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Experimental and Toxicologic Pathology 61 (2009) 363–370

www.elsevier.de/etp

Protective role of chickpea seed coat fibre on

N-nitrosodiethylamine-induced toxicity in hypercholesterolemic rats

Gaurav Mittala,�, Shyma Vadheraa, Apminder Pal Singh Brarb, Giridhar Sonia

aDepartment of Biochemistry and Chemistry, Punjab Agricultural University, Ludhiana 141 004, IndiabDepartment of Veterinary Pathology, Punjab Agricultural University, Ludhiana 141 004, India

Received 15 November 2007; accepted 8 July 2008

Abstract

N-nitrosodiethylamine (NDEA) is one of the important carcinogenic nitrosamines frequently present in humanenvironment and food chain that poses a significant human health hazard. This study was planned to investigate theprotective role of dietary fibre on NDEA-induced toxicity in hypercholesterolemic rats. Oral administration of NDEAat a dose of 100mg/kg diet to experimental rats under hypercholesterolemic conditions evoked severe biochemical andpathological changes. Supplementation of chickpea (Cicer arietinum L.) seed coat fibre in the diet along with NDEAreduced its biochemical and pathological effects. There was a reduction in the hepatotoxic effects of NDEA asevidenced by decreased hepatic degeneration and improved liver weight index. Administration of NDEA resulted in asignificant increase in the osmotic fragility of erythrocytes. The antioxidant activity of experimental animals decreasedin the NDEA-fed group, which was evident by increased in vitro lipid peroxidation (LPO) of erythrocytes. However,chickpea seed coat fibre considerably reduced the peroxidative damage done by NDEA. Administration of NDEA alsoresulted in a significant increase in LPO in all the tissues to a varying degree, although the effect on antioxidantpotential was variable in different tissues. However, chickpea seed coat fibre reduced the effect of NDEA on LPO andantioxidant potential of various tissues, providing reasonable protection against NDEA-induced oxidative stress andhence its toxicity. Histopathological analysis of different tissues (heart, liver and lungs) showed decrease in the severityof pathological changes among the experimental animals when they were given NDEA along with chickpea seed coatfibre in the diet as compared with giving NDEA alone. Our study therefore, emphasizes the importance of includingdietary fibre in the diet, in combating the ill-effects of nitrosamines such as NDEA, particularly on the antioxidantstatus of the body.r 2008 Elsevier GmbH. All rights reserved.

Keywords: N-nitrosodiethylamine; Chickpea seed coat fibre; Hypercholesterolemia; Lipid peroxidation; Antioxidant enzymes

e front matter r 2008 Elsevier GmbH. All rights reserved.

p.2008.07.006

ing author at: Institute of Nuclear Medicine and Allied

S.K. Mazumdar Marg, Delhi 110 054, India.

905125; fax: +91 11 23919509.

ess: gauravmittal23@gmail.com (G. Mittal).

Introduction

N-nitrosamines including N-nitrosodiethylamine(NDEA) are one of the important groups of carcinogensfrequently present in human environment and foodchain that pose a significant human health hazard (Aiubet al., 2003). Presence of these nitroso compounds,

ARTICLE IN PRESSG. Mittal et al. / Experimental and Toxicologic Pathology 61 (2009) 363–370364

