, and blood samples were
collected by cardiac puncture. The blood stranded
for half an hour and then centrifuged at 3,000 g for
15 min at 4ºC to separate serum and stored at -70ºC
until analysis. The left kidney was weighed and
homogenized immediately to give a 50% (w/v)
homogenate in ice-cold medium containing 50 mM
Tris-HCl, pH 7.4. The homogenate was centrifuged
at 3,000 g for 10 min at 4ºC. The supernatant (10%)
was used for the various biochemical
determinations. Right kidney was cut into small
pieces and kept for the histological and molecular
studies.
Biochemical estimations
Kidney function test
Serum uric acid, urea and creatinine contents
were determined colorimetrically by commercially
available diagnostic kits (Biodiagonstic-Egypt) as
per manufacturer’s instructions.
Oxidative stress markers
Lipid peroxidation (LPO) in kidney was
determined by using 1 ml of trichloroacetic acid
10% and 1 ml of thiobarbituric acid 0.67% and were
then heated in a boiling water bath for 30 min.
TBARS were determined by the absorbance at 535
nm and expressed as malondialdehyde (MDA)
formed (Ohkawa et al., 1979). Also, nitric oxide
was determined in acid medium and in the presence
of nitrite the formed nitrous acid diazotized
sulfanilamide is coupled with N-(1–naphthyl)
ethylenediamine. The resulting azo dye has a bright
reddish-purple color that can be measured at 540 nm
(Green et al., 1982).
In addition, the renal glutathione (GSH) was
determined by the reduction of Elman's reagent
(5,5` dithiobis (2-nitrobenzoic acid) DTNB) with
GSH to produce a yellow compound . The reduced
chromogen directly proportional to GSH
concentration and its absorbance can be measured at
405 nm (Ellman, 1959).
Enzymatic antioxidant status
The homogenates of kidney were used to
determine the activities of superoxide dismutase
(SOD) (Nishikimi et al., 1972), catalase (CAT)
(Aebi, 1984), glutathione peroxidase (GPx) (Paglia
and Valentine, 1967), glutathione-S-transferease
(Habig et al., 1974) and glutathione reductase (GR)
(Factor et al., 1998).
RENAL TOXICITY FOLLOWING COBRA ENVENOMATION 1721
Flow cytometry
Pieces of kidney were prepared by manual
disaggregation procedure. Briefly, a few drops of
RPMI were added to tissue and then minced until
complete tissue disaggregation was achieved.
Suspended cells were filtered using a 50 µm pore
size mesh and then centrifuged at 1000 g for 10 min.
Cells were resuspended in phosphate buffer,
counted and washed by calcium buffer then
centrifuged at 1500 g for 5 min. The pellet was
resuspended and then cells were counted. Annexin-
PI apoptotic assay was carried out using IQP-120F
Kit (IQ Products, Groningen, Netherlands). FAC
scan Becton-Dickinson (BD) flow-cytometer was
used and data were analyzed using cell Quest
software.
Western blot analysis
Total proteins were extracted using RIPA
buffer. Protein determination was applied by a
Lowry method (Lowry et al., 1951). Denatured
proteins (20 µg) were size fractionated by 12.5%
SDS-polyacrylamide gels. Proteins were transferred
to nitrocellulose membrane at 30 V for 1 h. The
blots were blocked for 1.5 h at room temperature in
fresh blocking buffer (0.1% Tween-20 in Tris-
buffered saline, pH7.4, containing 5% BSA). The
membrane was incubated overnight at 4°C with
primary antibodies against Bax, GPx, GR, SOD and
mitochondrial respiratory complexes at dilution of
1:200. β-actin was used as a loading control. The
membrane was incubated for 2 h with HRP-
conjugated secondary antibodies at a dilution of
1:1000. Chemiluminescent signals were captured
using an ECL-plus chemiluminescent kit (GE
Healthcare, UK) and Kodak Luminescent Image
Analyzer (Kodak In-Vivo Imaging Systems F).
Histopathology and p53 and caspase-3
immunohistochemistry
Small pieces of the kidney were quickly
removed, then fixed in 10 % neutral buffered
formalin. Following fixation, specimens were
dehydrated, embedded in wax, and then sectioned to
5 microns thickness. For histological examinations,
sections were stained with haematoxylin and eosin.
Immunolocalization techniques for p53 and caspase-
3 were performed on 3-4 µm thickness sections
according to Pedrycz and Czerny (2008). For
negative controls, the primary antibody was omitted.
