PROTECTIVE EFFECT OF GRAPE SEEDS EXTRACT AGAINST SODIUM NITRITE-INDUCED TOXICITY AND OXIDATIVE STRESS IN ALBINO RATS

Sodium nitrite (NaNO2) is important antimicrobial, flavoring, coloring and preservative agent in meat and fish product. However, nitrite may cause methemoglobinemia and other illness, and may react with certain amines to form carcinogenic nitrosamines. The study aimed to evaluate the efficacy of grape seeds extract (GSE) as a potential novel and useful strategy for the modulation of oxidative stress and toxicity induced by NaNo2 in male rats. Sodium nitrite was used at two dose levels 1 and 2g/L and orally administrated to rats in drinking water for 4 and 8 weeks. Ingestion of NaNO2, resulted in significant time and dosedependent reduction in RBCs count, WBCs count, hemoglobin (Hb) content, serum albumin and total protein (TP), plasma reduced glutathione (GSH), glutathione -Stransferase (GST), superoxide dismutase (SOD) contents in the treated rats. whereas, blood methemoglobin (MetHb), serum alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (GGT), creatinine, urea, triglycerides, total cholesterol and lipid peroxidation product (malondialdehyde, MDA) were significantly increased in dose and time dependant manner after implication of this preservative. Hyperglycemia was observed in NaNO2 ingested rats. Moreover, histopathological examination of NaNO2 treated rats at the end of experiment revealed marked alterations in liver, kidneys, brain, heart, lung and testes. The degree of severity of noted lesions increased with increase of administrated dose. Fortunately, synergistic administration of ethanolic GSE (150 mg/kg body weight (B.W)/day) and NaNO2 resulted in significant amelioration of negative effect of NaNO2 on the investigated biochemical parameters and improvement of investigated antioxidant especially with low NaNO2 dose and longer time. Moreover, GSE administration was able to protect most of tested organs against low NaNO2 dose and minimize the damage induced by high NaNO2 dose. It was concluded that GSE supplementation could be considered as a promising antioxidant in reducing oxidative stress and toxicity of NaNO2.


INTRODUCTION
Sodium nitrite (NaNO 2 ) is an inorganic salt used in the manufacture of dyes and considered as one of the important food additives for fishes and meat that has been used in vivo and in vitro experiments for decades to prevent growth of Clostridium botulinum, the bacteria which causes botulism (Luca et al., 1987).Also, it is used in drug industries and in medicine as antidote for cyanide poisoning (Filvo et al., 1993).Nitrites occupy a unique position in human toxicology.They ubiquitous in the environment and can be formed from nitrogenous compound by microorganisms present in the soil, water, saliva and gastrointestinal tract (Chow and Hong, 2002).Moreover nitrite (No 2 -) is a contaminant of some water supplies, extensive use of nitrogenous fertilizer, decomposition of plants and sewage wastes, followed by leaching of nitrates into ground water (Wolff and Wasserman, 1972).Nitrite is widely consumed from the diet by animals and humans.However the largest contribution to exposure results from the in vivo conversion of exogenously derived nitrate to nitrite.Because of its potential to cause methaemoglobin (MetHb) formation at excessive levels of intake, nitrite is regulated in feed and water as an undesirable substance.Forages and contaminated water have been shown to contain high levels of nitrate and represent the largest contributor to nitrite exposure for food-producing animals (Cockburn et al., 2013).A human health risk assessment was also carried out taking into account all direct and indirect sources of nitrite from the human diet, including carry-over of nitrite in animal-based products such as milk, eggs and meat products.The FAO/WHO, Joint Expert Committee on Food Additive (JECFA) established acceptable daily intakes (ADIs) of 0-0.07 mg kg -1 body weight for NaNO 2 (expressed as nitrite ion) (WHO, 2003).In order to protect animal and human health, the European Union, directive, 2002/32/EC on undesirable substances in animal feed, restricts the maximum content of nitrite in complete feeding stuff (with a moisture content of 12%) for livestock excluding birds and aquarium fish to 15 mg/kg, and the maximum content of fish meal to 60 mg/kg (EC, 2002).
The hazardous effect of NaNO 2 is derived from the reaction of nitrite with amines and amides to produce nitrosamines and nitrosamides, respectively.The toxic effects of nitrates and nitrites are well documented in mammalians, including impairment of reproductive function, hepatotoxicity and methaemoglobinemia, dysregulation of inflammatory responses and tissue injury, growth retardation and endocrine disturbance (Jahries et al., 1986).Nitrite is, however, also a natural constituent in the body, it is a potential nitric oxide (NO) donor and recent research has suggested that nitrite has important biological functions at low concentrations (Jensen, 2007).Nitric oxide free radical (NO • ) is generated from the nitrite by non-enzymatic method and O • formation is found in acidic environments such as the stomach and oral cavity (Lundberg et al., 1994 andMcKnight et al., 1997).Peroxy nitrite (ONOO -), the reaction product formed between NO • and superoxide plays a critical role in the induction of inflammatory reaction and apoptosis.O • is also associated with tumor promotion and/or progression (Radi et al., 1991).Anyway, nitrite when present at high concentration in blood, it can react with iron (III) of the hemoglobin, forming MetHb which has no oxygen-carrying ability.This fatal disease is called methemoglobinaemia (Zatar et al., 1999 andSanchez-Echaniz et al., 2001).The use of permitted colors has raised concern that they might be used in excess of the saturatory limit (100 mg kg -1 ) or in foods in which they were not permitted (Rao and Bhat, 2003).
An interest in natural antioxidants, especially of vegetal origin, has greatly increased in recent years.Natural antioxidants can protect the human body from free radicals that may lead to the aging process and cause some chronic diseases including cancer, cardiovascular diseases and cataract as well as retard lipid oxidative rancidity in foods (Craig andBeck, 1999 andLai et al., 2001).
Grape, one of the world's largest fruit crops, with more than 60 million tons is cultivated mainly as Vitis vinifera for wine production (Amico et al., 2004).It is estimated that around 13% of the total weight of grapes used for the wine and juice making results in grape pomace, which is a byproduct in this process (Torres et al., 2002).One way of using the potential of the grape pomace is the isolation of seeds and extraction of the polyphenols, since the total extractable phenolics in grape are a maximum 10% in pulp, 60 to70 % in the seeds and 28 to 35% in the skin (Shi et al., 2003).Phenolic compounds from grape seeds, as other phenolic compounds have pharmacological and nutraceutical benefits both antiviral and antimutagenic (Saito et al., 1998).
It has proved that, free radical scavenging capacity of GSE is 20 times more effective than vitamin E and 50 times more effective than vitamin C. GSE is beneficial in many areas of health because of its antioxidant effect to bond with collagen, promoting youthful skin, cell health, elasticity, and flexibility.Moreover, proanthocyanidins help to protect the body from sun damage, to improve vision, to improve flexibility in joints, arteries, and body tissues such as the heart, and to improve blood circulation by strengthening capillaries, arteries, and veins.The most abundant phenolic compounds isolated from grape seed are catechins, epicatechin, procyanidin, and some dimers and trimers (Shi et al., 2003).
Here, we investigated the role of GSE in ameliorating NaNO 2 -induced abnormalities in the hematological and biochemical parameters and injury of body organs associated with oxidative stress in male albino rats.

