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EVALUATION OF THE PROTECTIVE EFFECT OFASCORBIC ACIDAND/OR THYYMOQUINONE ON NITRATE TOXICITY IN RABBITS.

Byline: M.A. Hamouda F.A. Al Hizab and M. M. Hasseeb

ABSTRACT:

Background: Risk of nitrate toxicity among animal and human. The objective of this work isdesigned to study the capacity of thymoquinone and/or ascorbic acidfor detoxification of drinking water nitrate in growing New Zealand White rabbits.

Methods: In this experiment Hematological and serobiochemical parameters as well as histopathological examination of liver and kidney were estimated in 5 assigned groups as follows the 1st group served as a control the 2nd group was orally received sodium nitrate 1gm/L drinking waterfor 8 weeks. The 3rdgroup was orally received sodium nitrate 1gm/L drinking water and treated with ascorbic acid 300mg/L drinking water for 8 weeks.The 4thgroup was orally received sodium nitrate 1gm/L drinking waterfor 8 weeks and treated with thymoquinone(10mg/kg/day) via the intraperitoneal route for7 successive days. The 5th group was orally received sodium nitrate 1gm/L drinking water and treated with ascorbic acid 200mg/L drinking waterfor 8 weeksalong withthymoquinone(10mg/kg/day) via the intraperitoneal routefor 7 days.

Results:The leukocyte count and red blood cells were decreased significantly (Pless than 0.05) in the nitrate treated group compared to the control and other treated groups. Total protein and albumin concentrations were decreased significantly (Pless than 0.05) inthe nitrated treated group compared to the control and other treated groups.The activity of AST ALT as well as Cholesterol and glucoseconcentrations were significantly increased (Pless than 0.05) in the nitrate treatment group compared to thecontrol and other treated groups. The creatinine and blood urea nitrogen (BUN) were significantly increased (Pless than 0.05) in the nitrate treatment group compared tothecontrol and other treated groups. The supplementation of thymoquinone at the rate of 10mg/kg/day and/or ascorbic acid at the rate of 300mg/Litre lead to an improvement of the blood and biochemical parameters and also ameliorate hepatic and renal damage.Conclusion:The present study suggeststhe possible beneficial effects ofthymoquinoneeither alone or

withascorbic against sodium nitrate toxicity via an antioxidant mechanism.

Key words: Ascorbic acid thymoquinone nitrate histopathology. Liver kidney.

INTRODUCTION

The most common causes of high nitrate levels in water include shallow wells contaminated with surface water water containing animal wastes and surface runoff from heavy rain after fertilization with ammonium nitrate [1]. Water containing more than 500 ppm NO3 is potentially toxic [2].In the body nitrate is reduced to nitrite andthe absorption of nitrite leads to methaemoglobinaemiawhich cannot transport oxygen to bodytissues [3]. Exposure to nitrites has been reported to have an adverse effect on animals and humanhealth [4].Nitrate toxicity has been observed commonly in ruminants [5]. A high level of nitrate can lead to a change in blood constituents [6]. Vitamin C has potentials to scavenge free radicals and protect cells from oxidative damage[7]. Vitamin C have also been proved to have a hepatoprotective effect against nitrite toxicity [8].Thymoquinone (TQ)isthe major constituent of the volatile oil from Nigella sativaseeds. Its valuable effects are related to its

anti-oxidant anti-infective anti-tumor antiinflammatory properties [9].Previous studies have demonstrated that TQ has protective effect against oxidative injury induced bya variety of free radical generating agents [101112]. As far as we know there are no documented reports onthe protective effectsof TQ on nitrate toxicity. Thereforethe present study is designed to investigate whether oral supplementationofthymoquinoneand/orascorbic acid could ameliorate or protect against nitrate toxicity.

