Nano -Se delay the initiation of hepatocarcinogenesis in rats. [623377]

Biologically Produced Nano -selinum reduce oxidative stress induced by Experimental
Hepatocarcinogenesis in Rats
Doaa H.Abdelhady1 ,Walied Abdo2, Emad Ghazi1, Yahya Z. Eid3,Mustafa Shukry4
1Department of Clinical Pathology 2Department of Pathology,4Departm ent of physiology Faculty of Veterinary
Medicine,3Department of Poultry Production, Faculty of Agriculture, Kafrelsheikh University, 33516, Kafr –
Elsheikh, Egypt.
Running title: Nano -Se delay the initiation of hepatocarcinogenesis in rats.
*Corresponding au thors:
Yahya Eid, Ph.D.
Department of Poultry Production , Faculty of Agriculture , Kafrelsheikh University, 33516
KafrElsheikh, Egypt .
e-mail address: [anonimizat]
Phone number: +20-1111819340

Abstract
Biological Nano -selenium (Nano -se) considers one of the less toxic elements when
compared with the other forms of selenium. The present study evaluated the protective effect
of biologically Nano -se produced from bacteria against hepatotoxic effect induced by diethyl
nitrosamine (DEN) for 1 weeks in rats. Forty male wister rats were divided into four groups as
follows: Group (1) treated by intraperitoneal (i.p.) injection with physiological saline once and
kept as control; group (2) treated by DEN alone (200 mg/kg) by intraperitoneal (i.p.) injection
once; group (3) treated with both DEN and Nano -Se and group (4) treated by Nano -Se alone
(0.5mg/kg) orally by gastric intubation. DEN administration did not alter the erythrogram while
caused alter ation on leukogram represented by a significant (p≤ 0.05) increase in TLC,
neutrophil and monocyte count together with a significant reduction in lymphocyte count and
eosinophil count reflecting stress picture. DEN induced hepatotoxicity noticed through an
elevation of serum hepatic enzymes such as AST, ALT and γ -GT, additionally, an increase in
the oxidative stress marker as malondialdehyde content (MDA) and reduction of antioxidant
parameters such as catalase (CAT) and reduced glutathione (GSH) .Moreover , DEN was
associated with formation of putative hepatic foci indicated with over expression of GST -P
immunostaining. Interestingly, simultaneous supplementation of Nano -Se clearly alleviated the
negative impacts of DEN induced hepatotoxicity via returning leukogram to normal condition,
suppressed the elevated of serum hepatic enzymes, oxidative stress markers, decrease positive
GST -P foci induced by DEN and remarkably increased the antioxidant capacity. Therefore,
biological produced Nano -se might be consid ered as an effective strategy in hepatic cancer
chemoprevention.
Introduction
Hepatocellular carcinoma (HCC) represents one of the most lethal worldwide malignancies
in human .It is an environmentally related cancer. In Egypt, the incidence of HCC has been
increasing especially in the last ten years, and usually attributed to the increased prevalence of
numerous risk factors such as the higher prevalence of hepatitis B and C viral infection ,
aflatoxins, heavy metals and organochlorine pesticides and their combinations in contaminated
food and water (Abdo et al. 2013 ; Anwar et al. 2008 ; Shaker et al. 2013 ) Chemical carcinogens
with hepatitis viruses (B and C) are involved in a multistage process of hepatocellular
carcinogenesis involving genetic and epigenetic pathways (Gu 2012 ). There are two primary