In brief, mouse anti-p53 or mouse anti-caspase-3
(diluted1:200, Santa Cruz Biotechnology, Santa
Cruz, CA, USA), were incubated with sections for
60 min. Primary antibodies were diluted in Tris
buffered saline (TBS)/1% BSA. Then a biotinylated
secondary antibody directed against mouse
immunoglobulin (Biotinylated Link Universal–
DakoCytomation kit, supplied ready to use) was
added and incubated for 15 min, followed by horse
radish peroxidase conjugated with streptavidin
(DakoCytomation kit, supplied ready to use) for a
further 15 min incubation. At the sites of
immunolocalization of the primary antibodies, a
reddish to brown colour appeared after adding 3-
amino-9-ethylcarbasole (AEC)
(DakoCytomationkit, supplied ready to use) for 15
min. The specimens were counterstained with
hematoxylin for 1min and mounted using the
Aquatex fluid (Merck KGaA, Germany).
Scanning electron microscopic (SEM) study
Kidneys were removed, and cortical slices
were cut into big pieces, which were immersed in
Karnovsky’s solution (2% glutaraldehyde, 2%
paraformaldehyde in 0.1 M phosphate buffer, pH
7.4) at 4°C overnight. After rinsing in phosphate
buffer for 1 h, the specimens were post-fixed in
buffered 1% OsO4 at 4°C in the dark for 2 h and
then immersed in a 2.3 M sucrose solution at 4°C
overnight. The specimens were subsequently
immersed for 30 min in liquid nitrogen and then
fractured, washed in the same buffer, dehydrated in
a graded acetone series, and critical-point dried.
After identifying the fractured surface, specimens
were mounted on stubs, sputtered with gold for 120
s, and examined and photographed with a Zeiss
DSM 940A SEM operated at 10 kV.
Transmission electron microscopic (TEM) study
Kidneys were removed, and cortical slices
were cut into small pieces, which were immersed in
the same fixative with 0.1% tannic acid and 5%
sucrose for 3 h at room temperature. After rinsing in
a sugar-saline solution (0.15 M NaCl, 0.2 M
sucrose), the specimens were post-fixed with 1%
OsO4 at 4°C in the glucose-saline solution in the
M.A. DKHIL ET AL.
1722
dark for 2 h and then rinsed again in the glucose-
saline solution. The samples were dehydrated in a
graded ethanol series and embedded in Epon 812
resin at 60°C for 48 h. Thin sections (60–70 nm)
were double-stained with uranyl acetate and lead
citrate and were observed and photographed with a
LEO 906 TEM operated at 60 kV.
Statistical analysis
The obtained data were presented as means ±
standard deviation of the mean. Statistical analysis
was performed using an unpaired Student’s t-test
using a statistical package program (SPSS version
17.0). Differences between groups were considered
significant at P<0.05.
RESULTS
Kidney function parameters were affected by
a single i.p. injection of Egyptian cobra (0.025
mg/kg). The levels of serum urea and creatinine
were increased significantly (27.7 and 44.0%,
respectively) in envenometed rats (Table I).
However, the level of uric acid was significantly
reduced (22.2%; P<0.05) after 4 h of venom
injection compared to the control rats.
Table I.- Changes in kidney function of rats induced by
Egyptian cobra venom after 4 h.
Parameters Control
rats Intoxicated
rats
Serum uric acid (mg/dL) 69.63±9.81 54.14±6.61*
Serum urea (mg/dL) 3.21±0.40 4.1±0.46*
Serum creatinine (mg/dL) 0.50±0.03 0.72±0.03*
Values are means ± SD (n=6). *: significant change at P< 0.05
with respect to the Control group.
Table II shows the potential oxidative stress
effects of Egyptian cobra venom in the kidney
homogenate. This was evidenced by the significant
increase in the level of both of the lipid peroxidation
and nitric oxide by 78.5 and 82.2%, respectively.
The elevation in nitric oxide content of the kidney
could be explained by up-regulation in iNOS gene
that measured with RT-PCR (data not shown). Also,
the glutathione level was significantly reduced by
about 55% in the kidneys (Table II) of rats receiving
the venom. Moreover, there was a reduction in GR,
GST, and GPx by approximately, 28.6, 60.9 and
45.7%, respectively. Interestingly, the reduction in
GR and GPx activities were accompanied by
concomitant significant effect on its expression
(P<0.05) (Fig. 1). The reduction in GR and GPx
proteins expression were 24.1 and 15.1%,
respectively. The reduction in GPx enzyme activity
was also tested with RT-PCR method where GPx
gene was down-regulated as compared with that of
the control (data not shown).
Table II.- Changes in kidney oxidant/antioxidant state of
rats induced by Egyptian cobra venom after 4
h.