Chemicals
NaNO 2 were obtained from Sigma Chemical Co.(St Louis, MO, USA) and all other chemicals and solvents were of analytical grade and purchased from Merck (Darmstadt, Germany), unless stated otherwise.Kits used for the estimation of GSH, GST, SOD and MDA were purchased from Biodiagnostic, France.

Plant materials
Dried grape seeds were bought from Harrase Company Giza, Egypt.The dried seeds were ground to a fine powder with electric blender, and kept at 4 °C until further use.

Hydroalcoholic extract of dried grape seeds
About 500 g of the powder was mixed with 1000 ml of 70% ethanol in distilled water and kept for 3 days at room temperature.The extract was then filtered through a Buchner funnel.The filtrate was evaporated at 45 °C in a rotary evaporator to concentrate the solution, then lyophilized in order to obtain the dry extract and stored at 4 °C until use (Hemmati et al., 2008).
Enough amounts of the dried extract were suspended in water and administered orally by gavages to rats for 8 weeks (150 mg/kg/day).
At the 4 th week and 8 th week of the treatment period, blood samples were collected from the retro-orbital venous plexus under carbon dioxide anesthesia into three tubes.Two contain heparin, one used as fresh as possible for hematological parameters and other to obtain plasma.The third tube contains no anticoagulant to obtain serum.Plasma and serum separated and stored at −20°C until analysis.Rats were scarified at the end of experiment and livers, kidneys, brains, hearts, lungs and testes were collected and kept for histological examination.

Hematological analysis
Whole blood was analyzed for: Hb, RBCs and WBCs using an animal blood veterinary counter (ABC vet user Manual, RABOSIA Inc. A, France).

Estimation of methemoglobin (MetHb)
Aliquots of fresh blood were hemolyzed in water (1:5.5, v/v).After 3 min, phosphate buffer was added, and the samples were centrifuged at 14,000 rpm for 15 min.Measurements of MetHb in hemolysates was based on the absorbance of MetHb spectrophotometrically at 630 nm.Addition of cyanide eliminates the contribution of methemoglobin to the absorbance at 630 nm.The absorbance of the hemolysate at 630 nm was first determined in the absence of cyanide.To 100 µl of the hemolysate was added 50 µl of a neutralized cyanide solution containing 5.3% w/v sodium cyanide and 5.6% v/v acetic acid, and the absorbance of the cyanide-treated sample was reread at 630 nm.The absorbance in the absence of cyanide minus that in the presence of cyanide is a measure of the conversion of MetHb in the sample to cyanomethemoglobin which is proportional to concentration of MetHb (Kohn et al., 2002).

Biochemical assays
The ACE Alera Clinical Chemistry System Automatic Analyzer (Alfa Wasserman Corporation) was employed to measure the following parameters: ALT, AST, ALP, GGT, TP, albumin, creatinine, urea, total cholesterol, triglycerides and glucose in serum.

Estimation of antioxidant profile
The extent of lipid peroxidation in terms of thiobarbituric acid reactive substances (TBARS) which react with malondialdehyde (MDA) in acidic medium was measured in serum calorimetrically at 534 nm according to the method of Onkawa et al. (1979).Plasma GSH was estimated calorimetrically at 405 nm based on the reduction of 5, 5 dithiobis (2nitrobenzoic acid) (DTNB) with GSH to produce yellow compound which directly proportional to GSH concentration (Beutler et al., 1963).Plasma GST was determined spectrophotometrically by measuring the conjugation of 1-chloro-2,4-dinitobenzene (CDNB) with reduced glutathione.The conjucation is accompined by an increase absorbance at 340 nm.The rate of increase is directly proportional to the GST activity (Habig et al., 1974).Plasma SOD activity was estimated spectrophotometrically at 560 nm based on the method described by Nishikimi et al. (1972) which relies on the ability of the enzyme to inhibite the phenazine methosulphate-mediated reduction of nitroblue tetrazolium dye.

Histopathological studies:
Livers, kidneys, brains, hearts, lungs and testes from each group were fixed in 10% buffered formalin, routinely processed and embedded in paraffin.Sections of 4-6 µm thickness were prepared and stained with Haematoxylin and Eosin.Sections were then examined for histopathological findings (Banchroft et al., 1996).