driving forces behind the development and exacerbation of HCC incude persistent oxidative
stress and unresolved inflammati on are (Bishayee 2012 ).
Diethyl nitrosamine (DEN) is a potent hepatocarcinogen, has been detected in various
foodstuffs such as milk products, meat produ cts, soft drinks, and alcoholic beverages (Levallois
et al. 2000 ). Since DEN does not itself exert carcinogenicity, it needs to be bioactivated by
cytochrome P450 (C YP) enzymes in the liver, resulting in DNA -adducts that form through an
alkylation mechanism (Verna et al. 1996 ).(Kang et al. 2007 ; Williams 2001 ) detected that
CYP2E1 -deficient mice reveal lower tumor incidence and multiplicity compared with wild
type (WT) mice for DEN -induced hepatocarcinogenesis. This result potentially suggests that
CYP2E1 plays an essential role in the metabol ism of DEN, although several other CYP
enzymes are proposed to catalyze DEN bioactivation in vivo causing cell transformation
(Verna et al. 1996 ). Chemoprevention could be one of the strategies to decrease the risk of
HCC development especially in carriers of hepatitis B and C viruses (Stagos et al. 2012 ).
Selenium is an essential micronutrient that is important for the antioxidant effects of the
selenoproteins. At least two different enzyme families of selenoproteins are present:
glutathione peroxidases and thioredoxin reductases. Selenium takes part in scavenging free
radicals and being a cofactor of these antioxidant enzymes, thus, protecting cells, membranes
and cell organelles, enzymes and nucleic acids from the harmful effects of reactive oxygen
species (ROS) (Jia et al. 2015 ). The concentration of reactive oxygen species in various tissues
and cells such as neutrophil granulocytes and monocyte s is controlled by
Selenoproteins (Szuchman -Sapir et al. 2012 ). Which have an essential physiological role in
immune responses by producing huge amounts of ROS against microbes. These immune cells
antioxidant defense capacity can be exhausted by either elevated oxidative stress or depleted
protection by selenoproteins (due to selenium deficiency) causing immunotoxicity (Gust et al.
2013 ). Selenium is an important micronutrient at low concentration, but being toxic by
increased level (Wang et al. 2007 ). It also acts as a chemo preventive and chemotherapeutic
agent for human cancer (Feng et al. 2014 ).
Recently, Nano -materials have been received much m ore attention. They exhibit unique
superiority of its physical properties such as great specific surface area, high surface activity,
a lot of catalytic efficiency and potent absorptive ability due to the interaction between –NH 2,
C=O, −COO, and –C–N– groups of proteins and the nanoparticles of Se (Hassanin et al. 2013 ).
The unique absorptive ability which increase the bioavailability and lower toxicity compared

with selenite in chickens (Wang et al. 2009 ), rats(Jia et al. 2005 ). sheep (Shi et al. 2011a ), mice
(Wang et al. 2007 )and goats (Shi et al. 2011b ). Subsequent studies described that a novel
elemental Se source called nano -Se possessed a higher efficiency than selenite, seleno –
methionine, and methyl -selen ocysteine in up regulating selenoenzymes in mice and rats (Wang
et al. 2007 ). Elemental Nano selenium (nano -Se) is a bright red, highly stable, soluble and of
Nano meter size in the redox state of zero (Se0) (Zhang et al. 2001 ). Several mechanisms have
been postulated to elucidate the anticancer activity of selenium, including induction of cell
apoptosis, inhibition of cell proliferation, modulation of redox state, detoxificati on of
carcinogen, stimulation of the immune system and inhibition of angiogenesis (Zhao et al. 2006 ).
).Biologically produced Nano -Se could be safer and more efficient than the ones produced by
the chemical methods. (Saleh 2014 )found that there are no adverse effects of using biologically
produced Nano -Se in poultry nutrition. Selenium nanoparticles were found to be effective to
inhibit the proliferation of human breast cancer cell line MCF -7 in a dose dependent manner
(Ramamurthy et al. 2013 ). Although antioxidative and ant -iinflammatory properties of nano –
selenium might be the basic mechanism of the anti -cancer effect. The refore, the goal of the
present experiment was to assess whether the biologically produced Nano -Se may reduce
preneoplastic hepatic foci in rats exposed to diethylnitrosamine (DEN) .For this purpose,
hematological, serum biochemical and' lipid peroxidation and antioxidant biomarkers
,histopatholgical examination as well as assessment of glutathione S-transferase placental
form -positive (GST -P) initiated cells were evaluated in DEN intoxicated rat well as in Nano –
Se treated DEN -intoxicated rats.
2. Material s and methods
The animal experiment was approved and carried out according to the guidelines of animal
care and ethics in Kafrelsheikh University.
2.1. Chemicals :
DEN CAS No. 55 -18-5, purity > 99.0%) was purchased from Sigma -Aldrich Chemie GmbH
(Hamburg, G ermany).Selenium nano -particles (100 -500 nm range ) (sample ready for use) was
kindly obtained from Professor Mohsen Zommara, Department of Dairy Science, Faculty of
Agriculture, Kafrelsheikh University, Egypt and Prof. Jozsef Prokisch, Department of Anima l
Breeding, University of Debrecen, Debrecen, Hungary. Se nanoparticles of 100 to 500 nm were