including NDEA, has been widely reported in variousfoodstuffs such as milk products, meat products, softdrinks and alcoholic beverages (Tricker et al., 1991;Prasad and Krishnaswamy, 1994; van Maanen et al.,1998; Levallois et al., 2000). Although the occurrenceand concentration of these nitrosamines in commondietary items have been reduced considerably in theWestern countries due to changes in the manufactu-ring procedures, processing and/or ingredients, theirdietary levels in developing countries is still substantiallyhigh (Hotchkiss, 1989). In addition to this, tobaccousage in the developed as well as in the developingworld is also one of the biggest causes for individualexposure to nitrosamines (Wu et al., 2005). Presence ofthese nitroso compounds in the diet together with thepossibility of their endogenous formation in the humanbody by reaction of nitrite with amines and amides(Masuda et al., 2000; Ohsawa et al., 2003) has beenof great concern since they are suggested to causeoxidative stress and cellular injury due to the involve-ment of reactive oxygen species (Bartsch et al., 1989;Bansal et al., 2005). In vitro studies from our lab in ratand human erythrocytes have shown that NDEAexposure increases lipid peroxidation (LPO) and de-creases the activity of antioxidant enzymes (Bansalet al., 1996; Bansal and Bhatnagar, 1998). It is a well-known fact that oxygen-free radicals and related lipidperoxides also play a key role in the pathogenesisof normal senescence and of age-related chronicdegenerative diseases, including atherosclerosis (Stockerand Keaney Jr, 2004; Maxwell and Lip, 1997).Hypercholesterolemia is known to be one of the majorrisk factors of atherosclerosis, which plays an impor-tant role in the pathogenesis of coronary artery disease,a major cause of premature deaths in the world(Maxwell, 2000).

The preventive and therapeutic benefits of dietaryfibre including chickpea seed coat fibre against hyperch-olesterolemia are well known as a variety of epidemio-logical, animal and human studies have shown overthe years (Gardner et al., 2005; Pittaway et al., 2004;Jenkins et al., 2003; Chu and Hanson, 2000). Dietaryfibre, a component of food that resists the action ofdigestive enzymes in the gastrointestinal tract, reducesthe rate of diffusion of the products of digestiontowards absorptive surfaces (Bennett and Cerda,1996). Scavenger action of fibre may lead to removalor slowing down of absorption of nitroso compoundssuch as NDEA, which in turn may reduce the riskof their toxic effects. We have already shown inour previous work, the toxicity of NDEA underhypercholesterolemic dietary conditions (Mittal et al.,2006). We now report the protective effect of chickpeaseed coat fibre against this NDEA-induced toxicity andoxidative stress in hypercholesterolemic experimentalrats.

Materials and methods

All the chemicals used in the present study were ofanalytical grade. NDEA was purchased from SigmaChemical Company, St. Louis, MO, USA. Chickpea(Cicer arietinum L.) seed coat was procured from thelocal market. Fibre was prepared by the method asdescribed by Mann et al. (2001).

Animal experiments were approved by the SocialJustice and Empowerment Committee for the purposeof control and supervision of experiments on animals,Ministry of Government of India, New Delhi. Disease-free male Wistar rats (150–170 g) were procured fromthe Small Animal Colony of Punjab AgriculturalUniversity, Ludhiana. The rats were divided into threegroups of six rats each. Rats of group I were given ahypercholesterolemic diet consisting of 1% cholesterol,besides starch (63%), oil (10%), casein (15%), saltmixture (5%), and yeast powder (1%) that served as asource of vitamins and cellulose (5%). Group I served ascontrols for this study. Group II rats received the above-mentioned hypercholesterolemic diet along with NDEAat a dose of 100mg/kg diet (LD50 ¼ 280mg/kg), whilerats of group III were fed hypercholesterolemic diet withNDEA at a dose of 100mg/kg diet and 5% chickpea seedcoat fibre as well. Water and respective diets were suppliedad libitum to rats throughout the experimental period. Allthe rats were sacrificed after 4 weeks of feeding.

Blood samples were collected into heparinized tubesdirectly by cardiac puncture. Organs such as liver, heart,spleen, kidneys and lungs were removed and a portionof these tissues was also stored in 10% formalinfor histopathological examination. Erythrocytes werewashed with phosphate-buffered saline (pH 7.4) and 5%packed cell volume was prepared. A 10% tissuehomogenate was prepared in 0.1M potassium phos-phate buffer (pH 7.4) for estimating LPO.

