الفهرس 
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Abstract
Hypoxia refers to low concentrations of oxygen in the body
or body parts. The condition arises from a variance between the
amount of oxygen demanded by the body and the amount of
oxygen in circulating blood that is supplied. Sodium nitrite
(NaNO2) is a water soluble, inorganic salt widely used in various
industries including agricultural, chemical industry, textile
processing industry, disinfectants, coloring agents,….. etc.
Humans are exposed to nitrate and nitrite beside to the previous
things, also through food and drinking water, with a minor
contribution from the contaminate air. It has been found to inhibit
growth of microorganisms causing disease, and it is a common
food additive used as a color fixative and preservative mainly in
meats and fishes. So, It had a side effect on the body tissues.
The body has adaptive reacts to hypoxia with acclamation
responses, such as relaxation of smooth muscle, angiogenesis and
vasodilatation blood vessels , thus increasing blood supply to
tissues, compensating for the lack of oxygen. There are literature
data mainly for the effect of sodium nitrite induced hypoxia on
liver, lung, testes. But still subsequent hypoxia is poorly
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investigated and data about its influence on liver, lung
reproductive system are controversial. The widespread use of
NaNO2 in the food industry contributes to its potential health risk.
Stem cells have generated a great deal of excitement and
promise as a potential source for cell based therapeutic strategies.
Stem cells are undifferentiated biological cells that can
differentiate into specialized cells and can incubated (through
mitosis) to produce more stem cells. They are found in
multicellular organisms. In mammals, there are two broad types of
stem cells: embryonic stem cells, and adult stem cells, which are
found in various tissues. In adult organisms, stem cells and
progenitor cells act as a repair system for the body, replenishing
adult tissues. In a developing embryo, stem cells can differentiate
into all the specialized cells—ectoderm, endoderm and mesoderm
—but also maintain the normal turnover of regenerative organs,
such as blood, skin, or intestinal tissues.
Aim of the work:
The primary aim was to conduct the toxicological studies
by NaNO2 on the liver, testes and lungs of adult male rats. Also, to
investigate the effect of treated of sodium nitrite (NaNO2)
administration on Nitric oxide (NO), Malondialdhyde (MDA),
DNA fragmentation percentage (DNA F%), catalase (CAT) and
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total antioxidant activity (TAT), on the investigated tissues and to
show the histological investigation and assay the possible
pathological lesions could be found, and ultra-structure changes
occurred due to sodium nitrite toxicity in liver.
Also, to examine the possible protective effect of each of
the stem cells and recovery period against NaNO2 toxicity –
induced chronic hypoxia. Finally, comparing which is useful way
of treatment
Experimental animals:
A total number of ninety six adult male rats (weighing 150-180g)
were divided into six main groups; each consists of sixteen rats as
follows:
group 1: Rats served as controls being received a
subcutaneous injection of the drug solvent (distilled water) 8
rats for 3 weeks and 8 rats for 6 weeks daily at the morning,
and then were sacrificed in amount equivalent to that used
with the corresponding treated animals then they were
sacrificed (Control group).
group 2: Rats were administered NaNO2 subcutaneous
injection at dose of 35 mg/kg b.wt/ day for 3 weeks daily at
the morning, and then were sacrificed (Hpx group).
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group 3: Rats received NaNO2 subcutaneous injection at
dose of 35 mg/kg b.wt/ day for 2 weeks daily at the morning,
then injected with MSCs (2*106 cells intravenously, once)
according to Kebriaei et al. (2009) then scarification was
done after 4 weeks from the MSCs treatment (N-2WS
group).
group 4: Rats treated with NaNO2 subcutaneous injection at
dose of 35 mg/kg b.wt/ day for 2 weeks daily at the morning,
then injected with MSCs (2*106 cells intravenously, once)
followed by 1 week injection with NaNO2 at dose 35 mg/kg
b.wt/ day. The scarification was done after 4 weeks from
MSCs treatment (N-3WS group).
group 5: Rats treated with NaNO2 subcutaneous injection at
dose of 35 mg/kg b.wt/ day for 2 weeks daily at the morning,
then left to make recovery for 4 weeks before they were
sacrificed (N-2WR group).
group 6: Rats treated with NaNO2 subcutaneous injection at
dose of 35 mg/kg b.wt/ day for 3 weeks daily at the morning,
then left to make recovery for 3 weeks after that they were
sacrificed (N-3WR group).
The duration of the Hpx group and half of control group were
three weeks before decapitation. While, the duration of other
groups were six weeks.
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At the end of the experimental duration, the rats of all groups
were scarified. A part from liver, lung and testes, and
immediately excised on jacket ice, it used for estimation of
Nitric oxide (NO) contents, Malondialdhyde contents (MDA),
DNA fragmentation percentage (DNA F%), catalase (CAT)
Activity and total antioxidant activity (TAA). Also, a fresh
liver, lung and testes tissue were prepared for histological
investigation to detected the possible lesions can be seen. Also,
liver tissue was prepared for ultra-structure investigation.
The results:
A) The biochemical investigation of liver, lung and testes:
NO content: sodium nitrite administration caused a
significant increasing in liver, lung and testes NO content
comparing with the control. While, the treatment with MSCs
caused a significant decrease in liver, lung and testes NO
content comparing with the control.
MDA content: NaNO2 administration caused a significant
increase in liver, lung and testes MDA content comparing
with the control. While, the treatment with MSCs caused a
significant decrease in liver, lung and testes MDA content
comparing with the control.
