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Sodium nitrite (NaNO2) is an inorganic compound, slightly yellowish crystalline powder that is highly soluble in water. Its chemical formula is NaNO2. It is used in many industries such as rubber chemicals, in photography, bleaching fibers, dyeing and printing textile fabrics, as a corrosion inhibitor and a laboratory reagent. It has been reported that Sodium nitrite has many medical uses as in pulmonary hypertension, myocardial infarction and as an antidote for cyanide poisoning , an intestinal relaxant and a bronchial dilator.
It is also one of the common food additives in preserving of refrigerated meats and meat products to prevent botulism by inhibition of iron-sulfur clusters and hence inhibiting the growth of Clostridium botulinum spores in preserved meats. It also prevents rancidity by inhibition of lipid peroxidation.
Nitrate and nitrite can be found in soil from oxidation of ammonia which results from decomposition of inorganic nitrogenous fertilizers and organic nitrogenous wastes. Then, the plants absorb nitrite from soil during their growth. Nitrite can also be formed in distribution pipes of drinking water by action of Nitrosomonas bacteria in stagnant water or by chloramination of water.
Many cases of toxicities or even death in humans and animals from nitrate and nitrite have been reported . Hence, efforts for reduction of nitrite level in the environment are essential for protection of human health.
The aim of the present study was to test the role of pumpkin seed oil in prevention and/or reduction of toxicity associated with oxidative stress induced by NaNO2.
Sodium nitrite was purchased from Sigma Aldrich (Saint Louis, MO, USA) and was applied as a freshly prepared solution. Pumpkin seed oil was purchased from Nutra Manufacturing, Inc, USA and was applied as oil. Total antioxidant capacity (TAC) (Biodiagnostic, Egypt), Cholesterol kit (Biomed diagnostic, Egypt), Triglycerides kit (Biodiagnostic, Egypt), HDL kit (Spectrum, Egypt).
Forty eight adult healthy male Wistar albino rats weighing 180-200 gm, were purchased from the National Research Center, Cairo, Egypt. Rats were used after one week for proper acclimatization to the animal house conditions (12 h lighting cycle , 25 ± 2°C temperature and 45 ± 5% humidity) and had free access to standard rodent chow; drinking water and specific rats chow diet (obtained from El-Nile Company for chow and chicken). Procedures involving animals and their care were conducted in conformity with the protocols of the Research Advisory Ethical Committee of Faculty of Medicine, Minia University, Egypt.
The rats were randomly divided into 4 groups with 12 rats in each one:
Group I (control group): was received the standard diet only without any treatment.
Group II (pumpkin seed oil group): was received the standard diet with pumpkin seed oil at a dose of 1.5 ml/kg b.w. (body weight)/ day.
Group III (sodium nitrite group): was received the standard diet with sodium nitrite at a dose of 100 mg/kg b.w./day.
Group IV (treated group): was received the standard diet with sodium nitrite and pumpkin seed oil with the same previous doses. Sodium nitrite and pumpkin seed oil were given orally by gastric tube. The medications were given for a period of 5 weeks. Pumpkin seed oil was administered 6 hours after Sodium nitrite. The experimental protocol was approved by the institutional animal care and use committee at the department of pharmacology, Faculty of medicine, Minia University.
At the end of the experimental period, 6 rats / group were injected with 0.5 mg/kg of 0.5% colichicine intraperitoneally 2 hours prior to sacrifing the animals to arrest the mitotic division at metaphase stage to investigate chromosomal abnormalities. All rats were anesthetized with ether and sacrificed by cervical dislocation. Two blood samples were suitably collected after scarification from each rat (6 non dosed colichicine rats) one for biochemical analysis and the other for comet assay. The whole liver and heart were dissected out. Bone marrow cells were collected from both femur of each rat (6 IP dosed colichicine rats) by aspiration.
The blood samples were centrifuged (centrifuge Jantezki, T30, Germany) at 4000g for 10 minutes for serum collection. Sera were separated and freezed at -80°C until biochemical analysis. Total antioxidant capacity (TAC) and serum lipids profile (cholesterol, triglycerides and high density lipoprotein (HDL)) were determined using colorimetric assay kit according to the manufacturer`s instructions. Malondialdhyde (MDA) level as an indicator of lipid peroxidation was determined using the thiobarbituric acid method as described by Buege and Aust who measured the thiobarbituric acid reactive substances (TBARS) concentration.
The comet assay can identify low levels of DNA damage in tissue samples either in vivo or in vitro (any type of cells) and can evaluate the protective effect of antioxidants/micronutrients to the genetic material .
The comet assay under alkaline conditions was done as described by Singh et al., with some modifications according to Grover et al., . Briefly, The cell were embedded in agarose coated slide, their lysis in alkaline buffer 1 hour at 4ºC in dark, alkaline DNA unwinding occur, and finally electrophoresis at 25 V/ 300 mA for 30 minutes. Wash with neutralization buffer and staining with ethidium bromide. The extent of DNA damage was measured by quantitative method; measuring the tail length to measure DNA damage.
Chromosomal aberrations: Chromosome preparation from somatic cells (bone marrow cells) was performed as described by Alder . Femoral bone marrow cells were collected in physiological saline solution (0.9%) sodium chloride and centrifuged at 1200 RPM at 10 minutes. After centrifugation, the supernatant was discarded and potassium chloride solution 0.56% was added to the sediment as hypotonic solution and incubator at 37°C for 30 minutes. The cells were fixed in cold fixative (3:1 methyl alcohol: glacial acetic acid). Centrifugation and fixation were done for three times. Few drops of cell suspension were dropped on clean cold slide which was dropped in 70% ethanol. Alcohol was burnt on a burner to dry the slide.
After complete drying, slides were stained in 10% Geimsa stain in Sorenson buffer for 30 minutes. 50 metaphase for each animal was examined and structural chromosomal aberrations were recorded.
Discussion
Nitrite is a common contaminant present in natural water and the atmosphere . It is also an essential antimicrobial food additive that prevents the growth of botulinum toxin and inhibits rancidity during food storage. Nitrite is introduced to the environment from agricultural sources, the mishandling of inorganic fertilizers with the misuse of our natural resources that resulting in the disturbance of nitrogen cycles.
Conclusion
Chronic exposure to high level of sodium nitrite leads to biochemical effect, DNA damage and chromosomal aberrations. The pumpkin seed oil can ameliorate the toxic effect induced by sodium nitrite due to its antioxidant, antimutagenic and antifree radicals effects as it contains high amounts of antioxidant vitamins as α- and γ-tocoferol, β carotene and vitamin E. Moreover, it contains phenolic compounds such as vanillic acid, tyrosol and vanillin and high levels of selenium and lutein. Hence, we can use the pumpkin seed oil as a prophylaxis against NaNO2 toxicity, taking into consideration the dose and duration of treatment.
- Author : Dr. Walaa Yehia Abdelzaher, Minia University, EGYPT.