Parameters Control
rats Intoxicated
rats
Tissue MDA (nmol/g tissue) 729.70±50.21 1302.82±81.66*
Tissue NO (µmol/g tissue) 100.94±7.73 183.89±13.10*
Tissue GSH (mmol/g tissue) 67.33±2.89 39.71±1.33*
Tissue GPx (U/g tissue) 2075.03±317.67 1125.77±203.39*
Tissue GR (µmol/h/g tissue) 140.67±22.01 100.48±23.62*
Tissue GST (µmol/h/g tissue) 0.23±0.06 0.09±0.01*
Tissue SOD (U/g tissue) 2.52±0.04 1.21±0.16*
Tissue CAT (U/g tissue) 1.51±0.16 0.96±0.12*
Values are means ± SD (n=6). *: significant change at P< 0.05
with respect to the Control group.
Severe inhibition of SOD and CAT activities
(108.3 and 36.4%, respectively) were observed in
the kidneys (Table II) of Egyptian cobra venom-
injected rats compared to these of the controls. The
inhibition in SOD was accompanied by a significant
reduction in the expression of SOD after 4 h
Egyptian cobra envenomation by approximately
49% (P<0.05).
Figure 1 shows alterations in the activities of
mitochondrial respiratory complexes. Complex II,
III and IV activities in the kidney tissue from
envenomated rats were highly increased (40–100%
from control; P<0.05). However, complex V
showed a significant inhibition (32%; P<0.05) in its
activity.
In the present investigation, the percentage of
both of the apoptotic and necrotic cells in the kidney
of Egyptian cobra venom-injected rats were 28.3
and 32.06%, respectively, up to the control (Fig. 2),
while the viable cells were decreased down to -
21.4% compared to the control samples.
RENAL TOXICITY FOLLOWING COBRA ENVENOMATION 1723
Fig. 1: Western blots showing the effects of Egyptian cobra venom on proteins expression of β–Actin, GPx, GR,
SOD, Bax and complexes II, III, IV and complex V in the kidney after envenomation for 4 h.
Values are means ± SD (n=6). *: significant change at P< 0.05 with respect to the Control group.
M.A. DKHIL ET AL.
1724
Fig. 2: Changes in viable, early apoptotic
and late apoptotic and necrotic cells of kidney
cells of rats induced by Egyptian cobra venom
after 4 h.
Values are means ± SD (n=6). *: significant
change at P< 0.05 with respect to the Control
group.
Microscopic examination of the renal tissue
shows that the venom induced a severe glomerular
degeneration and coagulative necrosis. Also, the
urinary spaces appeared wider as compared with the
control one. Moreover, most of the renal tubules
were degenerated and filed with cellular debris (Fig.
3B). Immunohistochemical findings in control
kidneys showed a week immunoreactivity of both of
caspase-3 and p53 (Fig. 3C,E, respectively) while, a
strong positive reaction was detected in the sections
of the venom injected group (Fig. 3D and F,
respectively).
The ultrastructure of the glomeruli of the control
animals showed capillary loops which embodied blood
cells and precipitated plasma proteins. The capillaries
are lined with a thin layer of flattened and fenestrated
endothelial cells. The nuclei of such cells can be seen
bulging into the capillary lumina. Mesangial cells and
mesangial substance provides support for the capillary
loops. The podocytes form the outer layer of the
capillary wall, each has a cell body from which arise
several primary processes. Each primary process gives
rise to numerous secondary processes called pedicels
that embrace the glomerulus capillaries. The fenestrated
capillary endothelium is closely applied to the
glomerular basement membrane and on the opposite
side of the basement membrane are the podocytes
secondary processes (pedicels) separated from each
other by slight pores of approximately uniform width
(Fig. 4A). The electron micrographs of the convoluted
tubules of control animals reveal profuse, tall microvilli
which represent the brush border seen with light
microscopy. The plasma membrane at the bases of the
cells of the tubules exhibits deep basal infoldings into
the cells. These infoldings set up basal compartments
that embody elongated mitochondria (Fig. 4B).
In the venom injected group, the glomeruli
appeared with proliferated mesangial cells and most of
the foot processes of the epithelial cells were discrete
but few of these processes were fused together (Fig.
4C). On the other hand, the convoluted tubule showed
deformity of the nuclei, mitochondria and the enfolding
of the basement membrane. Extensive degenerative
changes represented by lysis of many cell contents were
notice (Fig. 4D).
SEM examination of the glomeruli tufts of
normal rats showed many erythrocytes with various
shapes, in the capillary networks and interdigitating
foot processes (Fig. 5A). The convoluted tubules of
normal rats contained basement membranes,
mitochondria, nuclei infoldings and lamina (Fig.