MATERIALS AND METHODS

Animals:

A total of 40 growing New Zealand White rabbits were obtained fromthe Animal Care Unit ofCollege of Veterinary Medicine and Animal Resources King Faisal University (KFU). The rabbits were assigned to fivegroups (8 rabbits/group) at 8 weeks of age. Animals were housed under good ventilation with free access to food and water. Chemicals:

Sodium nitrate (BDH)-Chemicals Ltd Poole England L-Ascorbic acidTechno-Pharmhem-HaryanaIndiathymoquinone-99%-Sigma-Aldrich Chemical Company St. Louis MO 63103 USA which dissolved in dimethylsulphoxide (DMSO) as 14 mg /ml and then diluted in PBS as required.

Experimental design:

The 1st group (n=8) served as a control the 2nd group (n=8) was orally received sodium nitrate 1gm/L drinking waterfor 8 weeks. The 3rdgroup (n=8) was orally received sodium nitrate 1gm/L drinking water and treated with ascorbic acid 300mg/L drinking waterfor 8 weeks.The 4thgroup (n=8) was orally received sodium nitrate 1gm/L drinking waterfor 8 weeks and treated with thymoquinone(10mg/kg/day) via the intraperitoneal route for 7 successive days. The 5th group (n=8) was orally received sodium nitrate 1gm/L drinking water and treated with ascorbic acid 200mg/L drinking waterfor 8 weeksalong withthymoquinone(10mg/kg/day) viathe intraperitoneal routefor 7 days.

Biochemistry and Hematology:

The rabbits were observed daily for clinical signs. Before necropsy the rabbits were euthanized with sodium pentobarbital (300mg/kg i.p.) according to Animal Welfare Advisory Committee (AWAC).Blood samples were collected via CardiacPuncture in EDTA tubes forhematologicalanalysis (WBC RBC Hb PCV MCV MCH and MCHC) using a coulter counter electronic analyzer (Vet Scan 5 HM-ABAXIS-USA). Serum samples were collected in plain tubes then separated and keptin -80c untilanalyzed fortotal protein albumin cholesterol glucose ALT AST BUN and creatinine usinga biochemical blood analyzer (ELLIPSE E0217 Italy 2003). Gross pathology and histopathology: Necropsy was performedat the end of an experiment and samples of liver kidney and lung were collected at 10% neutral buffered formalin for histopathology. Samples fixed in formalin were embedded within paraffin in 48 hours and processed routinely for hemotoxylin and eosin staining (HE) [13].

Statistical analysis:

Data were analyzed by the General Linear Model (GLM) procedure (SAS Institute Inc 2002). The least Square Mean (LSM) + standard errors for each group were calculated and tested for significance usingthe "t" test [14].

RESULTS

Clinical signs blood and biochemical parameters:

No mortality was recorded during the whole experiment. At 4 weeks lethargy and diarrhea were observed in the nitrate treated groupwhich can persist until the end of the experiment. These findings were not observed in control and other treated groups. The leukocyte count and red blood cells were decreased significantly(Pless than 0.05) in the nitrate treated group compared to control and other treated group (Table 1). Total protein and albumin concentrations were significantly decreased(Pless than 0.05)in the nitrated treated groupcompared to control and other treated groups (Table 2).The activity of AST ALT as well as cholesterol and glucose concentrations were significantly increased (Pless than 0.05) in the nitrate treatment group compared to a control and other treated groups (table 2). The creatinine and blood urea nitrogen (BUN) were significantly increased (Pless than 0.05) in the nitrate treatment group compared tothe control and other treated groups(Table 2).

Gross and histopathology findings:

Gross findings revealed enlargedpale livers and mild gastroenteritis in the nitrate treated group however in other treated groups these organs were more or less quite similar to the control group.

The liver sections of rabbits intoxicated with the nitrite showed disturbed lobular architecture and severe degenerative changes characterized by swelling and vacular appearance of hepatocytes with pyknotic nuclei ( Fig.1a). The vast majority of hepatocytes had significant scattered single necrotic cells (apoptoticcells). These necrotic cells were frequent in the periportal areas but they occurred to a lesser extent in mid zonal areas and were absent in centrilobular areas. Severe degrees of inflammatory reaction manifested with minute foci of macrophages and lymphocytes was noticed everywhere. The livers intoxicated with nitrate and treated with ascorbic acidshowed a mild degree of degenerative changes of some hepatocytes manifested with vacuolar cytoplasm ( Fig.1b). However The most hepatocytes appeared recovered and have an almost normal architecture. The livers intoxicated with nitrate and treated with thymoquinone revealed almost recovered and healthy hepatocytes except for occasional