Statistical analysis:
Statistical analysis of the obtained data was done using the least significant difference test (LSD) at the 5% level of probability as outlined by Snedecor and Cochran (1980).Using the Duncan test institute program used a computer in the statistical analysis.

RESULTS
In this study, the data as shown in tables (1-7) recorded that, the administration of GSE alone (G2) did not induce any obvious changes in all examined parameters along experimental periods (comparing to control (G1) (P<0.05)).

Hematological analysis:
Results presented in table (1) demonstrated that NaNO 2 administration for period of 4 and 8 weeks at both 2 dose levels (rats of G3 and G4) resulted in a significant dosedependent reduction (P<0.05) of blood WBCs, RBCs count and Hb concentration.However, supplementation of NaNO 2 -intoxicated rats with GSE (G5 and G6) ameliorated the adverse effect of both NaNO 2 doses on hematological parameters especially 1 g/L NaNO 2 and at 8 th week of experiment.Whereas, GSE administration restore RBCs count to normal in blood of rats treated with 1g/L NaNO 2 at 8 th week of treatment (comparing with control (P<0.05)).

Methemoglobin concentration:
MetHb concentration was significantly elevated (corresponding to control group (P<0.05)) in time and dose dependant manner in blood of NaNO 2 -treated rats of G3 and G4 as shown in table (1).Meanwhile, co-administration of GSE with 1g and 2 g/L of NaNO 2 (G5 and G6 respectively) resulted in attenuation of MetHb formation (compared to corresponding control (P<0.05)) in all experimental periods.Moreover, GSE was able to block MetHb formation in blood of 1g NaNO 2 -treated rats and restore it to normal level comparing with control group (G1).

Liver function parameters
Table (2) show the effect of GSE administration on liver function in serum of rats treated with NaNO 2 .Analysis of variance indicated that there was a significant dose dependant-increase in ALT, AST, ALP and GGT activities, whereas, a significant dose dependant-decrease in TP and albumin concentrations was detected in rats treated with both NaNO 2 doses for 4 and 8 weeks.However, GSE administration in NaNO 2 -treated rats ameliorated the NaNO 2 adverse effect as evidenced by a significant decrease of ALT, AST, ALP and GGT activities, and increase in TP and albumin concentration especially at low NaNO 2 dose and at 8 th week.The activity of GGT in serum of rats co-administrated with GSE and NaNO 2 was return to normal at 8 th week of treatment (compared with control (P<0.05)).

Kidney function parameters:
The kidney function parameters results summarized in table (3) revealed that there is a significant dose-dependent increase (P<0.05) in creatinine and urea concentrations in serum of NaNO 2 -treated rats (G3 and G4) while GSE intake caused desperation of this increase, in addition GSE administration restore creatinine in serum of 1g NaNO 2 -treated rats (G5) to normal and restore urea level in serum of both groups of NaNO 2 -treated rats (G5 and G6) to normal at 8 th week of treatment (compared to corresponding control (P<0.05)).

Lipid profile parameters:
The data in table (4) shows that NaNO 2 induced significant dose-dependent increase in serum total cholesterol and triglycerides concentrations comparing with control (P<0.05) at all experimental periods.Simultaneous administrations of GSE and NaNO 2 in G5 and G6 rats resulted in significant reduction of increased total cholesterol and triglycerides concentrations and restore cholesterol level at 4 th week of treatment and triglycerides levels along experimental period to normal in serum of 1g NaNO 2 -treated rats comparing with control (P<0.05).

Glucose concentration
As shown in table (5) NaNO 2 treatment induced significant dose-dependent increase in serum glucose level in respect to control group (P<0.05).The administration of GSE significantly alleviated the hyperglycemic effect of NaNO 2 .Such effect was more pronounced with 1g NaNO 2 and seemed to be time dependent as glucose level was return to normal in rats co-administrated with 1g NaNO 2 and GSE at 8 th week of treatment (P<0.05).

Antioxidant profile:
Data in table (6) illustrated that NaNO 2 treatment significantly reduce (P<0.05)plasma GSH, GST and SOD levels in dose-dependent manner in all experimental periods.GSE administration significantly elevate of GSH, GST and SOD levels in plasma of NaNO 2intoxicated rats especially with 1g NaNO 2 dose (G5) and at 8 th week of experiment comparing with corresponding control (P<0.05).GSH and GST levels were restored to normal level at 8 th week of experiment as a result of GSE intake in 1g NaNO 2 -treated rats.
Serum MDA level (lipid peroxidation product) was increased parallel to dose and time of NaNO 2 intake in G3 and G4 rats comparing with control (P<0.05).The administration of GSE ameliorated the NaNO 2 -induced elevation in lipid peroxidation as evidenced by a significant decrease (P<0.05) in MDA production in G5 and G6 rats.Such effect was more pronounced with 1g NaNO 2 and seemed to be time dependent as compared to control rats.The results were shown in table (7).

Histopathological Results:
Liver: Microscopically, examination of liver of rats from G1 (untreated, control) revealed the normal histological structure of hepatic lobules.Also no histopathological changes were noticed in liver of rats from G2 which treated with GSE (fig.1).Conversely, the examined sections from G3 which treated with 1g/L NaNO 2 showed focal hepatic necrosis associated with inflammatory cells infiltration (fig.2), kupffer cells activation and sinusoidal leucocytosis (fig.3).While the liver of rats from G4 which treated with 2g/L NaNO 2 showed dilatation and congestion of hepatic sinusoids (fig.4), portal infiltration with mononuclear cells, focal hepatic necrosis associated with mononuclear cells infiltration (fig.5), cytoplasmic vacuolization of hepatocytes and necrosis of sporadic hepatocytes (fig.6).Meanwhile, some examined sections of liver of rats from G5 which treated with 1g/L NaNO 2 and GSE showed kupffer cells activation (fig.7), while other sections from same group revealed no histological changes (fig.8).Moreover, liver of rats from G6 which treated with 2g/L NaNO 2 and GSE showed cytoplasmic vacuolization of hepatocytes (fig.9).