prepared according to (Eszenyi et al. 2011 ). Selenium nanoparticles were prepared from pure
yoghurt cultures of Lactobacillus delbrueckii subsp. bulgaricus (L.bulgaricus) (NCAIM B
02206),Streptococcus thermophilus (S. thermophilus) (CNCM I -1670) got from the National
Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary (El-Baz and
Zommara 2007 ). The obtained selenium nanospher es were within range 100 -500 nm range as
mentioned by (Prokisch et al. 2011 ) and (Eszenyi et al. 2011 ). MRS media were supplemented
with 20 ppm of filter -sterilized (SARTORIUS AG, Germany) selenium Se (IV) (sodium
selenite, Na2SeO3. 5H2O, SIGMA -ALDRICH, Switzerland) and incubated at 40°C up to 24
h. The media centrifuged for 20 min at 4500 g at room temperature to spin down the bacteria
cells. The cell pellets washed two times by Tris -HCl buffer (50mM, pH 7.5) and finally with
ultra-pure water to get the Nano – Se particles fortified cell fraction. The Se content analyzed
by inductively coupled plasma mass spectrometer (ICP -MS) (X series, THERMO Fisher
Scientific, Germany). The obtained selenium Nano spheres were within range 100 -500 nm
range as mentioned by (Eszenyi et al. 2011 ; Prokisch et al. 2011 )
2.2. Animals :
Forty male Wist ar rats (age, 6 weeks) (150- 160g) were obtained from the Animal House
Colony of the National Research Center, Dokki, and Cairo, Egypt. The animals fed a standard
diet (Al Wadi Co., Giza, Egypt), had access to water ad libitum, and provided water ad libidum .
The animals housed in a plastic cage in an air -conditioned room at 25 °C with a 12 -h light/dark
cycle. The animals used for the experiments after one-week acclimatization period. All animals
handled in accordance with the regulation for care and use of experimental animals guidelines
for the National Institutes of Health (NIH) and the Animal Ethics Committee of Kafrelsheikh
University approved the expe rimental protocol (Kafr el -Sheikh, Egypt) .
2.3. Experimental protocol
The experimental protocol summarized in table (1). The experimental protocol was done
according to (Sayed -Ahmed et al. 2010 ). Forty adult male Wister rat were divided into 4 groups
(n=10). Control group received a single I/P injection of saline at the 1st day of experiment. DEN
group received a single (I/P) of DEN (200 mg /kg body weight) according to (Basak et al. 2000 )
at the 1st day of experiment. DEN+Nano -Se group which administrated Nano -selenium (0.5
mg/kg body weight) dispersed in 0.5 mL PBS orally by stomach tu be was given 1 hour before
DEN administration from the beginning of the experiment for one week (Peng et al. 2007 ) and

received a single (I/P) of DEN (200 mg/kg body weight) at the 1st day of experiment. Nano -Se
group received a daily oral administrati on of Nano -Se at dose of (0.5 mg/kg body weight of
dispersed in 0.5 mL PBS orally by stomach tube.