Hemoglobin (Hb) and osmotic fragility of erythro-cytes were estimated as described by Dacie and Lewis(1968). To assess the oxygenic potential of erythrocytes,in vitro LPO of erythrocytes was determined by exposingerythrocytes to 40mM H2O2 and determining themalondialdehyde (MDA) produced using thiobarbituricacid (TBA) as described by Stocks and Dormandy(1971). Plasma aminotransferases, namely, aspartateaminotransferase (AST) (EC 2.6.1.1) and alanineaminotransferase (ALT) (EC 2.6.1.2) were assayed asdescribed by Bergmeyer (1974). LPO of tissue homo-genates was determined by the method of Jamall andSmith (1985) using MDA–TBA reaction. Superoxidedismutase (SOD) (EC 1.15.1.1) activity was determinedby the ability of enzyme to inhibit auto-oxidation ofpyrogallol as described by Marklund and Marklund(1974). Catalase (CAT) (EC 1.11.1.6) activity wasassayed by the decomposition of H2O2 as described byAebi (1983). Peroxidase (Px) (EC 1.11.1.7) activity was

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measured by the method of Claiborne and Fridovic(1979). Plasma creatinine was estimated using thealkaline picrate method while urea in plasma wasestimated by the diacetylmonoxime method, bothdescribed by Wootton (1974). Histopathological studieswere carried out by the method of Sheenan (1973).

All data were expressed as mean7 standard deviation,and Student’s t-test was used for comparative analysis.The results were considered significant if pp0.05.

Results

Oral administration of NDEA to experimentalrats under hypercholesterolemic conditions resulted insubstantial decrease in feed intake, which improvedwith addition of chickpea seed coat fibre in the diet.

Table 1. Effect of chickpea seed coat fibre on oral toxicity of NDEA

body weight and organ weight indices

Parameter Group

I (control)

Feed intake/day (g) 11.5070.99

Change in body weight (g) Nil

Organ weight� indices

Heart 0.3370.01

Lungs 0.6070.05

Liver 4.2670.12

Spleen 0.2070.01

Kidneys 0.6170.04

Values are mean7S.D., n ¼ 6.aPo0.01.bPo0.05 with respect to control group.cPo0.01 with respect to NDEA-treated group.�g/100 g body weight.

Table 2. Effect of chickpea seed coat fibre on oral toxicity of ND

transaminases, urea and creatinine

Parameter Group

I (control)

ALT (U/L) 8.9670.64

AST (U/L) 5.7770.62

Urea (mg/dL) 45.4172.50

Creatinine (mg/dL) 0.4170.05

Values are mean7S.D., n ¼ 6.aPo0.01.bPo0.05 with respect to control group.cPo0.01 with respect to NDEA-treated group.

A decrease in body weight was observed, which can beattributed to the decreased food intake (Table 1).NDEA administration also resulted in a significanthepatic degeneration as evident by a significant decreasein liver weight index (Table 1). However, the degen-erative effects of NDEA were partially but non-significantly decreased by the addition of chickpea seedcoat fibre. Heart showed marginal enlargement whenNDEA was fed to the experimental rats. This may bedue to accumulation of fluid as supported by oedemaobserved in histopathological studies in heart.

Activities of plasma aminotransferases, namely,AST and ALT increased significantly and substantiallyupon NDEA administration as compared with thecontrol (Table 2). Effect on the activities of plasmatransaminases, especially ALT was partially reducedwhen chickpea seed coat fibre was also given in the diet.

under hypercholesterolemic conditions – effect on feed intake,

II (NDEA) III (NDEA+fibre)

6.0070.50a 9.0070.75a,c

�(4.0070.54) �(2.0070.49)

0.3570.02b 0.3470.01b

0.6070.06 0.5970.04

3.9670.15a 4.0570.11a

0.2170.03 0.2170.01

0.6270.07 0.6170.07

EA under hypercholesterolemic conditions – effect on plasma

II (NDEA) III (NDEA+fibre)

12.6770.94a 11.0070.82a,c

11.3370.94a 10.6670.94a

76.6170.43a 58.6172.59a,c

0.5570.09a 0.4870.06b

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Administration of NDEA in the hypercholesterolemicdiet also resulted in a significant and substantial increasein the urea and creatinine content as compared withthe control, indicating its nephrotoxicity. However,the effect was reduced considerably in the presence ofchickpea seed coat fibre, as evident by significantreduction in the urea content.

