Indian Journal of Experimental Biology Vol. 42, June 2004, pp. 595-600

Effect of amaranth leaves on dimethylhydrazine-induced changes in multicomponent antioxidant system of rat liver

K R Anilakumar*, Farhath Khanum, K R Sudarshanakri shna & K Santhanam

Biochemistry and Nutrition Di scipline, Defence Food Research Laboratory, Mysore 570 011. India

Received 22 May 2003; revised 17 February 2004

Effect of prefeeding dehydrated amaranth (A. gallgeliclIs) leaves at 10 and 20% level s on a chemical toxicant, d imethylhydrazine (DMH)-induced free radical stress in rat liver was evaluated. DMH-induced ri se in hepatic malondialdehyde (MDA), was dimini shed by AL. AL intake resulted in a significant increase in hepatic glu tathione (GS H). The feeding of AL at 10%level increased the hepatic glucose-6- phosphate dehydrogenase (G-6-PDH) activity, while that at 20% level increased the hepatic glutathione reductase (GSSGR) as well, in addition to G-6-PDH. Amaranth leaves at 10 and 20% levels of feeding diminished the hepatic superoxide di smutase and glutathione peroxidase (GSH-Px) activities. DMH influenced adversely the hepatic antioxidant enzyme activities. Simultaneous administration of DMH and feeding of AL enhanced the DMH-induced decrease in hepatic GSH-Px. DMH enhanced formation of micronuclei was reverted significantly by AL intake. Hence, it was concluded that the consumption of AL at 20% level reduced DMH-induced impaired antioxidant status in rat li ver.

Keywords: Amaranth leaf, Antioxidant, Liver, Multicompone nt antioxidant system , Rat

IPC Code: Int CI7 A61 K

Amaranth leaves (Amaranthus gangeticus Linn .) are widely consumed as a vegetable in India and are rich in carotenoids. The carotenoids and perhaps non­nutrients like dietary fibre and other phenolic constituents influence the enzymes involved in the activation and detoxification of xenobiotics including carcinogens. This effect cou ld primarily be mediated through alterations in levels of phase I cytochrome P-4S0 enzyme-dependent metabolism of carcinogens and induction of phase-II enzymes i.e. glutathione S­transferase, glutathione reductase, g lutathione peroxidase, catalase etc. I

-4

. Therefore, vegetables are important source of antioxidant nutrient and non­nutrients and are being advocated for use in the dietary management of degenerative diseases. The antioxidant compounds have been well recognized to have effective role in reducing the oxidation stress and thereby, reduce the risk of cancer. National Research Counci I, therefore, has recommended an increased consumption of fruits and vegetables5

.

There are several reports on cabbage6, garlic7

, carrotS, I 9 B I 10 h . . I curry eaves russe s sprouts etc. on t elr potentia

beneficial effects per se as well as against chemically induced carcinogenesis. Fibre ll

, calcium l 2 and folic

*Corrcspondent author: Tel : +91-821-473290. Fax:+9 1-82 1-474282. E-mail: dfoodlab @sancharnet.in

acid 13 are some of the food components, currently in the centre of research to ascertain their effects. Amaranth leaves contain dietary fibre, fo lic acid 14 and perhaps other bioactive nutrients such as bioflavonoids. Flavonoids are also reported to possess antioxidant potency 15. Further, amaranth leaves contain Mg, an antimutagen and chlorophyllin, a proven efficient anti mutagen and antioxidant l6

. The knowledge presently available on the potential for protective capacity of dietary components like green leafy vegetables in many instances is sparse and insufficient to make dietary advice on food selection. This study was, therefore, undertaken to investigate whether the consumption of dehydrated leaves of amaranth have any beneficial effect in preventing or protecting against the toxicity of a cancer causing xenobiotic, dimethylhydrazine (DMH), which is known to induce oxidati ve stress 17 .

Materials and Methods Preparation of dehydrated amaranth leaves

diet--Good quality fresh amaranth (Amarallthus gallgeticus) leaves procured from local market were washed, blanched for 2 min with 0.12% potass ium metabisulphite and dried in a cross flow drier till the moisture content was reduced to 9%. The dehydrated amaranth leaves (AL) thus prepared were powdered

596 INDIAN J EXP BIOL, JUNE 2004

and found to contain 41.9% carbohydrates, 25.7 % prote in , 2.7 % fat, 14% ash and 6.7 % crude fibre . The content of carbohydrates was calculated by difference. The dehydrated AL was added at 10 and 20% levels to prepare the experi mental diet. The control and experimental diets were made nearly isoenergetic by adj usting corn starch, casein and o il.

Allilllal lreallllell/-Thirty-six male wistar rats ( I 10- 130 g) were a llocated rando mly to 6 groups of each. Groups I and 2 were fed the control diet, groups 3 and 4 received 10% AL incorpo rated control die t and groups 5 and 6 received AL at 20% level in control diet for 12 weeks. Groups 2, 4 and 6 were administered 10 weekly injections of DM H (60 mg/kg body weight; sc) and groups I , 3 and 5 received saline. All the rats were hou sed in indi vidual sta inl ess

stee l wire-bottomed cages at o f 27°±2° C. They were fed ad IibilulIl w ith free access to water. Weekly food intake and weight gain were monitored. At the end of 12 weeks, all the rats were sacri ficed under mi Id anaes thes ia (sod ium pentobarbitone) and organs / ti ssues were qui ck ly exci sed and stored in liquid nitrogen until analy ses.