2.4. Body and liver weight :
Total body weight was daily measured from the beginning to the end of the experiment.
2.5. Blood sample :
At the end of experim ental period 7days (24 h after the last treatment of Nano -Se), blood
samples were collected via retro -orbital Venus plexus under light ether anesthesia and
immediately divided into two aliquots .The 1st one containing (EDTA) as anticoagulant for
hematolo gical analysis and kept at ( -20°C) and for estimation of reduced glutathione (GSH) .
The 2nd part without anticoagulant left to clot then centrifuged at 3000 rpm for 15 minutes. Sera
were then, separated into aliquots for individual biochemical and antio xidant biomarkers
(MDA and CAT ) estimations and stored at -20°C.After blood collection, the rats were
sacrificed by decapitation. The liver, were excised and weighed.
2.6. Hematological analysis :
The aliquot contained EDTA (1 mg/ml) used for asses sing red blood corpuscles
(RBCs), hemoglobin (Hb), packed cell volume (PCV), mean corpuscular volume (MCV),
mean corpuscular hemoglobin(MCH), mean corpuscular hemoglobin concentration
(MCHC), Total leucocytic count (TLC) and differ ential leucocyte count (Weiss and Wardrop
2011 )
2.7. Serum biochemical assay :

The serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) evaluated
according to (Reitman and Frankel 1957 ) and gamma glutamyl transferase (γGT) was evaluated
according to (Szasz 1969 ) were measured spectrophotometrically according to manufacturers'
instructions.
2.8. Estimation of antioxidants and lipoperoxidation markers :
Measurement of MDA content as an indicator for lipid peroxidation was performed
according to (Mihara and Uchiyama 1978 ). Antioxidant biomarkers as catalase (CAT)
according to (Aebi 1984 ) and reduced glutat hione (GSH) according to (Beutler et a l. 1963 )
were measured spectrophotometrically according to manufacturers' instructions .
2.9. Histological and immunohistological analyses :
Liver sections got from the right and median lobes fixed in 10% neutral -buffered formalin.
The specimens then dehydrated in a graded series of ethanol, cleared in xylene and embedded
in paraffin wax. Next, the paraffin ‑embedded specimens were sectioned into4 -µm samples for
hematoxylin and eosin staining ( Sigma -Aldrich Chemie GmbH) and immune staining .
Immunohistochem ically staining for placental glutathione S -transferase done as follows. The
serial slices dewaxed, rehydrated, immersed in 0.05 M citrate buffer (pH 6.8) and heated at
100˚C for 8 min in an autoclave. The slides gently rinsed with distilled water or
phosp hate‑buffered saline (PBS; pH 7.2) and treated by hydrogen peroxide 0.3% in absolute
methanol at room temperature for 30 min to inactivate the endogenous peroxidase. The sections
then incubated for 30 min with Protein Block Serum ‑Free (Dako, California, US A), then
overnight at 4˚C with a polyclonal rabbit anti ‑rat GST ‑P antibody (cat. no. 311, Medical &
Biological Laboratories Co., Ltd., Nagoya, Japan; 1:500 dilution). The sections were then
incubated with a goat anti -rabbit secondary antibody (cat. no. K40 03, EnVision+™ System
Horseradish Peroxidase Labelled Polymer; Dako) at room temperature for 30 min. The sections
rinsed with PBS containing 0.1% Tween 20 (Sigma ‑Aldrich, St. Louis, MO, USA) between
each step. Then signal detection by a freshly prepared so lution of 3 , 3'-diaminobenzidine
tetrahydrochloride (Liquid DAB+ Substrate Chromogen System; Dako), then were washed in
distilled water, counter stained with Mayer's hematoxylin, and dehydrated. As a control
procedure, the primary antibody replaced with no rmal rabbit serum. The analysis of GST -P-
positive foci was then done as previously described by (Abdo et al. 2013 )