Administration of NDEA had a marginal butinsignificant effect on the Hb content of the experi-mental animals, but increased the osmotic fragility oferythrocytes significantly (Table 3). The effect waspartially reduced when chickpea seed coat fibre wasalso supplemented in the diet along with NDEA. The in

vitro LPO of erythrocytes too showed considerableincrease as compared with control. However, chickpeaseed coat fibre considerably reduced the peroxidativedamage done by NDEA. Table 3 also represents theeffect of NDEA administration on the activities ofantioxidant enzymes viz., CAT, Px and SOD. NDEAadministration decreased the CAT activity as comparedwith control. The decrease in activity was however lesswhen chickpea seed coat fibre was also given in the diet.The activity of Px increased but the effect was similarlyreduced in the presence of chickpea seed coat fibre.Marginal decrease in SOD activity upon NDEAadministration returned to normal when chickpea seedcoat fibre was also supplemented in the diet.

The effect of oral administration of NDEA underhypercholesterolemic dietary conditions on LPO andantioxidant enzymes in different tissues is shown inTable 4. Administration of NDEA increased LPO in allthe tissues studied (viz., liver, lungs, heart, spleen andkidney) to a varying degree under hypercholesterolemicdietary conditions. Inclusion of chickpea seed coat fibre,

Table 3. Effect of chickpea seed coat fibre on oral toxicity of NDEA

(Hb), osmotic fragility, in vitro lipid peroxidation (LPO) and antiox

Parameter Group

I (control)

Hb (g/dL) 11.7671.16

Osmotic fragility (% hemolysis) 24.7272.60

LPO (nmol of MDA formed/g Hb/h) 214.8726.2

Antioxidant enzymes (units/mg Hb)

Catalase� 82.84710.19

Peroxidase�� 0.3070.02

SOD��� 11.1270.65

Values are mean7S.D., n ¼ 6.aPo0.01 with respect to control group.cPo0.01.dPo0.05 with respect to NDEA-treated group.�Expressed as standard international units.��1 unit ¼ increase in O.D./min.���1 unit ¼ amount of enzyme that inhibits 50% of auto-oxidation of py

however, considerably reduced the peroxidative damagecaused by NDEA in all the tissues. As for the effecton the antioxidant enzymes’ activities, there was asignificant decrease in CAT activity in liver and lungs byNDEA. The effects were however, reduced with theinclusion of chickpea seed coat fibre in the diet. Theactivity of Px on the other hand, showed significantincrease in liver, lungs, heart and kidneys upon NDEAadministration. The effects were however reduced withthe inclusion of fibre in the diet for all the organs, exceptfor the heart. SOD enzyme activity was not altered inany of the organs, except heart, which showed asignificantly increased activity. Chickpea seed coat fibrehowever, did not provide any significant protectionagainst this NDEA induced increase in SOD activity. Inspleen, activities of none of the antioxidant enzymeswere significantly affected with any of the treatments ascompared to control group.

The toxicity of NDEA under hypercholestrolemicconditions, on the histopathology of various tissueswas studied along with the effect of dietary fibre. NDEAadministration resulted in severe granular degenerationalong with infiltration of fibroblasts in liver. Moreover,accumulation of lipid droplets was also seenin hepatocytes (Fig. 1). In presence of dietary fibre,however, the liver was almost normal and verymild granular changes were seen (Fig. 2). In heart,administration of NDEA resulted in accumulationof lipid droplets in myocardium fibres along withdegenerative changes (Fig. 3) but the additionof chickpea seed coat fibre resulted in almost normalmyocardium fibres of heart (Fig. 4). NDEA treatmentalso caused accumulation of fat droplets in the wallsof coronary vessel along with periarterial oedema and

under hypercholesterolemic conditions – effect on hemoglobin

idant enzymes in erythrocytes

II (NDEA) III (NDEA+fibre)

11.4270.12 11.5570.15

64.6774.47a 59.3274.20a,d

280.0717.9a 250.7733.5

46.3476.34a 54.6777.95a

0.3370.01a 0.3170.01c

10.5670.40 11.8171.29d

rogallol.

