The aim of this study was to determine the nitrite levels in the sub-cellular fractions of cassava (Manihot esculenta crantz) leaves containing chloroplast, mitochondria and in the cytosol respectively and to their contribution in the causation of liver toxicity in rats exposed to N-nitrosamine precursors. The design of this experiment consisted of three animal groups. The first group was administered with dimethylamine hydrochloride plus sodium nitrite (DMA.HCL + NaNO2), the second group was administered with sodium nitrite (NaNO2) only and the third group (control) was given distilled water. The methods used included cell fractionation, tissue homogenization and centrifugation, spectrophotometric analysis, enzymatic determination and histopathology. Nitrite levels were estimated at 6.08 ± 0.92, 4.06 ± 1.65 and 1.29 ± 1.66μg/200g of cassava leaf tissue in chloroplasmic, mitochondrial and cytosolic sub-cellular fractions respectively. Both the NaNO2 dose regime and the combined dose of DMA.HCL and NaNO2, at P- value 0.05, caused significant increases in GGT, ALP, AST and ALT levels in serum. The histopathological study of the rat liver for DMA.HCL + NaNO2 administration showed severe portal and central venous congestion while the NaNO2 administration revealed a mild periportal cellular infiltration. This study shows that there is a correlation of nitrite in the chloroplast, mitochondria and cytosol sub-cellular fractions of cassava leaves and administration of nitrite dietary level in cassava leaves and dimethylamine hydrochloride produced acute synergistic toxicity in the liver

Akpabio, C. J.

Efuruibe, A. A.

Maduagwu, E. N.