5B). On the other hand, glomeruli of the venom
injected rats were enlarged due to loss of mesangial
cells and matrix. Also, the glomeruli appeared with
a segmental ballooning lesion with honeycomb-like
appearance on the surface (Fig. 5C). In addition, the
convoluted tubules were damaged (Fig. 5D).
DISCUSSION
Several works dealing with the effects of
snake venoms in blood cells, marrow cells and in
cells from other organs of animals, like muscle,
liver, kidney and skin, showed varying results,
depending on the experimental concentrations,
RENAL TOXICITY FOLLOWING COBRA ENVENOMATION 1725
Fig. 3. Histological structure of kidney of A) Control rat show intact architecture of the glomerulus and renal
tubules, B) Egyptian cobra venom injected rats with severe glomerular degeneration, wide urinary space, and
degenerative renal tubules (H&E X 150), C) control rat appearing with no immunoreaction of caspase-3 in the cortical
regions, D) Egyptian cobra venom injected rat shows strongly caspase-3 expression in inner cortical and outer
medullary areas especially in the proximal convoluted tubules, whereas there is less caspase-3 expression in the
glomerular structure (caspase-3 immunostaining X 300), E) control rat shows weak immunoreactivity of p53, and F)
Egyptian cobra venom treated rat shows strong immunoreactivity of p53 (p53 immunostaining X 300).
exposure time, site of injection, and type of toxin
(Maria et al., 2003; Fox and Serrano, 2008; Tohamy
et al., 2014). In the present investigation, we
explored the systemic physio-pathological changes
induced by the Egyptian cobra venom in kidney as a
vital organ. The high levels of creatinine and urea
indicate an impairment of renal function. Similar
observations were reported in rats following
administration of various snake venoms (Omran and
Abdel-Nabi, 1997; Schneemann et al., 2004). Such
increased vascular permeability, together with renal
damage would further aggravate the accompanying
M.A. DKHIL ET AL.
1726
Fig. 4: Electronmicrographs of kidney of A) a portion of glomerulus of control rat shows two capillary loops (C). Notice
the erythrocyte (RBC). The capillaries lined with thin fenestrated endothelial cells (EC). The primary podocyte processes (P1)
give rise to numerous secondary foot processes (P2) which rest on the basement membrane of the capillary (BM), B): a part of
distal convoluted tubule of control rat shows the enfolding of the basal membrane (BM), a large number of mitochondria (Mi)
and a few number of microvilli (Mv). Notice the wide lumen of such tubule (LU), C) a portion of glomerulus of rat showing
shows proliferation of the mesangial cells. Most of the foot processes of the epithelial cells were discrete but few of these
processes were fused together, D) a part of convoluted tubule shows deformity of the nucleus (N), mitochondria (M) and the
enfolding of the basement membrane (DI). Notice extensive degenerative changes represented by lysis of many cell contents
(TEM, Micron bar, 10 µm)
hypoproteinemia. Furthermore, the rise in serum
urea and creatinine associated with the reduction of
serum uric acid level observed, in the present study,
supports the proposed impairment of renal function.
Similar observations were reported following
various viper envenomation of rats (Sant and
Purandare, 1972; Omran and Abdel-Nabi, 1997).
The proteolytic and phospholipase A2 activities of
Egyptian cobra venoms have important cytotoxic
effects (Kerns et al., 1999; Rowan et al., 1991;
Barrington et al., 1986; Stefansson et al., 1990) and
could contribute to the nephrotoxicity seen in the
current study.
The ultrastructural alterations induced by the
snake venom in the trilaminar structure of the
glomerular basement membrane (GBM) suggest the
involvement of physicoelectrostatic barrier sites.
Podocytes and endothelial cells retain heparin
sulfate (Stow et al., 1985) in the lamina rara
external and internal of the GBM, and the feet
processes are coated with sialoproteins that
contribute to the anionic charges of the filtration
RENAL TOXICITY FOLLOWING COBRA ENVENOMATION 1727
Fig. 5: Scanning Electron Microscopy micrographs of A): glomerular tufts of normal rats showing many
erythrocytes (RBC) with various shapes, in the capillary (Cap) networks and interdigitating foot processes (FP) (Bar,
20 µm), B): convoluted tubule of normal rat showing basement membrane (BM), mitochondria (M), nucleus (N)
infoldings (I) and lumen (Lu) (Bar, 10 µm), C): Glomerulus from venom treated rat showing capillaries are markedly
distended and the glomerulus is enlarged due to loss of mesangial cells and matrix. Notice glomerulus shows
segmental ballooning lesion with honeycomb-like appearance on the surface (H) (Bar, 8 µm), D) convoluted tubule
from venom treated rat showing disturbance of the normal structure (Bar, 10 µm).
barrier (Kerjaschki et al., 1984). This ionic barrier
of glomerular filtration was shown to be destroyed
by Vipera russelli venom in isolated rat kidneys
(Willinger et al., 1995). Furthermore, since the
glomerular and renal tubule epithelial cells are
strategically interposed between the extra and
intramilieu, they are potential targets for numerous
nephrotoxic agents, and the glomeruli are the first
structure of the nephron to come in contact with
circulating venom.