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Table 4. Effect of chickpea seed coat fibre on oral toxicity of

NDEA under hypercholesterolemic conditions – effect on LPO

and antioxidant enzymes in different tissues

Parameter Group

I (control) II (NDEA) III (NDEA+fibre)

Liver

LPO4 200.9716.4 269.1719.9a 213.7712.1c

Antioxidant enzymes#

Catalase� 372.6753.7 293.4724.7a 356.6753.7d

Peroxidase�� 0.1670.03 0.4070.03a 0.3370.01a,c

SOD��� 9.5770.56 9.1370.78 9.2971.60

Lungs

LPO4 62.6074.57 94.02714.18a 72.7378.09b,c

Catalase� 52.0174.90 44.2973.90a 49.0174.90

Peroxidase�� 0.3670.07 0.5870.12a 0.4070.07a

SOD��� 18.9973.50 17.6470.58 18.1471.52

Heart

LPO4 103.9714.1 170.9716.0a 149.6715.5a,d

Catalase� 34.0174.90 36.2474.70 38.0174.90

Peroxidase�� 0.4970.07 0.6770.12a 0.6170.11b

SOD��� 11.3771.90 16.4871.85a 15.1571.41a

Spleen

LPO4 114.0716.8 172.0712.1a 145.3716.0a,c

Catalase� 52.8276.40 58.2075.50 56.8276.40

Peroxidase�� 0.8470.10 0.8070.10 0.8170.10

SOD��� 9.8371.60 8.7571.57 9.7872.24

Kidneys

LPO4 110.1719.8 154.2712.1a 129.9716.0c

Catalase� 196.6718.6 209.2723.1 200.6718.6

Peroxidase�� 0.1270.05 0.1570.02 0.1370.02d

SOD��� 11.6371.70 10.5971.95 11.3070.89

Values are mean7S.D., n ¼ 6.aPo0.01.bPo0.05 with respect to control group.cPo0.01.dPo0.05 with respect to NDEA-treated group.4n Moles of MDA formed/g tissue.#Activities expressed as units/mg protein.�Expressed as standard international units.��1 unit ¼ increase in O.D./min.���1 unit ¼ amount of enzyme that inhibits 50% of auto-oxidation

of pyrogallol.

Fig. 1. Section of liver showing accumulation of lipid droplets

in hepatocytes in the presence of NDEA – (H.E. 40�

magnification).

Fig. 2. Section of liver showing very mild granular changes on

addition of chickpea seed coat fibre along with NDEA – (H.E.

40� magnification).

Fig. 3. Section of heart showing accumulation of lipid droplets

upon administration of NDEA – (H.E. 40� magnification).

G. Mittal et al. / Experimental and Toxicologic Pathology 61 (2009) 363–370 367

degenerative changes in muscle fibres. In lungs, chronicinterstitial pneumonia along with infiltration of leuko-cytes was seen upon NDEA treatment (Fig. 5). Theeffects were however milder, and the lungs were almostnormal when chickpea seed coat fibre was also added tothe diet (Fig. 6).

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Fig. 4. Section of heart showing normal myocardium fibres on

addition of chickpea seed coat fibre along with NDEA – (H.E.

20� magnification).

Fig. 5. Section of lung showing chronic interstitial pneumonia

along with infiltration of leukocytes in the presence of NDEA

– (H.E. 20� magnification).

Fig. 6. Section of lung in almost normal condition on addition

of chickpea seed coat fibre along with NDEA – (H.E. 10�

magnification).