Chelllical assay--For the assay of MDA I8, li ver

ho mogenate (0.5 g) was precipitated wi th tri chloroacetic ac id (10%) and reac ted with thi obarbituric acid reaction mixture 0.35 % consisting of sodium dodecy l sulphate, fer ri c chloride and butylated hydroxy to luene in 0.1 M of g lyc ine-H C I buffer. After bo iling and cooling, OD was taken at

A.m:!x 532 and MDA was calcu lated using a molar

ex tinction coefficient o f 1.56 x 105/M/cm. Hepat ic glutath io ne (GS H) content was determined by the method of Ellman l9 using 5,5'-dithiobis-2-nitrobenzoic acid reagent. For the assay of catalase

2o,

li ver (0. 5 g) was homogeni zed in phosphate buffer (5 M:pH 7.4) and the homogenates were centrifuged at 700 xg . The supernatant was assayed using hydrogen perox ide as a substrate. G lutathione reductase (GSSGR) and g lutathi o ne peroxidase (GS H-Px) activ iti es were detennined by the method of We iss et aP I in the supernatan t of li ver ho mogenate prepared in phosphate bu ffer (0.5 M pH 7.0) using H20 2 and NAD PH as substrates . Hepatic g lutathi one S­transferase (GST) activity was determined by the procedure of Habig el 0[22 in the supernatant of li ver homogenate in phosphate buffer (0. 1 M pH 6 .5), centrifuged at 700xg using l-chloro-2,4-dinitrobenzene as substrate. Superoxide di smutase (SOD) was measured by the inhibition o f cy tochrome

C reductio n medi ated via superoxide anions generated by xanthine-xanthine oxidase and mon itored at 550 nm. One unit of SOD was de fined23 as the amount required to inhibit the reduction of cytochrome C by 50%. The assay mixture for the es timation of hepat ic g lucose-6-phosphate dehydrogenase (G-6-PDH) consisted of the enzyme source prepared in 0 . 1 mM of Tri s buffe r, 2 mM of g l cose-6-phosphate, 0 .3 mM of NA DP and I'1A was monitored at 340 nm2~ . Gamma g lutamyl transpeptidase (GGT) was est imated in

kidney by the method of M eister el 0125 using L-Y­g lutamy l-p-nitroan ilide as the subst rate in a homogenate prepared with 0.1 M of T ris- HCI buffer. Micronuclei in fe mur bone marrow was est imated essentiall y according to Countryman and Heddle25

.

Prote in in ti ssues was de termined accordi ng to Lowry et aP7. The sign ificance of differences among mean va lues was calculat ed according to Student' s I test28

.

Results Effecl 0 11 daily food ill lake, weighl gaill alld li ver

weight of rats-The e ffect of consumption of AL for 12 weeks with or without DMH injection on daily food intake and weight ga in of rats are g iven in Fig. I A and

>. ro u ---OJ

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20 (A L a 18 16 14 12 10 8 6 4 2 0

Food intake

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6

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Fio 1- Effec t o f AL with or without the treatme nt o f DMH o n (A)' food intake; and (B) weight gai n o f rats. [Va lues bearing d iffere nt superscripts in the fi gure are s ign ificanlly different

(P<O.OS ): Va lues are Mean±S D for 6 rats).

ANILA KUMAR el al.: EFFECT OF AMARANTH LEA VES ON DIMETHYLHYDRAZINE INDUCED C HA NGES 597

I B respecti vely. Dehydrated amaranth leaves were fed to rats at 10 and 20%. DMH injection did not appear to influence the food intake and weight gain pattern of rats. The consumptio n of AL containing diet was found to be reduced s igni ficantly both at 10 and 20% levels as compared to control irrespec ti ve of DMH admini strat ion.

Effect 0 11 hepatic lipid peroxides alld alltioxidallts ill li ver-The data for effect of AL ingesti on and DMH treatment o n cytotox ic product viz., MDA and ti ssue antiox idant (GSH ) are g iven in Table 1. DMH induced a sig nificant ri se in MDA with a concomitant decrease in GSH as compared to contro l. An increase in the hepati c GSH level was seen in the group fed AL at LO and 20% levels. Dimethylhydrazine did not influence the hepati c lipid perox ides and the combination o f AL intake and DMH injection did not bring about any change in these parameters.