Snakebites are most often accompanied by
signs of inflammation and local tissue damage
(Nelson, 1989). Neutrophils and macrophages are
induced to produce superoxide radical anion which
belongs to a group of reactive oxygen species (ROS)
and this reacts with cellular lipids leading to the
formation of lipid peroxides and the observed
necrosis (Valko et al., 2007). As the origin of
oxidative stress is the mitochondrial respiratory
electron transport chains (Fletcher et al., 1991), it is
possible that mitochondrial death mediates venom-
induced cellular damage (Haffor and Al-Sadoon,
2008; Abdel Moneim et al., 2014a).
It is generally accepted that a marked increase
in Ca2+ could lead to ROS production. In many
models of cell death it has also been proposed that
ROS production is an early event in the process of
apoptotic cell death. Furthermore, it had been
proposed that ROS can also modulate gene
expression by acting on transcription factors in a
M.A. DKHIL ET AL.
1728
variety of families like NF-κB, activator protein 1
(AP-1) and AP-2 (Ray et al., 2012). Also, N. haje
venom contains cardiotoxins (CTX), a group of
highly basic polypeptides of approximately 60
amino acids present in the many snakes, have ability
to increase H2O2 production. All of these findings
suggested that the renal cytotoxic effects of venom
were apparently triggered by oxidative stress
mediated by H2O2 and superoxide anion production.
Generation of H2O2 in the mitochondria may result
in mitochondrial peroxidation (Al-Quraishy et al.,
2014).
Superoxide dismutase and catalase are
considered as the primary antioxidant enzymes,
since they are involved in the direct elimination of
active oxygen species (Abdel Moneim et al.,
2014b). Dai et al. (2012) have reported that, Naja
sp. venoms have the SOD activity. Apart from this,
in the past few years several peptides have been
reported to exert deferent mechanisms of action in
free radical mediated oxidative sequences by radical
scavenging and metal ion chelation (Sri
Balasubashini et al., 2006; Tohamy et al., 2014).
Many L-amino acid oxidase demonstrate
apoptosis-inducing activity (Zhang and Wu, 2008)
and it is partially due to the generation of hydrogen
peroxide. Hydrogen peroxide belongs to ROS and it
is widely accepted that mitochondrial perturbation
has been associated with the increased production of
ROS (Othman et al., 2014).
Expression of p53 as proapoptotic protein
was significantly activated by Egyptian cobra
venom. Moreover, our results showed that caspase-3
was most potently activated by the venom.
Moreover, the present data suggest that the
activation of caspase-3 is critical in snake venom-
induced kidney damage. Also, p53 plays multiple
roles in cell cycle control, differentiation, genomic
stability, angiogenesis, and apoptosis (Maxwell and
Davis, 2000). Miao et al. (1999) showed that the
mRNA of p53 increased within 3 and 6 h after
vascular endothelial cells were treated with rattle
snake venom. Our results indicated that p53 plays
an important role in apoptosis induced by Egyptian
cobra venom in the kidney and they are in the same
pathway in apoptotic signal transduction in the
kidney.
CONCLUSIONS
The data presented herein suggested several
aspects of the mechanisms of Egyptian cobra
venom-induced renal toxicity. We proposed that: (i)
Egyptian cobra venom induced apoptosis in kidney
via activation of caspase-3 and modulation of the
protein levels of Bax and Bcl-2; (ii) Egyptian cobra
venom-activated p53 induced apoptosis; (iii)
oxidative stress involved in transmitting apoptotic
signals in Egyptian cobra venom-treated rats. These
results provide valuable insight into the toxicity of
Egyptian cobra venom and deepen our previous
understanding of its molecular mechanisms of
action.
Conflict of interests statement
The authors declare that there is no conflict of
interests regarding the publication of this article.
ACKNOWLEDGEMENTS
The authors would like to extend their sincere
appreciations to the Deanship of Scientific Research
at King Saud University for its funding this
Research group NO. (RG -1435-198).
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(Received 16 August 2014, revised 16 September 2014)