Covenant University Repository

Available online on www.ijtpr.comInternational Journal of Toxicological and Pharmacological Research 2013; 5(3): 59-62ISSN: 0975-5160*Author for correspondence: E-mail:comfortsamonline@yahoo.comResearch ArticleSub-Cellular Correlation of Nitrite in Cassava (Manihot EsculentaCrantz) Leaves and Nitrosamine Toxicology in Wistar RatsEfuruibe A. A., *Akpabio C. J., Maduagwu E. N.Department of Biochemistry, Basic Medical Sciences, University of Ibadan, Oyo State Nigeria.ABSTRACTThe aim of this study was to determine the nitrite levels in the sub-cellular fractions of cassava (Manihot esculenta crantz)leaves containing chloroplast, mitochondria and in the cytosol respectively and to their contribution in the causation ofliver toxicity in rats exposed to N-nitrosamine precursors. The design of this experiment consisted of three animal groups.The first group was administered with dimethylamine hydrochloride plus sodium nitrite (DMA.HCL + NaNO2), the secondgroup was administered with sodium nitrite (NaNO2) only and the third group (control) was given distilled water. Themethods used included cell fractionation, tissue homogenization and centrifugation, spectrophotometric analysis,enzymatic determination and histopathology. Nitrite levels were estimated at 6.08 ± 0.92, 4.06 ± 1.65 and 1.29 ±1.66µg/200g of cassava leaf tissue in chloroplasmic, mitochondrial and cytosolic sub-cellular fractions respectively. Boththe NaNO2 dose regime and the combined dose of DMA.HCL and NaNO2, at P- value 0.05, caused significant increases inGGT, ALP, AST and ALT levels in serum. The histopathological study of the rat liver for DMA.HCL + NaNO2administration showed severe portal and central venous congestion while the NaNO2 administration revealed a mildperiportal cellular infiltration. This study shows that there is a correlation of nitrite in the chloroplast, mitochondria andcytosol sub-cellular fractions of cassava leaves and administration of nitrite dietary level in cassava leaves anddimethylamine hydrochloride produced acute synergistic toxicity in the liver.Keywords: Cassava, Chloroplast, Cytosol, Dimethylamine hydrochloride, Mitochondria, Nitrite.INTRODUCTIONNitrosamines are present in water, soil and air and they canbe found contaminating food, feeding stuff, drugs,cosmetics, and pesticides.1 There is evidence that nitroso-compounds may be generated in vivo from nitrites ornitrates and primary, secondary and tertiary amines inorgans of people who apparently were not exposed to thesecompounds.2 The formation of nitrosamines depends onthe pH of the environment, alkalinity of amine precursorand temperature. The rate of formation of N-nitrosocompounds from secondary amines increasesproportionally with a decrease in alkalinity of the amines.Nitrosamines are formed by the interaction of nitrous acidas a nitrosating agent with secondary or tertiary amines.Nitrite is known to be a precursor of toxic and carcinogenicN-nitrosamines3 and it induces cancer in experimentalanimals.4,5 Nitrite can also interact with haemoglobin byoxidation of ferrous ion (Fe2+) to the ferric state (Fe3+)thereby preventing or reducing the ability of blood totransport oxygen in a condition known asmethaemoglobenaemia.6,7Cassava (Manihot esculenta) is extensively cultivated asan annual crop in tropical and subtropical regions for itsedible starchy, tuberous root and a major source ofcarbohydrates. It is high energy food obtained with lowinputs and little effort.8,9 Cassava can be classified intotwo varieties namely, toxic or ‘bitter’ and non-toxic or‘sweet’ varieties, based on cyanide content. Varietiesyielding less than 50mg HCN/kg of fresh root areclassified as non-toxic, whilst those yielding more areconsidered toxic.10 Nitrate content is an important qualitycharacteristic of vegetables, the concentration of nitrate invegetables varies considerably, and may be up to 3-4g/kgfresh weight, and these levels could have potential healthimpacts, especially in cassava eating populations.11 One ofthe toxicology implications of nitrate and nitrite ingestionin cassava-eating populations is that thiocyanate, which ispresent in high amounts in the stomach of such individuals,may act as catalyst for the nitrosation of amines in thestomach to form carcinogenic nitrosamines, arising frommicrobial conversion of nitrate to nitrite which acts as thenitrosating agent enzymatically or spontaneously.12The liver is the primary site of chemical