Testes:
Microscopically, examination of testes of rats from G1 (control, untreated) and rats from G2 (GSE-treated rats) showed normal seminiferous tubules and complete spermatogenesis (fig.43).However, testes of rats from G3 which treated with 1g/L NaNO 2 revealed degeneration of spermatogoneal cells lining seminiferous tubules and necrosis leydig cells (fig.44).Also, examination of testes of rats from G4 which treated with 2g/L NaNO 2 revealed interstitial edema (fig.45) and degeneration of spermatogoneal cells lining seminiferous tubules (fig.46).Meanwhile, testes of rats from G5 and G6 which treated with 1and 2g/L NaNO 2 respectively in combination with GSE revealed no histological changes (figs.47 and 48).(2002) predicts that 10% of the Hb is oxidized to the ferric form after oral doses of 15.9 mg/kg NaNO 2 in male rats and 11.0 mg/kg NaNO 2 in female rats and after intravenous doses of 8.9 and 7.1 mg/kg in male and female rats, respectively.The chemical reactivity of NaNO 2 with Hb may enhance heme-or iron-mediated toxicities.Nitrite is known to cause free radical generation, as it can stimulate oxidation of ferrous ions in oxyhemoglobin to form MetHb as well as various ROS (reactive oxygen species) (Gladwin et al., 2004 andBaky et al., 2010).The nitrite ion, its metabolites, and lipid peroxidation products are supposed to react with sulfhydryl groups of the lipid bilayer and protein components of erythrocyte membrane and change its structure (Maeda et al., 1987).Nitrite-promoted Ca 2+ influx in blood cells activates phospholipases, which increase the proportion of phospholipids with a rigid structure in the membrane (Kaya and Miura, 1982).Moreover, WBCs decrease is associated with the failure of the hematopoietic tissues to produce new WBC (Tan et al., 1992).It is suggested that the decrease in RBCs and Hb concentration may be attributed to microcytic and/or hypochromic anemia possibly as a consequence of the toxic effect of NaNO 2 on bone marrow, spleen and liver (Aboel- Zahab et al., 1997).
This study has shown that GSE alone does not have any negative impact on hematological parameters and MetHb formation.In the current study, the decrease in hematological parameters and increase in MetHb formation was reversed near normalcy by the administration of GSE in all experimental periods, moreover RBCs and MetHb were return to normal at end of experiment in 1g NaNO 2 -treated rats.Similarly, no significant changes in hematological parameters in male and female Sprague-Dawley rats provided with GSE in the diet at levels of 0.5, 1.0, or 2.0%, for a period of 90 days (Wren et al., 2002) and in male and female rats provided with GSE in the diet at levels of 0.63, 1.25 or 2.5%, for a period of 3 months (Bentivegna and Whitney, 2002).While the relative improvement of these parameters may be due to suggested antioxidant activity of GSE in vivo includes stimulating enzyme production of nitric oxide, oxygen radical scavenging and inhibition of nitrositive stress (Bagchi et al., 2000 andRoychowdhury et al., 2001).
The current study revealed that NaNO 2 induced dose-dependent liver damage as evidenced by changes in liver function parameters and histopathological examination.The administration of NaNO 2 for 8 weeks induced dose-dependent increase in ALT, AST, ALP and GGT activities and significant dose dependant-decrease in TP and albumin.Thus increased concentrations of these enzymes in serums normally indicate hepatocellular damage and leakage of these enzymes from liver cells into the blood stream (Carey, 2000).Similar to our results, Helal and Elsaid (2006) reported increased activities of ALT, AST, ALP and GGT and decrease concentrations of TP and albumin in NaNO 2 -treated rats.Also,