2.10. Statistical analysis
The data analyzed by one -way analysis of variance (ANOVA) with the General Linear
Model using SPSS Sta tistics 17.0 (Statistical Packages for the Social Sciences, released 23
August 2008). The significant differences among means of treatments compared by Tukey’s
multiple test. P<0.05 was set as limit of significance.
3. Results
3.1. Body and liver weight :
In all the experimental groups , the rats survived throughout the study period with no
observed severe adverse clinical effects. The final body and liver weights of the rats from the
different treatment groups presented in Table (2 ). The rats treated with DEN revealed increased
absolute and relative liver weights; however, it was non -significant when compared with
control group. Notably, supplementation with Nano –Se inhibited the increase in the absolute
and relative liver weights that induced by the DEN. Similarly, Nano -Se alone treated group
were also within the normal limits.

3.2. Effect on Hematological Parameters
The effect of DEN intoxication as well as the protective effects of Nano -Se on
hematological parameters are shown in (Table 3).There was no significant difference on
erythrogram in all the examined groups, while the leukogram, revealed a significant (p<0.05)
increase in lymphocyte count ( about 117.2% ) in rat group received only Nano -Se compared
to the control group . Administration of DEN significantly (p<0.05) increase in the total
leucocytic count (TLC) ,neutrophils and monocyte counts ( about 159.8,245.5 and 361.5%

respectively ).Contrary to this , a significant (p<0.05) decline in lymphocyte and eosinophile
counts were reco rded ( about 71.1 and 47.4% respectively) compared to the control group.
Nano -Se co supplementation significantly (P≤ 0.05) ameliorated the stress picture of
leukogram and restored the elevated TLC ,neutrophil and monocyte counts toward the normal
value ( about 66.9,43.8 and 49.2% respectively) ,improving the declined lymphocyte and
eosinophil counts (about 138.5 and 198% respectively) compared to DEN treated group . Thus,
Nano -Se at a dose of (0.5mg/kg bw) can effectively ameliorate the adverse effects of DEN on
hematological parameters.

3.3. Serum biochemical analysis :
The impact of N ano-Se supplementation against DEN induced toxicity on serum enzymes
shown in (Figure.1 ).There was no significant difference in serum levels of liver function
marker enz ymes in rats received only N ano-Se compared to the control group. However, DEN
treated group showed a significantly (P<0.05) elevate d the serum activities of ALT, AST, and
γ-GT enzymes (about 303.2, 300.9 and194.7 % respectively) compared to the control group.
Moreover , Nano-Se co- administration significantly (P<0.05) ameliorated the changes in the
measured serum parameters (about 46.4, 61.02 and 58.6 5 % respectively) compa red to the
DEN -treated group. These results indicated that Nano -Se could effectivel y abolish ed DEN –
induced hepatic toxici ty.

3.4. Oxidative stress marker and antioxidant activities:
The influences of nano -Se supplementation against DEN on serum MDA content as well as
antioxidant activity presented in (Figure.2 ). Rat group supplemented by Nano-Se only did not
affect MDA values however, revealed a significantly (P<0.05) enhanced GSH and CAT
activities (about 134.8 and 110.8% respectively) compared to control group . DEN
adminestration significantly (P<0.05) increased serum content of MDA (about 213.9%) , while
significantly (P<0.05) depleted GSH and CAT activities (about 45.3 and 63.3% respectively )
compared to the control group . Nano-Se supplement ed group with DEN restored the altered
serum MDA (about 62.4%) and enhanced GSH and CAT activi ties (about 197.9 and 153.9%
respectively) compared to the DEN group.
3.5. Histopathological and immunohistochemical findings
Histopathological and immunohistochemical of DEN and Nano -Se treated groups illustrated
in (Figure. 3 and 4). Liver of control an imals showed normal hepatocytes, which arranged cords
around the central vein. DEN -treated rats showed congestion of central vein and blood
sinusoids, centro -lobular hepatocytes hypertrophy associated with vacuolation of their
cytoplasm as well as periduct al oval cell proliferation and an increase in mitosis. Many of
preneoplastic foci also observed such as clear and eosinophilic types. GST -P immunostaining
markedly expressed in DE N treated group within the hepatic lobules, associated with marked
periportal oval cells hyperplasia. Nano -Se co -administration with DEN revealed a significant
decrease the neoplastic -GST -P positive cell.