biotransformation.Serum tests are often used to detect the liver injury causedby certain chemicals. These tests are based uponidentifying and measuring the serum activity of enzymespresent within liver cells. Thus increased concentrations ofthese enzymes in serums normally indicate hepatocellulardamage and leakage of these enzymes from liver cells intothe blood stream.13The objective of this study is to investigate the correlationof nitrite level in the chloroplast, mitochondria and cytosolsub-cellular fractions of cassava leaves, to evaluate thetoxicity of the liver and the effects on the serum enzymesof wistar rats on exposure to sodium nitrite anddimethylamine hydrochloride (precursors of N-nitrosamine).Akpabio C. J et al. / Sub-Cellular Correlation of…IJTPR, September2013– November 2013, 5(3),59-62mageSMMATERIALS AND METHODSChemical and reagents: Sodium nitrite (NaNO2, Mol.wt69) Dimethylamine hydrochloride (CH3)2NH.HCL),Mol.wt 81.55), were obtained from Sigma (USA). Othersreagents such as alanine aminotransferase (ALT), aspartateaminotransferase (AST), alkaline phosphatase (ALP)  andgamma- glutamyl transferase (GGT) were from RandoxLaboratories Ltd, United Kingdom. All the otherchemicals and test kits used were of analytical grade.Experimental Animals: The experimental animals used inthis work were healthy male albino rats (Rattusnorvegicus) of the Wistar strain. They weighed between180g and 200g, and were obtained from the animal houseof Veterinary Physiology Department, University ofIbadan, where they had been fed commercial rat pellets adlibitum and allowed access to clean drinking water. Onlythose certified free of infection by the Veterinarypathologist were used. They were kept at roomtemperature (approximately 28oC) and all test animalswere acclimatized to their environment before experimentswere begun.Animal treatment: The doses of precursors of nitrosaminegiven to the rats were:A single dose of 6.20mg NaNO2/kg.A concurrent dose of 50mg of dimethylaminehydrochloride and 6.20mg NaNO2/kg.Both compounds were dissolved in distilled water andadministered orally by intubation using a cannula tubes.Control animals were given drinking water and food only.All animals were starved over night prior to theadministration of toxin compounds. The rats were sacrified24 hours after dosing.Collection of blood samples for serum preparation: Therats were sacrificed within 24 hours after the oral dose ofsodium nitrite and Dimethylamine Hydrochloride, all therats were sacrificed by cervical dislocation. Blood wascollected with the capillary tubes from the eyes. The bloodwas collected in dry plastic or glass centrifuge tubes. Theblood was allowed to clot and immediately transferred toan ice water bath prior to centrifugation. The clotted bloodsamples were centrifuged at 2000 rpm in a portable generallaboratory centrifuge for about 15 minutes. The resultantsupernatant sera were collected and preserved in arefrigerator at 40C for a short time.  The activity of Alanineamino transferase (ALT) and Aspartate Amino transferase(AST) were estimated using the method of Reitman andFrankel.14 The activities of Alkaline Phosphatase (ALP)and Gamma – glutamyl Transferase (γ-GT) weredetermined in the serum samples using the method ofKlein et al and Szasz.15,16Histopathological analysis: Liver samples wereimmediately collected and fixed in 10% buffered formalsaline solution for a period of at least 24 h beforehistopathological study. Samples were then embedded inparaffin wax and five-micron sections were prepared witha rotary microtome. These thin sections were stained withhematoxylin and eosin (H&E), mounted on glass slideswith Canada balsam (Sigma, USA) and observed forpathological changes under a binocular microscope.Extraction of Sub-cellular Fraction of Chloroplast,Mitochondria and Cytosol from Cassava Leaves: Freshcassava leaves (sweet species) were obtained from a farmin University of Ibadan, washed and cut into small pieces.50g -80g from each of the cultivars was homogenized in250ml -350ml of 0.1% orthophosphoric acid (0.2μl) pH 7.4at 40C was blend for 3 minutes. The homogenate wasfiltered at 40C through four layers of muslin. The filtratewas centrifuge at 2960xg for 5 minutes at 40C. Chloroplastformed a soft green pellet at the bottom of the centrifugetube. The supernatant was centrifuged at 17,700g forTable 1: Nitrite Concentration in the sub-cellular fractions of the cassava leaf tissueChloroplast (µg) Mitochondria (µg) Cytosol (µg)6.08 ± 0.92 4.06 ± 1.65 1.29 ± 1.66Table 2: Distribution of Nitrite in the sub-cellular fractions of the cassava leaf tissueSub-Cellular Fractions ChloroplastvsMitochondriaChloroplastvsCytosolMitochondriavsCytosolRegression Equation Y=7.34-0.54X Y= -4.48+0.95X Y=2.07-0.19XStandard Error 1.85 13.50 1.55Correlation Coefficient 0.69 0.91 0.54Number of samples=30Table 3: Sub-cellular Correlation of nitrite in 50g of cassava leaf on different days of consumptionDays Chloroplast (µg) Mitochondria (µg) Cytosol (µg) Total (µg)1 day 6.08 4.06 1.29 11.437 days 42.56 28.42 9.03 80.0128 days 170.24 113.68 36.12 320.04Table 4: Serum enzyme activities in rats following oral administration with dimethylamine hydrochloride and sodiumnitriteGroup GGT (U/L) ALP (U/L) AST (U/L) ALT (U/L)Control 7.46 ± 1.40 5.45 ± 1.16 2.63 ± 1.12 27 ± 0.73DMA.HCL+ NaNO2 19.20 ± 0.36 18.42 ± 1.78 69.84 ± 1.77 35.83 ± 1.31NaNO2 13.82 ± 0.60 8.19 ± 0.26 44.90 ± 0.85 31.85 ± 0.59Values are mean ± SD of 5 determinantsAkpabio C. J et al. / Sub-Cellular Correlation of…IJTPR, September2013– November 2013, 5(3),59-62mageS115minutes in a cold centrifuge. A policeman rod was usedto re-suspend the pellet in a small volume of 1%orthophosphoric acid and diluted to a final volume of 10mlwith the acid. The supernatant was centrifuged at 19400xgfor 10 minutes. The pellet was gently suspended in a smallvolume of 1% cold orthophosphoric acid. (The supernatantis the cytosol extract and the pellet is the mitochondriaextract.17) The extracts were stored at 4OC and the amountof nitrite and nitrate present in the different sub-cellularextract were determined.18STATISTICAL ANALYSISData were analysed using student’s T-test analysis and wasexpressed as mean ± standard deviation. A level of p<0.05was considered statistically significant.RESULTS AND DISCUSSIONThe mean values of nitrite obtained in the chloroplast,mitochondria and cytosol sub-cellular fraction were 6.08±0.92, 4.06 ±1.65 and 1.29±1.66µg respectively (Table 1).There was a significant deviation between the three sub-cellular fractions with chloroplast showing the highestconcentration.There was a strong positive correlation relationship whenchloroplast sub-cellular fraction was compared to thecytosol sub-cellular fraction. There was a positivecorrelation relationship when chloroplast andmitochondria were compared to mitochondria and cytosolsub-cellular fraction respectively (Table 2). These valuesfall into the normal range because according to the JointExpert committee on food Additives (JECFA), establishedacceptable daily intake of sodium nitrite is 0-0.07mg/kgbody weight. This indicates that nitrite level in cassava issafe if minimum amount of cassava leaf are consumed. Ifthis amount is consumed for 7 days and for 28 days thetotal concentration of nitrite in the sub-cellular fractionswill be 0.08mg and 0.320mg/kg (Table 3) and this can leadto toxicity in the system.ALP, AST, ALT and GGT levels are widely used inanimal’s studies to diagnose and observed thedevelopment of hepatocarcinogenesis. In the present study,the values of the analytes showed sharp significantlyFigure 1: Serum enzyme activities in rats following oral administration with dimethylamine hydrochloride and sodiumnitriteHistopathology Section of the Liver of RatsFigure 2: Photomicrograph ofHepatocyte of rat following oraladministration of 50mg of DMA.HCLand 6.2mg NaNO2/kg and showing avery severe portal and central venouscongestion. (Mag. x 40)Figure 3: Photomicrograph ofHepatocyte of rat following oraladministration of 6.2mg  NaNO2/kgand showing the portal areasmarkedly infiltrated bymononuclear cells.(Mag. x 40)Figure 4: Photomicrograph of liversection of control rats showing novisible legion. (Mag. x 40)M1MOMPM4MRMSMTMUMApq Aiq ddq AimConcentration of perum bnzymesEu/lFperum  bnzymesaMAKeCi + kaklOkaklOControlAkpabio C. J et al. / Sub-Cellular Correlation of…IJTPR, September2013– November 2013, 5(3),59-62mageSOincrease at P-value < 0.05 in the group administereddimethylamine hydrochloride plus sodium nitrite andsodium nitrite, respectively as compared with that of thenormal control rats (Table 4).The photomicrography of liver section in DMA.HCL +NaNO2 group and NaNO2 group shows that the rat’s liversin these two groups were damaged when compared to thecontrol. DMA.HCL + NaNO2 group shows very severeportal and central venous congestion (Fig.2), while NaNO2group shows markedly infiltration by mononuclear cellsand