Krishnamoorthy and Sangeetha (2008), Hassan et al. (2009) and Efuruibe et al. (2013)
reported increase of ALT, AST, GGT and ALP activities in rats as a result of treatment with NaNO 2 (300 mg/kg B.W for 3o days, 80 mg/kg B.W for 3 months and single dose of 6.20mg, respectively).El-Sheikh and Khalil (2011) fed rats with 0.2% NaNO 2 for 6 weeks and recorded decrease of TP and albumin.The increase in the activity of AST, ALT, GGT and ALP enzymes in the serum of NaNO 2 -treated rats could be attributed to the toxic effect of nitroso-compounds, formed in the acidic environment of the stomach, in causing severe hepatic necrosis Hassan et al. (2009).However, the indicated inhibitory effect of NaNO 2 on the biosynthesis of protein could be attributed to stimulation of the thyroid and the adrenal glands by NaNO 2 which can lead to a blockage in protein synthesis, fast breakdown, and increased rate of free amino acids, and decreased protein turnover (Eremin and Yocharina, 1981).In addition, nitrite interactions results into nitric oxide release, which can inhibit total protein synthesis (Kolpakov et al., 1995).The severity of hepatic lesions in our study was found to be parallel to NaNO 2 concentration in water.The liver of 1 g/L NaNO 2 -treated rats showed focal hepatic necrosis associated with inflammatory cells infiltration, kupffer cells activation and sinusoidal leucocytosis while the liver of 2 g/L NaNO 2 -treated rats showed the same previous lesions besides dilatation and congestion of hepatic sinusoids, portal infiltration with mononuclear cells, cytoplasmic vacuolization of hepatocytes and necrosis of sporadic hepatocytes.Such hepatic damage by NaNO 2 was previously recorded by Gomaa and Abd Elaziz (2011) in rats by 80 mg/kg NaNO 2 oral administration for 2 months.Also, Abebe et al. (2013) recorded severe haemorrhage within the sinusoid and the portal triad was infiltrated by inflammatory cells in liver of NaNO 2 -treated rats (25 and 50mg/ kg).
While, Efuruibe et al. (2013) were reported mild periportal cellular infiltration as a result of NaNO 2 administration in rats.Such liver damage could be explained by formation of free radicals (ONOO -) from nitric oxide.Both NO and oxygen radicals could react further to produce other oxidant and nitro compounds such as peroxynitrite to induce liver injury (Liaudet et al., 2000 andChow andHong 2002) and it play an important role in death of liver cells (Krishnamoorthy and Sangeetha, 2008).
This study has shown that GSE alone does not have any negative impact on liver function parameters and histology of liver.As revealed previously in toxicological studies on GSE by Wren et al. (2002) and Bentivegna and Whitney (2002).The current results revealed that co-treatment with GSE and NaNO 2 resulted in a significant improvement in all liver function parameters and liver histology as indicated by significant alleviation of toxic effect of NaNO 2 on ALT, AST, ALP, TP and albumin levels in serum.Especially with low NaNO 2 dose and at 8 th week of treatment, but it failed to normalize them except GGT, which restored to normal.Moreover the hepatic structure of 1g NaNO 2 and GSE-treated rats was reversed to normal.The protective effect of GSE on liver against oxidative damage was previously recorded by Dulundu et al. (2007) who reported that GSE administration was significantly able to decrease the increase of AST and ALT levels as a result of biliary obstruction induced by bile duct legation in serum of rats due to its antioxidant and antifibrotic prosperities.Chis et al. (2009) suggested that the GSE enhanced the antioxidant defense against reactive oxygen species produced under hyperglycaemic conditions, hence protecting the liver cells.Yousef et al. (2009) reported that GSE antagonized cisplatininduced increase in the activities of AST and ALT and decrease concentrations of TP and albumin in rats.Furthermore, El-Beshbishy et al. (2010) observed that oral intake of GSE to tamoxifen-intoxicated rats resulted in attenuation of histopathological changes in liver and corrected all liver function parameters (ALT, AST, ALP and GGT).More interesting Hassan (2012) noted that the ethanol grape seed extract (EGSE) was more effective than water grape seed extract (WGSE) against hepatotoxicity of alcohol in rats.Hepatoprotective activity of the grape seed extracts could be attributed to the antioxidant effect of the constituents and enhanced antioxidant defenses.
Kidneys represent the major control system maintaining body homeostasis.The serum concentration of urea and creatinine determine renal function and are thus biomarkers for kidney disease (Levey et al., 1999).The present results revealed dose-dependent increase of creatinine and urea concentration in serum of NaNO 2 -intoxicated rats.Such finding was previously reported in rats by Hassan et al. (2009) and El-Sheikh and Khalil (2011).These effects could be attributed to the changes in the threshold of tubular re-absorption, renal blood flow and glomerular filtration rate (GFR) (Zurovsky and Haber, 1995).The elevation in kidney function biomarker was synchronized with observed damage in kidney.The noticed lesions in kidneys of both NaNO 2 -treated groups (1g and 2g/L) showed the same degree of severity.Kidney of NaNO 2 -intoxicated groups revealed vacuolization of endothelial lining glomerular tuft with presence of protein cast in the lumen of renal tubules, hypertrophy of glumerular tuft, periglomerular fibroblasts proliferation, vacuolization and congestion of glumerular tuft, perivascular edema and atrophy of glumerular tuft.Such kidney damage by NaNO 2 was previously recorded by Gomaa and Abd Elaziz (2011) and Abebe et al. (2013).
This study has shown that GSE alone does not have any negative impact on kidney function parameters and histology of kidney.As revealed previously in toxicological studies on GSE by Wren et al. (2002) and Bentivegna and Whitney (2002).The current results revealed that co-treatment with GSE and NaNO 2 resulted in a significant improvement in all kidney function parameters and Kidney histology which indicated by significant alleviation of toxic effect of NaNO 2 on creatinine and urea levels in serum especially with low NaNO 2 dose and at 8 th week of treatment.Both of them were restored to normalcy level at the end of experiment.Moreover the GSE administration reversed toxic effect of both NaNO 2 dose and restore kidney histology to normal.The protective effect of GSE on kidney against oxidative damage was previously recorded by Yousef et al. (2009) and Saad et al. (2009) they reported that GSE antagonized cisplatin-induced increase in concentration of creatinine and urea in serum of rats and damage in Kidney genomic DNA.The mechanism underlying GSE nephroprotection may be due to the marked radical scavenging ability of GSE proanthocyanidins (Sato et al., 2005).
Lipids play an important role in cardiovascular disease, not only by way of hyperlipidaemia and the development of atherosclerosis, but also by modifying the composition, structure, and stability of cellular membranes.Excess lipids in the blood are considered to accelerate the development of arteriosclerosis and are the major risk factor in myocardial infarction.High levels of circulating cholesterol and its accumulation in heart tissue are well associated with cardiovascular damage (Salter and White, 1996).The results of the present study revealed time and dose-dependent increase in total cholesterol and triglycerides concentration in serum of NaNO 2 -treated rats.Such results have been provided by Helal and Elsaid (2006) in NaNO 2 -intoxicated rats.These biochemical changes were coincident with the histopathological changes in heart tissue induced by NaNO 2 in the present study which represented by congestion of myocardial blood vessels and myolysis of focal myocytes in 1g/L NaNO 2 -treated group.And congestion of myocardial blood vessels and zenker's necrosis of sporsdic myocytes in 2g/L NaNO 2 -treated group.In an early chronic toxicity study (Gruener and Shuval, 1973), rats were given nitrite in the drinking water at doses equivalent to 0, 10, 100, 200 and 300 mg/kg B.W/day) corresponding to 0, 6.7, 67, 124, 201 mg/kg B.W per day for nitrite).At the highest dose, focal degeneration and fibrosis of the heart muscle as well as dilatation of coronary arteries were also observed.More recently Gomaa and Abd Elaziz (2011) reported necrosis of myocardial muscle cells in NaNO 2 -treated rats.