DNA F%: sodium nitrite administration caused a significant
increase in liver, lung and testes DNA fragmentation
percentage comparing with the control. While, the treatment
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with MSCs caused a significant decrease in liver, lung and
testes DNA fragmentation percentage comparing with the
control.
CAT: sodium nitrite administration caused a significant
decrease in liver, lung and testes CAT activity comparing
with the control. While, the treatment with MSCs caused a
significant increase in liver, lung and testes CAT activity
comparing with the control.
TAA: sodium nitrite administration caused a significant
decrease in liver, lung and testes TAA activity comparing
with the control. While, the treatment with MSCs caused a
significant increase in liver, lung and testes TAA activity
comparing with the control.
B) Histological investigation:
The structural changes in rat’s liver tissue in experimental
induced hypoxic effect, after daily injection with NaNO2 (35
mg/kg b.wt/ day sc) in (Hpx group), manifested in
hypertrophied, hydropic degenerated hepatocytes, other noticed
changes were lymphocytes inflammation around bile duct and
portal areas and fibrous bands around portal vein, cytoplasmic
vacuolation hepatocytes induced faintly stain, hyaline material
scattered in blood sinusoids. Also, showed a massive number of
apoptotic bodies, was obviously with activated Kupffer cells
acting phagocytes in sinusoidal spaces, rupture of endothelial
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lining of central vein. Fibrous and around the central vein
congested with foam cells. The treatment with MSCs showed
repairing and reforms of liver cells; some areas appeared nearly
normal, with persistent focal degenerative vacuoles within
dilated blood sinusoids in some areas in the liver tissue, and
eventually with regenerative capacity of centrilobular areas,
accompanied significant interstitial lymphocytes inflammation.
As regards to the liver tissue of rats in each of groups N-2WR
and N-3WR groups, there were slightly improvement occurred
among the hepatocytes, due to re-oxygenation which had
ameliorative effects on the tissue activity.
The structural changes in lung tissue induced by NaNO2
administration, in (Hpx) group revealed, inflammation common
around bronchi and bronchioles, hyaline material in lung alveoli
walls and fibrosis alveolar septa, collapse of alveolar with loss of
normal architecture, degenerative features of pneumocytes type I
and II, congestion of the blood vessels and thickening of their
walls could be seen with extravasations of RBCs. Fibrosis of
thickened lung alveoli wall filled with granular necrotic debris and
minimal fatty degeneration. In addition, hypoxic lung tissues
manifested hyaline material in the lung bronchioles pink
membranes are partly hyperplasia of their lining epithelium,
included increasing of the alveolar macrophages. Under the
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positive impact of stem cells (MSCs), lung tissue revealed
alveolar wall lined by flat pneumocyte type I and cubical
pneumocyte type II, with vascular tissue resembling an alveolar
wall. A few macrophages are present within the inter alveolar
septa. These features nearly to normal structural arrangements.
The relatively previous regenerations were noticed also due to reoxygenated
tissue in each of N-2WR and N-3WR groups.
The testes tissue from hypoxic group after daily
administration of NaNO2 (35 mg/kg b.wt/ day sc), illustrated
that the majority of somniferous tubules exhibited
vacuolization, lipid accumulations in interstitium space with
gelatinous material. Moreover, rupture of the semniferous
tubules membrane with hyperplasia in its germinal epithelial
layer cells and karyorrhexis in some nucleus of primary
spermatocytes, Also, dilated interstitial blood vessels with
absence the leydge cells and sertoli cells, disappearance of
many spermatogoneic cells and maturation arreast. On the other
side the treatment with MSCs was apparent relatively normal
spermatogonesis, with significantly loss of different cells of
spermatocytes. Besides, some seminiferous tubules appeared
lack spermatids, other tubules displayed hyaline materials
between spermatogenic layers with necrotic spermatocytes and
other germinal epithelium cells with pyknotic nuclei. Mild
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improvement in the testicular structure tissue of rats were clear
in each of the groups N-2WR and N-3WR groups.
C- Ultra structural investigations of Liver:
Electron microscope findings of the liver tissue from rats
treated with NaNO2 showed plasma membrane blebs and
activated lysosomal body. The nuclear membrane appeared
destroyed, lysed swollen mitochondria, with empty
degenerative areas in mitochondria matrix. and ill-defined
endoplasmic reticulum. A fibrous capsule showed with
nucleolar segregation. Also, there are dilated smooth
endoplasmic reticulum cavities (SER), degenerated hepatocyte
nucleus note faint appearance of destroyed mitochondria.
Variable size of lipid droplets, condensed nucleus with
disintegrated of its envelope were obvious.
Ultra structural investigation of liver tissue from Hpx group
followed by stem cell treatment showed marked improvement
of mitochondrial cristae and represented by its self division,
RER lamella and nuclear envelope were reformed with changes
in hepatocyte. In addition, granularity of numerous per
chromatin was noticed at periphery of condensed chromatin
swelling of internal compartment with fragmentation of cristae.
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D- It can be concluded that:
Administration of sodium nitrite has impact on liver, lung
and testes tissues evidenced by biochemical and histological
investigation. It revealed as the increasing in NO, MDA and DNA
F% in the liver, lung and testes tissues. On the other side, there are
decreases in the CAT and TAA parameters. Nevertheless, the
treatment with MSCs gives a sign up of improvement in
biochemical and histological features, after a recovery period
recorded a mild improvement of the biological parameters and
investigated tissues too. It can be concluded that MSCs may
reduce oxidative stress and improve the hazard effects of NaNO2.