infiltration ofmononuclear cells in the portal areas( Fig.1c).The livers intoxicated with nitrate and treated with ascorbic acid and/ or thymoquinone revealed recovered and healthy hepatocytes which have an almost normal architecture( Fig.1d). Kidney of the rabbitsintoxicated with the nitrate showed moderate tubular degeneration predominantly of the distal tubules manifested with cellular swelling and fine granular appearance of the cytoplasm. Vacuolar degeneration was also noted but the severe degree characterized by desquamation of cells in almost all distal tubules. The most common lesion was interstitial nephritis manifested by focal aggregations of mononuclear cell between the tubules ( Fig.2a). The kidneysintoxicated with nitrate and treated with ascorbic acidshowed a mild degree of vascular degeneration of some tubules with cellular casts in some distal tubules (Fig.2b).The kidneysintoxicated with nitrate and treated with thymoquinoneshowed only a mild degree of vacuolar

degeneration of some tubules( Fig.2c). Apart from very mild degree of tubular cellular degeneration in rabbits intoxicated with nitrateand treated with ascorbic acidand /or thymoquinonethe majority of convoluted tubules as well as the glomeruliappeared more or less quite similar to the control rabbits ( Fig.2d).

DISCUSSION

Nitrate is a health hazard because of its conversion to nitrite. Once ingestedtheconversion of nitrateto nitrite takes place in the saliva and in the gastrointestinal tract of animals and human.The present study indicates that excess nitrate in drinking water leads to gastrointestinal disturbance. These signs might be due to the irritant effects of nitrite on the mucous membranes of the gastrointestinal tract [15].The leukocyte count decreasedsignificantly in the nitrate treated group compared to control and other treated groups. Furthermore the number of red blood cells decreasedsignificantly in the nitrate treated group compared to control and other treated groups. This finding might be due to the effect of nitrite on the Ca Mg and ATPs activity of the cell membrane of RBC3.Dehydration due to diarrhea might be another factor for this decrease in RBC. Hemoglobindecreased significantly in the nitrate treated groupcompared to control compared and other treated groups.

These results might be due to the decrease number of RBC or due to the conversion of nitrate to nitrite ions which can convert ferrous ions of hemoglobin into ferric form (methaemoglobin) resulting in tissue anoxia which may be responsible for renal and hepatic damage[16].Total protein and albumin concentrations were significantly decreased in the nitrated treated group. The decrease of total protein and albumin concentrations could be attributed to impair liver and kidney functions[1718] or might be due tothe formation of nitric oxide or peroxynitrite which oxidizes proteins and lipoproteins [19]. The activity of AST and ALT weresignificantly increased in the nitrate treatment group.These results indicate hepatic damage [20]. Cholesterol concentrations were significantly increased inthe nitrate treatment group. These findings might be due to the endothelial damage of blood vessels by nitric oxide and superoxide [19].The creatinine and blood urea nitrogen (BUN) were

significantly increased in the nitrate treatment group. These findings could be attributed to the renal damage. The addition of ascorbic acid to nitrateamendedtissues changesblood andbiochemical parameters as well.These results indicated that ascorbic acid can reduce methaemoglobin [21] ormay augment the function of endogenous free radical scavengers and decreases the adverse effect of nitrates on body cells [7]. The addition of thymquinone either alone or along with ascorbic acid to nitratealso ameliorate tissues and blood changes.It has been shown that thymoquinone (TQ) works as a scavenger of various reactive oxygen species including superoxide radical anion and hydroxyl radicals1022.

ACKNOWLEDGMENTThe authors are gratefulto the Deanship of Scientific Research King Faisal University Saudi Arabia for support this work with a financial grant forthe annual project No: 140066

Table 1: Blood parameters in control group nitrate group nitrate and ascorbic acid group nitrate and TQ group

###and nitrate ascorbic acid and TQ group.