It worthy to note that GSE were had no negative effect on total cholesterol, triglycerides and heart tissue.The hypercholesterolemic, increased triglycerides and heart injury induced by NaNO 2 in the present study were significantly alleviated by GSE administration.GSE administration restores cholesterol level at 4 th week of treatment, triglycerides levels along experimental period in serum and heart histology to normal of 1g NaNO 2 -treated rats.These results are in agreement with finding of Karthikeyan et al. (2007) showed that the administration of grape seed proanthocyanidins significantly maintained the cholesterol, phospholipids, triglycerides, and free fatty acids levels in serum and heart tissue of the isoproterenol -induced myocardial injury in rats.The experiments conclude that grape seed proanthocyanidins possess cardioprotective and hypolipidemic effect on the treatment of ISO-induced myocardial injury.Also, Yousef et al. (2009) reported that GSE antagonized cisplatin-induced increase in cholesterol and triglycerides.Furthermore Lipid peroxidation, a type of oxidative degeneration of polyunsaturated fatty acids (PUFA), has been linked with altered membrane structure and enzyme inactivation (Karthikeyan et al., 2007).Consequently, lipid peroxidation is known to induce cellular damage and is responsible for ROS induced organ damage.Lipid peroxidation of membranes is regulated by the availability of substrate in the form of PUFA, the availability of inducers, such as free radicals and excited state molecules, to initiate propagation, the antioxidant defense status of environment, and the physical status of the membrane lipids (Anandan et al., 1998).GSH is one of the essential compounds for regulation of variety of cell functions.It has a direct antioxidant function by reacting with superoxide radicals, peroxy radicals and singlet oxygen followed by the formation of oxidized glutathione (GS-SG) and other disulfides.GST is GSH-dependent antioxidant enzymes (Karthikeyan et al., 2007).GST catalyses the conjugation of GSH via the sulfhydryl group, to electrophilic centers on a wide variety of substrates.This activity is useful in the detoxification of endogenous compounds such as peroxidised lipids (Valavanidisa et al., 2006).The enzyme SOD catalyzes dismutation of the superoxide anion (O -2 ) into hydrogen peroxide (H 2 O 2 ), which is then detoxified to H 2 O by catalase (Mukherjee et al., 2003).
Results of this study showed that NaNO 2 induce oxidative stress in rats as evidenced by dose-dependent increase of serum MDA and dose-dependent decrease of plasma GSH, GST and SOD.Oxidative stress induced by NaNO 2 was previously fully described by Krishnamoorthy and Sangeetha (2008); El-Sheikh and Khalil (2011) and Gomaa and Abd Elaziz (2011).
The present data indicated that treatment with GSE alone didn't alter any of oxidative stress parameters and significantly alleviated the toxic effect of NaNO 2 on antioxidant defense system especially on lower dose and longer time as GSH and GST levels were restored to normal level at 8 th week of experiment as result of GSE intake in 1g NaNO 2treated rats.Such enhancement in antioxidant profile by GSE intake against oxidative stress induced by many chemicals were previously recorded such as isoproterenol Present histopathological results revealed that NaNO 2 treatment for 8 weeks induced dose-dependent neurotoxicity.Brain of 1g/L NaNO 2 -treated rats showed pyknosis of neurons, neuronophagia, menengial hemorrhage associated with inflammatory cells infiltration while brain of 2g/L NaNO 2 -treated rats showed focal hemorrhage, hemorrhage in Virchow space, necrosis of neurons and congestion of cerebral blood vessels.NaNO 2 induced neural toxic effects by exerting oxidative stress and retrograde the endogenous antioxidant system (Hassan et al., 2010).Moreover hypoxic neurotoxicity was induced by 30 mg/kg of NaNO 2 intake dissolved in normal water for 14 day and 75 mg/kg of subcutaneous injection NaNO 2 (Premanand and Ganesh, 2010 and Zaidi, 2010, respectively).
Meanwhile, present study revealed that GSE intake significantly reversed neruotoxic effect of both NaNO 2 dose which induced oxidative stress and restore brain tissue to normal.This suggestion run with data of Balu et al. (2005) demonstrated that GSE enhanced the antioxidant status and decreased the incidence of free radical induced protein oxidation in aged rats thereby protecting the central nervous system from the reactive oxygen species.
In the present investigation, treatment of rats by NaNO 2 for 8 weeks resulted in lung injury as revealed by histological study.The degree of severity of pathological lesions was correlated to concentration of NaNO 2 .Microscopically, lungs of 1g/L NaNO 2 -treatrd rats showed atelectasis and bronchitis.Whereas the examined lungs of rats treated with 2g/L NaNO 2 showed perivascular inflammatory cells infiltration, interstitial pneumonia and pulmonary hemorrhage.Histopathologic changes in lung as a result of NaNO 2 toxicity were also observed by Gruener and Shuval (1973) who stated that, the changes in the lungs consisted of dilatation of the bronchi with infiltration of lymphocytes and alveolar hyperinflation.U.S. EPA (1995) reported dilated bronchi, fibrosis and emphysema at 1000 ppm NaNO 2 or above.
Present histopathological results revealed that GSE was able to protect lung against low NaNO 2 dose and minimize damage of lung induced by high NaNO 2 dose.The beneficial effect of GSE on lung tissue was previously reported by Hemmati et al (2008) revealed that the use of GSE can provide a protective action against the harmful and fibrogenic effect of silica on lung tissue.
Present histological examination revealed that NaNO 2 induce dose-dependent testicular damage.Testes of rats administrated with 1g/L NaNO 2 revealed degeneration of spermatogoneal cells lining seminiferous tubules and necrosis leydig cells.Also, examination of testes of rats treated with 2g/L NaNO 2 revealed interstitial edema and degeneration of spermatogoneal cells lining seminiferous tubules.The effect of NaNO 2 on testis was previously reported by many studies.Akintunde et al. (2013) suggested that nitrite may change the testicular oxidative status and play a role in testicular dysfunction that causes infertility.Testes of rats received 0.04mg/kg B.W, 0.06mg/kg B.W and 0.08mg/kg B.W nitrite by oral route for 30 days showed Seminiferous tubules with focal areas of lost germ cells, hyperplasia of Leydig cells and at 0.06mg/kg dose level, there was arrest of spermatogenesis while there was no evidence of spermatogenesis at 0.08mg/kg nitrite dose.Pavlova et al. (2013) reported that intraperitoneal injection of male Wistar rats with NaNO 2 at dose of 50 mg.kg-1B.W and sacrificed at different time intervals (1 h, 5 h, 24 h and 48h) following the administration resulted in disorganization of seminiferous epithelium and assemblance of undifferentiated germ cells in the luminal area of the tubules.
Present histopathological results revealed that GSE was able to protect testes against both NaNO 2 dose and restore testicular tissue to normal.Such protective effect of GSE was recorded against sodium fluoride induced testicular damage (El-Demerdash et al., 2008).
Generally the cytotoxic and oxidative stress activity of NaNO 2 may be due to NO• and ONOO -formed from nitrite.Such product may increase lipid peroxidation which can be harmful to different organs (Chow and Hong, 2002).The high oxidative stress indicator lipid peroxidation could be attributed to the oxidative cytotoxicity of nitrite (Patsoukis and Georgiou, 2007).Moreover, nitrite can react with secondary and tertiary amines forming Nnitrosocompounds (nitrosamines) (Walker, 1990), which are associated with high risk of stomach, liver and esophagus carcinomas (Mitacek et al., 1999 andKim et al., 2002).Nitrosamines have strong toxic, mutagenic, neuro-and nephrotoxic and carcinogenic effect.These may interpret the distortion in lung, testis, brain, heart, liver and kidney and reflected by the changes in the investigated parameters.
Meanwhile, GSE could reduce organ injury through its ability to balance the oxidantantioxidant status, and to regulate the release of inflammatory mediators (Sehirli et al., 2008).GSE has been shown to have conjugated structures between the B-ring catechol groups and 3-OH free groups of polymeric skeleton allowing being effective free radical scavengers and metal chelators (Yilmaz and Toledo, 2004).As GSE scavenges free radicals, the resulting aroxyl radical formed has been shown to be more stable than that generated from other polyphenolics to prevent DNA damage (Bradham et al., 1998).Also, Devi et al. (2006) found that intake of proanthocyanidin which is a naturally occurring antioxidant from GSE in low quantity is effective in up-regulating the antioxidant defense mechanism by attenuating lipid peroxidation.
Conclusion: Sodium nitrite (preservative) has harmful effects on all tested parameters and induces oxidative damage in liver, kidney, brain, heart, lung and testis tissues in dosedependent manner.GSE not only has antioxidant properties that can enable it to scavenge the reactive oxygen species resulted from the ingestion of the food additives, but also it can normalize the changes induced by low NaNO 2 dose in most examined organs.It has hypoglycemic and hypochlesterolemic actions.