G. Mittal et al. / Experimental and Toxicologic Pathology 61 (2009) 363–370368

Discussion

Although nitrosamines are known carcinogenicagents, their mechanism of action is not very wellunderstood. Nitrosamines such as NDEA have beensuggested to cause oxidative stress and cellular injurydue to involvement of free radicals (Bansal et al., 2005;Bartsch et al., 1989). The free radicals initiate LPO,causing oxidative deterioration of the membrane lipidsand other biomolecules (Gebicki et al., 2000; Spickettet al., 2000; Horton and Fairhust, 1987). NDEA hasbeen shown to be metabolized to its active ethyl radicalmetabolite, and the reactive product interacts with DNAcausing mutation leading to carcinogenesis (Sundaresanand Subramanian, 2003). Experimental, clinical andepidemiological studies have provided evidences sup-porting the role of reactive oxygen species in the etiologyof cancer (Devasagayam et al., 2004; Droge, 2002). Asalready mentioned earlier, though there are severalreports where acute doses of NDEA, varying between100 and 200mg/kg body weight (LD50 ¼ 280mg/kg)were used to show its toxic effects (Kaushal et al., 2003;Bansal et al., 2000; Peto et al., 1991), there is however,no report that looks into the NDEA toxicity underhypercholesterolemic condition, a condition that mayco-exist in a sizable human population, both indeveloping as well as in the developed world. Therefore,a study that investigates the toxicity of NDEA underhypercholesterolemic dietary conditions and the effectof supplementation of dietary fibre was thought to bequite relevant in this context.

Inclusion of dietary fibre such as chickpea seed coatfibre is known to induce a number of physiologicaleffects like increasing the faecal bulk, decreasingnutrient availability and reducing levels of plasmacholesterol. The hypocholesterolemic effect of chickpeaseed coat fibre has already been reported from our laband elsewhere (Singh et al., 1983; Hunninghake et al.,1994). Chickpeas have been a staple part of Indian,Mediterranean and African diets for many thousands ofyears but are a relatively novel addition for the Westerncuisine (Pittaway et al., 2004). Although there is noreport that suggests the presence of antioxidant proper-ties in chickpea seed coat fibre, the lowering of NDEA-induced toxic effects in the presence of chickpea seedcoat fibre could be attributed to the reduced rate ofabsorption of NDEA and hence lesser degree of stress.Chickpea seed coat fibre might be providing protectionagainst NDEA-induced toxicity by interfering with itsentero-hepatic cycle leading to its reduced bioavailabil-ity to the tissues and hence lowering its toxic effects.

Studies carried out elsewhere with other nitrosamineshave shown that they induced LPO in their target tissue,and slightly decreased antioxidant enzyme activities andGSH contents in the liver (Taniguchi et al., 1999;Ahotupa et al., 1987). This gives support to our findings

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wherein the administration of NDEA resulted inincreased LPO in all the tissues to a varying degree.Increased LPO was also associated with significantlyincreased activity of Px in most tissues studied. Theincrease in Px activity may probably be an adaptiveresponse towards NDEA-induced oxidative stress.Decreased activity of other antioxidant enzyme (CAT)indicates that NDEA administration interferes withantioxidant potential resulting in increased generationof active oxygen species and hence increased peroxi-dative damage. However, the effects of NDEA onantioxidant enzymes were reduced considerably oninclusion of chickpea seed coat fibre in the diet,indicating its importance in providing reasonableprotection against NDEA-induced oxidative stress andhence its toxicity.

The results indicate an association between theobserved changes in biochemical parameters, notablyoxidative stress due to oral administration of NDEAunder hypercholesterolemic conditions. The study alsoemphasizes the importance of inclusion of dietary fibrein the diet, in combating the ill-effects of nitrosaminessuch as NDEA, particularly on the antioxidant status ofthe body. However, the underlying mechanisms in-volved at the molecular level need to be furtherexplored.

Acknowledgements

The authors are grateful to the Head, Department ofBiochemistry and Chemistry for providing necessaryfacilities for the study, and to Mr. Avtar Singh fortechnical help.

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