###Control group###Nitrate treated###Nitrate and###Nitrate and TQ###Nitrate ascorbic

###group###ascorbic acid###group###acid and TQ

###WBC 103/ml###6.200.15###a

###3.000.26###b###group

###5.560.14 c###5.150.12###d###groupe

###5.750.06

###a

###LYM %###52.952.10###43.230.27b###50.100.16 c###49.740.40 c###50.370.47 c

###a###b###c###cd

###MON %###26.220.21###20.150.11###24.880.26###24.700.33###25.120.13ce

###NEU %###19.240.07 a###13.320.13 b###17.320.15 c###17.980.08 d###18.060.09 d

###6###a###b###c###c

###RBC 10 /ml###6.910.08###3.470.19###5.970.07###6.060.13###6.130.12 c

###Hb g\dl###12.700.09 a###8.750.45 b###10.32.39 c###10.700.26 d###11.180.17 e

###PCV %###39.510.24 a###30.270.41 b###37.170.23 c###38.040.09 d###38.120.07 d

###a###b###c###d

###MCV fl###68.220.06###58.020.27###66.220.12###66.820.09###67.100.14 e

###MCH pg###22.140.04 a###15.740.22 b###20.860.18 c###20.930.10ce###21.030.11de

###a###b###c###ce

###MCHC g/dl###32.250.27###23.900.17###30.870.11###31.010.11###31.140.08de

Table 2: Biochemical biomarkers in control group nitrate group nitrate and ascorbic acid group nitrate and TQ group

###and nitrate ascorbic acid and TQ group.

###Control group###Nitrate treated###Nitrate and###Nitrate and TQ###Nitrate ascorbic

###group###ascorbic acid###group###acid and TQ

###Albumin (g/dL)###5.560.21 a###2.100.07 b###group

###4.850.10###c

###4.960.08 ce###5.090.14

###groupde

###T protein (g/dL)###7.610.06 a###3.150.13 c###6.380.21 c###6.300.15 c###6.400.16 c

###AST (IU/L)###30.180.07 a###40.410.30 b###31.350.23 c###31.940.12 d###32.120.12 d

###ALT (IU/L)###50.170.08 a###63.520.38 b###51.370.33 c###51.950.21 d###52.150.11 d

###a###b###c###d

###Cholesterol mg/dL)###40.350.34###74.070.18###41.900.18###42.520.32###41.040.52 e

###Creatinine (mg/dL)###0.810.02 a###3.330.27 b###0.980.04 cd###0.990.04 cd###0.880.04 ad

###a###b###c###d

###BUN###25.190.08###49.680.13###26.590.34###26.330.19###25.990.10 e

###Glucose###137.20.04 a###2560.66 b###139.30.49 c###139.10.26 c###138.20.81 d

###(mg/dL)

REFERENCES

1. Wahaab R.A. and M.I. Badawy 2004. Water quality assessment of the River Nile system:an overview. Biomed. Environ. Sci 17:87-100.

2. Gupta S.K. R.C. Gupta A.B. Gupta A.K. SethJ.K. Bassin and A. Gupta 2001. Recurrent acute respiratory tract infection in areas with high nitrate concentration in drinking water. Environmental Health Perspectives 108(4):363-66.

3. Shahid Mahboob A.N Sheri A.R. Shakoori S.H. Raza and S. Andleeb 2001. Effect of nitrate and nitrite pollution on some haematological parameters of rabbits. Pak l Agri. Sci Vol.38:44-46.

4. Raaz K. Maheshwaria A.K. Chauhanb Lal Bhanwar and A.K. Sharmad 2013. Nitrate toxicityin ground water: its clinical manifestations preventive measures and mitigation strategies.Oct. Jour. Env. Res Vol. 1(3):217-230.

5. Tokarnia C.H. J.DAlbereinerand P.V.Peixoto 2002. Poisonous plants affecting livestock in Brazil. Toxicon40(12):1635-60.