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Fig. (1): Liver of rat from G2 showing the normal histological structure of hepatic lobule (H and E X 400).

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Fig. (2): Liver of rat from G3 showing focal hepatic necrosis associated with inflammatory cells infiltration (H and E X 400).

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Fig. (3): Liver of rat from G3 showing kupffer cells activation and sinusoidal leucocytosis (H and E X 400).

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Fig. (4): Liver of rat from G4 showing dilatation and congestion of hepatic sinusoids (H and E X 400).

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Fig. (5): Liver of rat from G4 showing portal infiltration with mononuclear cells, focal hepatic necrosis associated with mononuclear cells infiltration (H and E X 400).

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Fig. (6): Liver of rat from G4 showing cytoplasmic vacuolization of hepatocytes and necrosis of sporadic hepatocytes (H and E X 400).

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Fig. (7): Liver of rat from G5 showing kupffer cells activation (H and E X 400).

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Fig. (8): Liver of rat from G5 showing apparent normal hepatocyte (H and E X 400).

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Fig. (10): Kidney of rat from G2 showing the normal histological structure of renal parenchyma (H and E X 400).

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Fig. (11): Kidney of rat from G3 showing vacuolization of endothelial lining glomerular tuft with presence of protein cast in the lumen of renal tubules (H and E X 400).

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Fig. (12): Kidney of rat from G3 showing hypertrophy of glumerular tuft and periglomerular fibroblasts proliferation (H and E X 400).

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Fig. (14): Kidney of rat from G4 showing eosinophilic protein cast in the lumen of renal tubules (H and E X 400).

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Fig. (15): Kidney of rat from G4 showing hypertrophy and congestion of glumerular tuft, perivascular edema (H and E X 400).

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Fig. (16): Kidney of rat from G4 showing atrophy of glomerular tuft (H and E X 400).

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Fig. (17): kidney of rat from G5 showing apparent normal renal parenchyma (H and E X 400).