4. Discussion
This current experiment designed to elucidate the effect of biologically produced nano
elemental selenium agains t the oxidative stress induced by hepatocarcinogenesis using DEN .
DEN considers ROS -generating carcinogen inducing preneoplastic foci. ROS generated inside
the mammalian bodies are normally neutralized by the endogenous antioxidants (Ibrahim and
Abdel -Daim 2015 ). Antioxidants consider the cellular housekeeper’s through mopping up free
radicals before they cause DNA damage.
The antioxidants may act as free rad ical scavengers, reducing agents, chelating agents
for metals transition , quenchers of singlet oxygen molecules and activators of anti -oxidative

enzymatic defense system to suppress free radical damage in biological systems (Abdel –
Ghaffar 2013 ) ). Cancer in human and animals mostly arise from increased cellular oxygen –
free radicals, which could induce direct chromosomal mutations and abnormalities causing a
blockage of DNA replication (Valavanidis et al. 2013 ).
Selective and efficient targeting of cancer cells is the foremost goal in the improvement
of anti -cancer therapeutics (Pabla and Dong 2012 ). Biologically produced Nano -Se could be
safer and acceptable than the ones produced by chemical process. Selenium detected to be
importantly substitute vitamin E in the diets for preventing and treating of vascular, muscular
and hepatic lesions. Similarly, nan o-particulate form of selenium has hepatoprotective and
anticarcinogeneic effects (Zhang et al. 2004 ). Hematological parameters can be used to
monitor interactions of chemicals with the biological system i n vivo in order to assess
their systemic effects.
In the present investigation, no alteration was detected on the erythrogram in any of the
tested animal groups, however, it was found that, DEN administration caused significant
leukocytosis , marked neutrophilia, and monocytosis with marked lymphocytopenia and
eosinopenia compared to control group as shown in (table.3).DEN also caused significantly
elevated MDA with increased liver biomarker enzymes as ALT , AST and GGT.
The association between the pres ence of elevated neutrophil count and MDA as an
oxidative stress marker with depletion of antioxidant activity of CAT and GSH level can be
supported by finding of (Amulic et al. 2012 ), who explained the neutrophil -mediated pro –
tumor mechanism as being multifactorial giving numerous inflammatory mediators, cytotoxic
molecules, and mitogeni c factors released by neutrophils . Neutrophils induce oxidative DNA
damage in bystander cells by intercellular transfer of ROS (Jaeschke 2006 ). These data was
potentiated by finding of (Wilson et al. 2015 ) who examined human HCC tissue intrigued by
the juxtaposition of neutrophil rich inflammatory foci with hepatocytes positive for 8 –
hydroxyguanosine, a biomarker for oxidative DNA damage.
Neutrophils induce bystander ROS and DNA damage in hepatocytes. Moreover, when
primary hepatocytes were co -cultured either by direct contact with neutrophils or indirectly
exposed to neutrophil -derived diffusible factors by means of trans well culture these exposures
were sufficient to elevate hepatocellular ROS. Thus, providing an in vivo support for ROS as
a mediator of neutrophil -stimulated HCC in mice providing a clear in vivo correlation between
neutrophil -derived ROS , oxidative DNA damage and development of HCC.