6. Rawat S.k R.K. Singh F.W. Bansode Poonam Singh and Rana P.Singh 2013. Nitrate induced toxicity on some haematological parameters of Charles foster rats. Journal of Recent Advances in Applied Sciences 28:35-38.

7. Mendiratta S. Z.C. Qu and J.M. May 1998. Enzyme dependent ascorbate recycling in human erythrocytes: role of thioredoxinreductase. Free Rad Biol Med25(2):221-8.

8.KrishnamoorthyP.andM.Sangeetha2008. Hepatoprotective effect of vitamin C on sodium nitriteinduced Lipid peroxidation in albino rats. Indian Journal of Biochemistry andBiophysics Vol.45:206-208.

9. Ragheb A. A. Attia W.S. Eldin F. Elbarbry S. Gazarin and A.Shoker 2009. The protective effect of thymoquinone an anti-oxidant and anti-inflammatory agent against renal injury: A review. Saudi J. Kidney Dis. Transpl 20: 741752. 10. Badary O.A. R.A. Taha A.M. Gamal el-Din and M.H.Abdel-Wahab 2003.Thymoquinone is a potent superoxide anion scavenger. Drug Chem. Toxicol26:87-98.

11. Nili-Ahmadabadi A. F. Tavakoli G.R. Hasanzadeh H.R. Rahimi and O. Sabzevari 2011. Protective effect of pretreatment with thymoquinone against Aflatoxin B1 induced liver toxicity in mice. DARU Vol.19No4.

12. Amina E.E.M.A.M.AshrafI.K.Latifaand A.E.Aglal 2012. Nigella sativa seeds protect against hepatotoxicity and dyslipidemia induced by carbon tetrachloride in mice. Journal of applied Pharmaceutical ScienceVol.2(10):021-025.

13. Bancroft J.D and A.Stevens 1996.Theory and Practice ofHistological Techniques. 4th Ed. Churchill Livingstone New York Edinburgh London.

14. Steel R. G. D. and J. H. Torrie 1960. Principles and Procedures of Statistics. (With special Reference to the Biological Sciences.) McGraw-Hill Book Company New York Toronto London.

15. Manoj Kumar Sharma Hemlata Sharma and Neelam Bapna 2013a. Toxic effects of high nitrate in oesphagus and stomach of rabbits. Int J Med Res Heath Sci 2(3):407-411.

16. Kammerer M. and B. Sillart 1993. Midterm toxicity ofnitrates: Experimental evaluation of the effects onreproductive functions of the female rabbit. Vet. Res24: 434-444.

17.BassunyS.M. S.A.Shehata L.B.Bahgat S.I.A.Mohamed 2004. Nitrate toxicityin rabbits: Effect of nitrate in drinking water on digestion some blood constituents and growth performance of growing rabbits. Egyptian J. of Rabbit Sci 14: 147-158.

18. Zraly Z.J. Bendova D. Svecova L. Faldikova Z. Vezeznik and A. Zajicova1997. Effectof oral intake of nitrates on reproductive functions of bulls. Vet. Med. (Praha)42: 345-354.

19. Guzik T.J. N.E.J.West E.BlackD. McdonaldC. Ratnatunga R.PillaiandM.Channonk2000. Vascular superoxide production by NAD(P)H oxidase: Association with endothelial dysfunction and clinical risk factors. Circulation Res 86: 85.

20. Manoj Kumar Sharma Hemlata Sharma and Neelam Bapna2013b. Histopathological changes in the liver of rabbits exposed to high nitrate ingestion in drinking water. Journal of Clinical and Diagnostic Research 7(8):1552-1554.

21. Nahid Atyabi SeyedehParastooYasini SeyedehMissagh Jalali and Hamid Shaygan 2012. Antioxidant effect of different vitamins on methemoglobin production: An in vitro study. Veterinary Research Forum 3(2):97-101.

22. Zeinab Solati Badlishah Sham Baharin and Hossein Bagheri 2014. Antioxidant Property Thymoquinone Content and Chemical Characteristics of Different Extracts from Nigella sativa L. Seeds. J Am Oil ChemSoc 91:295300
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