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Fig. (18): Kidney of rat from G6 showing apparent normal renal parenchyma (H and E X 400).

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Fig. (19): Brain of rat from G2 showing the normal histological structure (H and E X 400).

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Fig. (21): Brain of rat from G3 showing menengial hemorrhage associated with inflammatory cells infiltration (H and E X 400).

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Fig. (22): Brain of rat from G4 showing focal hemorrhage (H and E X 400).

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Fig. (23): Brain of rat from G4 showing hemorrhage in Virchow space (H and E X 400).

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Fig. (24): Brain of rat from G4 necrosis of neurons (H and E X 400).

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Fig. (25): Brain of rat from G4 showing congestion of cerebral blood vessels (H and E X 400).

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Fig. (26): Brain of rat from G5 showing pyknosis of neurons (H and E X 400).

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Fig. (28): Heart of rat from G2 showing the normal cardiac myocytes (H and E X 400).

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Fig. (29): Heart of rat from G3 showing congestion of myocardial blood vessels (H and E X 400).

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Fig. (30): Heart of rat from G3 showing myolysis of focal myocytes (H and E X 400).

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Fig. (31): Heart of rat from G4 showing congestion of myocardial blood vessels (H and E X 400).

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Fig. (33): Heart of rat from G5 normal cardiac myocytes (H and E X 400).

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Fig. (34): Heart of rat from G6 showing congestion of myocardial blood vessels (H and E X 400).

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Fig. (35): Lung of rat from G2 showing the normal histological structure (H and E X 400).

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Fig. (37): Lung of rat from G3 showing bronchitis.Note mononuclear cells infiltration in bronchial mucosa (H and E X 400).

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Fig. (38): Lung of rat from G4 showing perivascular inflammatory cells infiltration (H and E X 400).

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Fig. (41): Lung of rat from G5 showing no histopathological changes (H and E X 400).

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Fig. (43): Testis of rat from G2 showing the normal seminiferous tubules and complete spermatogenesis (H and E X 400).

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Fig. (44): Testis of rat from G3 showing degeneration of spermatogoneal cells lining seminiferous tubules and necrosis leydig cells (H and E X 400).

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Fig. (46): Testis of rat from G4 showing degeneration of spermatogoneal cells lining seminiferous tubules (H and E X 400).

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Fig. (47): Testis of rat from G5 showing normal seminiferous tubules and complete spermatogenesis (H and E X 400).

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Fig. (48): Testis of rat from G6 showing no histological changes (H and E X 400).
Jiao et al. (2010) concluded that the hypocholesterolemic activity of grape seed proanthocyanidins was most likely mediated by enhancement of bile acid excretion and upregulation of cholesterol-7α-hydroxylase (CYP7A1).On the other hand, potential mechanisms by which grape polyphenols may exert cardioprotective effects include a reduction in oxidative stress, modulation of the inflammatory cascade, improvement in vascular endothelial function (eg, flow-mediated dilatation [FMD]), and protection against atherothrombotic episodes including myocardial ischemia and inhibition of platelet aggregation(Cui et al., 2002Shanmuganayagam et al., 2002and Delmas et al., 2005).Our results clearly showed that there was a significant dose dependent-increase in serum glucose concentration of NaNO 2 -treated rats in all experimental periods.The same results was obtained by Hassan et al. (2009) and Gomaa and Abd Elaziz (2011) in rats and by Abdul-Ameer and Abed (2012) in mice.The findings suggest nitrite-stimulation of gluconeogenesis (Wiechetek et al., 1992), and glucose shift from tissue to blood or an impairment of glucose mobilization.Furthermore, nitroso-compounds can alter the antioxidant system causing disturbance in the metabolic processes leading to hyperglycemia(Anil et al., 2005).However, in our study serum glucose levels were ameliorated upon GSE supplementation and return to normal level in serum of 1g NaNO 2 -treated rats at 8 th week.The hypoglycemic effect of grape seed proanthocyanidins was previously reported by El-Alfy et al. (2005) and Terra et al. (2009).The mechanism underlying such protection is mediated via prevention and restoration of pancreatic antioxidant defense systems and stimulating insulin release by pancreatic β-cells.An additional mechanism of the antihyperglycemic action of grape seed may be through stimulating the surviving pancreatic cells to release more insulin (El-Alfy et al. 2005).A recent study by Pinent et al. (2004) has also shown that grape-seed procyanidins possess an insulinomimetic activity on insulin sensitive cell lines.

Table ( 1): Hematological
parameters and methemoglobin concentration in blood of control and treated rats during experimental periods (means ± SE).
Within the same column, various superscript letters indicate significant differences (Duncan, P <0.05).

Table ( 2
): Liver functions parameters in serum of control and treated rats during experimental periods (means ± SE).
Within the same column, various superscript letters indicate significant differences (Duncan, P <0.05).

Table ( 3
): Kidney functions parameters in serum of control and treated rats during experimental periods (means ± SE).
Within the same column, various superscript letters indicate significant differences (Duncan, P <0.05).

Table (
4): Total cholesterol and triglycerides concentrations in serum of control and treated rats during experimental periods (means ± SE).Within the same column, various superscript letters indicate significant differences (Duncan, P <0.05).

Table ( 5
): Glucose concentrations in serum of control and treated rats during experimental periods (means ± SE).
Within the same column, various superscript letters indicate significant differences (Duncan, P <0.05).

Table ( 6
): Antioxidants parameters in plasma of control and treated rats during experimental periods (means ± SE).
Within the same column, various superscript letters indicate significant differences (Duncan, P <0.05).

Table (
7): Lipid peroxidation (Malondialdehyde levels) in serum of control and treated rats during experimental periods (means ± SE).Within the same column, various superscript letters indicate significant differences (Duncan, P <0.05).