Our result showed that co administration of Nano -Se relief the adverse effect of DEN and
return the different value of leukocyte, neutrophils, monocytes , lymphocytes and eosinophils
to the control level. Presence of significant (p<0.05) differences on TLC, lymphocytes and
neutrophil between treatment group is in line with (Shlig 2009 )and (Surai 2006 ) who detected
enhancement of the growth rate of broiler s fed an organic Se in their diet which could be
related to the increased concentrations of the active form of thyroid hormone in their
serum as well as to the immunomodulating properties of selenium (McKenzie et al. 1998 ),
who found that, supplementing experimental animals with selenium was associated with
enhancing in natural killer cell activity, T -cell proliferation, lymphokine -activated killer cell
activity, delayed -type hyper sensitivity skin responses, and vaccine -induced immunity.
Several researchers implied that, selenium deficiency as an effective factor on the reduction
of the lymphocyte reproductive potency a nd mention that the importing receptor of transferrin
(is effective in the reproduction of lymphocytes) will be depleted in the animals with selenium
deficiency (Pighetti et al. 1998 ).Similarly, (Lessard et al. 1991 )who believed that se lenium
played a role as an additive factor in enhancing the lymphocyte reproduction . Similarly,
(Kumar et al. 2008 )who stated that Se had no effect on serum total proteins and
albumin/globulin ratio, but could increase the capacity of the immune system to protect cells
from free radical injuries .
The liver transaminase enzymes ALT and AST are considered markers for assessing
liver function, elevated serum lev els of these enzymes are indicating presence of hepatic
cellular damages and loss of functional integrity of the cell membrane (Kumar et al.
2005 ).The current study clarified that , supplementing Nano -Se alone, did not reveal change
in serum enzymes this find ing was supported by (Kumar et al. 2008 )who found that Se had no
effect on serum SGPT and SGOT activities but could increase the capacity of the immune
system to protect cells from free radical injuries . However, DEN administration to rats
provoked marked elevation in serum ALT, AST and γGT activities which indicated
hepatocellular damage.
In our results, the biochemical alterations were confirmed by the histopathologi cal
and immune -histochemical findings which detected that DEN treated group revealed centro –
lobular hepatocytes hypertrophy associated with vacuolation of their cytoplasm as well as an
increase in mitosis ,together with diffusely GST -P immunostaining withi n the hepatic
lobules.This elevation could potentially be attributed the production of ROS which enhanced

the lipid peroxidation and more production of toxic aldehydes as MDA. Furthermore, reduction
and exhaustion of antioxidant defense mechanism enzymes a nd finally occurrence of
hepatocellular injury and necrosis with a result of releasing the intracellular enzymes as ALT,
AST and γGT as shown in (Figure.1). The increase in the activity of these biomarkers reflects
active liver dysfunction is matched with that of (Nyblom et al. 2006 ). In the current study,
DEN -induced significantly increased MDA levels ( product of lipid peroxidation) together with
reduce antioxidant enzymatic system as catalase and non -enzyme antioxidant GSH as shown
in (Figure.2). Glutathione considers one of the most abundant tr i-peptide, biological non –
enzymatic antioxidant present in the liver. It removes free radical species such as hydrogen
peroxide , superoxide radicals and maintains membrane protein thiols. Decreased level of GSH
is associated with an enhanced lipid p eroxidation. Imbalance between production and
neutralization of ROS results in serious disturbances in cells metabolism and induces oxidative
damage to biomolecules, DNA, lipids and proteins (Eldahshan and Abdel -Daim 2015 ).DEN
may initiates a series of mutagenic environment which lead to cancer.
An explanation for the oxidative stress in livers of HCC -induc ed rats may be attributed to
that DEN is mainly metabolized in the liver by the action of cytochrome p450 enzymes
specifically CYP -(2E1) to ethyldiazonium ion which alkylates DNA bases to form
promutagenic adducts such as O6 -ethyldeoxyguanosine and O4and O6-
ethyldeoxythymidine,these reactive metabolites are primarily responsible for its hepatotoxic
effects by inducing oxidative stress and cytotoxicity by damaging biomolecules such as DNA,
lipids and proteins (Verna et al. 1996 ).Our results are in line with (El Mesallamy et al. 2011 )
who stated that DEN altered antioxidative defense as indicated by a significant elevation in the
level of oxidative stress marker (MDA) and a significant impairment of free radical scavenging
antioxidants (GR, GPx, SOD and GSH). Depletion in GSH level and GSH dependent enzym es,
glutathione peroxidase (GPx) and glutathione reductase (GR) in DEN treated rats attributed to
the reduction in their biosynthesis during hepatocellular damage or their excessive utilization
in scavenging the free radicals formed during the metabolism o f DEN . Additionally, the
decreased levels of cellular GSH might have caused a reduction in the activities of GSH –
dependent enzymes, GPx and GR, as GSH is a vital co -factor for these enzymes (Pradeep et al.
2007 )Nano -SE supplementation succeeded in re storing hepatic integrity by lowering oxidative
stress, as the administration of Se nanoparticles caused a significant increase in antioxidant
activities (GSH and CAT ) and depletion of MDA level. Thus, counter act the DEN adverse
effect on oxidative stress and antioxidant status .