periportal cellular infiltration (Fig.3).Nitrites have been observed by researchers to occur widelyin foods of animals and plant origin as well as communitywater supply.19 Nitrite levels in food are very low(generally well below 10mg/kg and rarely exceed100mg/kg). Exceptions to this are vegetables that havebeen damaged, poorly stored, or stored for extendedperiods as well as prickled or fermented vegetables.20CONCLUSIONThis study indicates low level of nitrite in sub-cellularfractions containing chloroplast, mitochondria and cytosolof cassava leaves. It also shows that dimethylaminehydrochloride and sodium nitrite (precursors of N-nitrosamine) can cause acute synergistic toxicity in theliver and could also make the serum enzymes level to risedue to the damage that it has done to the liver.It is concluded that frequent consumption of vegetableswhose nitrite contents are high by cassava-eating peoplemight put them at risk of developing stomach cancer andother possible result of nitrite toxicity.REFERENCES1. [1]. Wang, J., W.G. Chan, S.A. Haut, M.R. Krausss,R.R. Izac, W.P. Hempfling. Determination of total N-nitroso compounds by chemical denitrosation usingCuCl. J. Agric. Food Chem.,2005, 53(12): 4686-4691.2. [2]. Brendler S.Y., Tompa A., Hutter K.F.,Preussmann R., Pool-Zobel B.L. In vivo and In vitrogenotoxicity of several N-nitrosamines in extrahepatictissues of rat. Carcinogenesis, 1992, 13, 2435.3. [3]. Bassir O, Maduagwu EN. Occurrence of nitrate,nitrite, dimethylamine and dimethylnitrosamines insome fertilized Nigerian beverages J. Agric. FoodChem. 1978, 26(1): 200-203.4. [4]. Sen NP, Baddoo PA. Trends in the levels ofresidual nitrite in Canadian cured meat products overthe past 25 years. J. Agric. Food. Chem. 1997, 45:4714-4718.5. [5]. Mirvish SS. Role of N-nitroso compounds (NOC)and nitrosation in etiology of gastric, esophageal,nasopharyngeal and bladder cancer and contributionto cancer of known exposures to Noc. Cancer Lett.1995, 93:17-48.6. [6]. Phillips WEJ. Naturally occurring nitrate andnitrite in foods in relation to infantilemethemoglobinaemia. Food and Cosmetics Toxicol.1971, 9:219-228.7. [7]. Tannenbaum SR and Young VR. Endogenousnitrite formation in man. J. Environ. Pathol. andToxicol.1980, 3, 357-368.8. [8]. Claude F. and Denis F. "African Cassava MosaicVirus: Etiology, Epidemiology and Control" PlantDisease 1990, Vol. 74(6): 404–11.9. [9]. Jalaludin, S. Cassava as feedstuffs for livestock.Proceeding of the Symposium on feedstuffs forlivestock in South-East Asia Ed.1977, 105-106 and158-159.10. [10]. Grace. M .R. Cassava Processing. FAO PlantProduction 1977, Series No 3. FAO, Rome.http:/www.fao.org/inpho/vlibrary/x0032/X0032E00.htm11. [11]. Okoh, P. N. The metanolictate of cyanide carbonin animals. Nig. J. Phs. Sci.1992, 8(1 – 2):1 – 9.12. [12]. Maduagwu, E. N. and Umoh, I. B. BiliaryExcretion of Linamarin in Wistar rats after a singledose. Biochem. Pharmacol. 1986, 35: 3003-3006.13. [13]. Carey WD. How should a patient with anisolated GGT be evaluated? Cleve Clin J Med. 2000,67: 315-316.14. [14]. Reitman,S. And Frankel S. A colorimetricmethod for the determination of serum oxaloaceticand glutamic pyruvic transmnases. Am. J. Clin. Pathol1957, 28: 56-63.15. [15]. Klein, B., Read, P.A and Balson, L.A, Rapidmethod for the quantitative determination of serumalkaline phosphatase. Clin. Chem., 1960, 6:269-275.16. [16]. Szasz, G., A kinetic photometric method forserum gamma- glutamyl transpeptidase. Clin.Chem.,1969, 15: 124-136.17. [17]. Montgomery, H.A.C., Dymock, J.F. Thedetermination of nitrite in water. Analyst 1961, 86:414-416.18. [18]. Alyson K.T. Subcellular Fractionation of PlantTissues, Methods in Molecular Biology™Volume1996, 59, pp 57-68.19. [19]. Anyana, V., Umar S., Iqbal M., Abrol y. Arenitrite concentrations in leafy vegetables within safelimits? Proceeding of the workshop on nitrogen inEnvironment, industry and Agriculture, New Delhi,India.2006, Pp.81-84.20. [20]. Onyesom I, Okoh P.N. Quantitative analysis ofnitrate and nitrite contents in vegetables commonlyconsumed in Delta State, Nigeria. Br J Nutr. 2006Nov; 96(5):902-5

Sub-Cellular Correlation of Nitrite in Cassava (Manihot Esculenta

Crantz) Leaves and Nitrosamine Toxicology in Wistar Rats

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Sub-Cellular Correlation of Nitrite in Cassava (Manihot Esculenta Crantz) Leaves and Nitrosamine Toxicology in Wistar Rats

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