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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 Summary 174 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 Summary 175 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). Summary 176 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. Summary 177 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 Summary 178 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 Summary 179 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 Summary 180 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 Summary 181 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. Summary 182 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. |