Our histopathological studies provide a supportive evidence for this. Our findings are in
harmony with (Sadeghian et al. 2012 )who clarified that when Nano -selenium was
administered to sheep as feed supplements, it reduce d the levels of thiobarbituric acid reactive
substances (TBARS) in plasma indicating a decrease in lipid peroxidation . Similarly,
antioxidant effects of inorganic, organic, and elemental nano -selenium were studied in growing
weaned Taihang black male goats in a 90-day experiment ,higher antioxidative activities were
observed in nanoparticle -treated animal compared to control (Shi et al .,2011b) .Similarly,
improved antioxidant status were monitored when they were applied as dietary
supplementation in a fish like crucian carp (Carassius auratus Gibelio ) (Zhou et al. 2009 )
Treatment with Nano -Se prevented the depletion of antioxidant enzyme activity and
restored the activities of these enzymes toward normal, thus, providing protection of bio –
membrane from oxidative attack. Our observations are in line with reports of (Zarei and
Shivanandappa 2013 ) who stated GSH depletion leads to lowering cellular defense against free
radical -induced cellular injury resulting in necrotic cell death.
Our result support that the hathepatoprotective effect of Nano -Se against DEN -induced
toxicity and hepatic damage involve d inhibition of lipid peroxidation and enhancement of
antioxidant enzyme levels in addition to free radicals scavenging action. This could indicate
the potent antioxidant acti vity possessed by elemental -Se.
On the other hand, Nano -Se co -administration with DEN revealed significant decrease the
neoplasti c-GST -P positive cell , which could be indicate the ability of nano -Se to decrease this
foci .Our result is in line with (Abaker et al. ).For explaining, the mechanism for inhibiti ng the
proliferation in cancer cells treated with drugs could be duo to induction of apoptosis or cell
cycle arrest or a combination of these two actions . (Luo et al. 2012 )recorded that HeLa cells
treated with nano -se arrested in the S phase. When cancer cells arrested in the S phase, the
mitosis and proliferation of cancer cell s are inhibited. Thus, nano -se could significantly inhibit
the growth and proliferation of HeLa cells. On the same line, (Ahmed et al. 2014 ) who detected
that nano -se enhanced the necrotic/apoptotic rate in liver tissues of HCC -rats. Thus , nano -se
could be able to inhibit the proliferation in cancer cells due to induction of apoptosis and arrest
the cell cycle or a combination of these two modes.
Conclusion

Our results clearly indicate that Nano -Se was able to enhance the level of the defense
system of the animals, leading to neoplastic resistant as shown i n histopathological and
immunehistochemical figures as well as its beneficial effects upon hematological,
biochemical ,oxidative stress indicating that Nano -Se has chemo -protecti ng efficiency
against HCC induced by DEN in rats .
Acknowledgements.
The authors are grateful to Professor Mohsen Zommara Department of Dairy Science,
Faculty of Agriculture,Kafrelsheikh University, Egypt and Professor Jozsef Prokisch, Centre
for Agricultural Sciences and Engineering, H -4032 Debrecen ,Institute of Bio and
Environmental Energetics ,University of Debrecen, , Böszörményiút 138, Hungary, for the
supply us with selenium nano -particles.

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