Effect 0 11 hepatic alltioxidallt / detox(fy illg ell ZY1l1es ill liver-T able 2 presents the effec t o f feeding of dehydrated AL on DMH-induced changes in hepatic anti ox idant/detoxify ing enzymes . DMH influenced adversely the hepatic enzy me activiti es viz. , GSH-Px , catalase and SOD activiti es as compared to co ntro l

Table I- EfFect o f AL w ith or witho ut DMH on GS H and li pid perox ides in rat li ve r

(Values are mea n ± SD of 6 rats)

Rat group GSH MDA

mll1o le/g nmole/g

Control 9.0 ± 1.0 1 "c 7.9 ±0.8 1 " DM H 7.1 ± 0.89 h 10.6 ± 1.00 h

AL 10% 10.4 ± 1.46 cJ 7 .6±0.91 " AL 10%+ DMH 7.9±0.9 I hf 7.8 ±0.74" AL 20% 12.4± 1.05 J 6.5 ±0.82 c

AL 20%+ DMH 8.2 ± 0.93 cf 8.0 ±0.75 "

Va lues bearing differe nt supersc ripts in the same co lumn are significantly d ifferent at p<0.05

group of rats. Amaranth leaves at 20% level enhanced the hepatic GSSGR activity as compared to control. At the same time the simultaneous admini strati on of DMH and feedi ng o f AL did not alter the DMH­induced decrease in hepatic GSH-Px and SOD activities. The activity of GSSGR in 20% of AL fed rats was signifi cantly hi gher than control and DMH ad ministratio n had no effect.

Gamma g lutamyltranspeptidase in kidney was increased signi ficantly by DMH , and AL at both the levels of feeding reduced it (Fig. 2A).

Effect 0 11 DMH-incluced alteratioll s ill colollic

alltioxidall t status ill rats-Table 3 shows the effect of feeding of dehydrated AL with or without DMH treatment in colonic GSH , MDA and antioxidant enzy mes. An increased GSH content was observed in co lon by AL at both the levels of feeding . The superox ide dismutase activity was decreased with AL (at both the leve ls) with a si multaneo Li s increase in GSH-Px and GSSGR activities (at 20% level on ly). As seen earl ier, DMH produced signifi cant decrease in coloni c GSH and GSH-Px , GST and SOD activities. At the same time, the colonic catalase activity was enhanced by DMH . The AL feeding per se increased colo nic GSH and the co-admini stration of AL at 10 and 20% levels in DMH treated rats impro ved the DHM-induced reduction in GSH. The colo ni c MDA showed a differe nt pattern . AL feed ing increased the colonic MDA o n co-admini stration with DMH . Here, the MDA values remained higher than the control. Catalase was increased afte r DMH insu lt which re mained unaltered afte r inclusion of AL in the diet.

E.ffect 0 11 bOll e lI1a rrow 1I1icrolluclei-Fi g. 28 presents the effect of AL o n DMH- inducecl a lte ratio ns in micro nuclei formation in fe mur bone marrow. The results showed the DMH-inducecl

Table 2- EfTect of AL with or without DMH in hepatic an tiox idant/detox ifying e nzy mes

(Va lues are mea n ± SD 01' 6 rats)

Rat group GSH -Px GSSG R Catal ase G-6-PDH x 10.3 # X 10') # x 1 0~ * @

Cont ro l 2.30 ± 0.30" 3.4 ± 0.45 " 0.91 ± 0. 124" 66.4 ± 5.52 " DMH 1.6 1 ± 0. 16" 3.3 ± 0.30" 0.50 ± 0 .1 05 b 89.5 ± 4 .9 1 b

AL IO% 1.90 ± 0 .20 c 3.2 ± 0.44" 0.96 ± 0. 180" 73 .9 ± 9.84 c

AL IO%+DMH 1.4 1 ± 0. 18 b 3.5 ± 0.54" 0.54 ± 0.1 IS b 84.4 ± 5.35 b

AL20% 1.94 ± 0.34 c 5 . 1± 0.78 b 1.00 ± O. 125 " 89.2 ± 8.35 b

AL20%+DM H 1.94 ± 0. 19 c 5.2 ± 0.83 b 0.70±0. 11 2h 86.3 ± 6.42 b

Values bearing differe nt s upe rsc ript s in the same column are signifi cantl y d ifferent at p<0.05 *f'..A ofO . llmin/mg protein; # mmo le NADP formed/min/ mg prote in : ** units/min/mg protein ; @fll1lo le NADP+ reduced /m in/mg prote in ; $ mmo le conjugate formed/min/mg prote in

SODx l02 GSTx l02

** $

1.6 1 ±0. 13 1" 2.0 ± 0.29 " 0.92 ± 0. 139 b 1.9 ± 0.2 3 " 1.29 ± 0.252 c 2.3 ± 0.28" 1.02 ± 0.287 c 2.2 ± 0.3 1 " 1.35 ± 0.256 c 2.0 ± 0.3 3 " 1.04 ±0.23 3 c 2.4 ± 0 .39 "

598 INDIAN J EX? BIOL, JUNE 2004

enhanced formation of micronucl ei and the reversion by AL at both levels.

Discussion Effect of amaranth leaves on biomarkers of

oxidative stress was studi ed in order to demonstrate antiox idative potentials of phytochemicals, if any , in vivo. Our earlier work6

.g

and the present paper describe the onset of oxidative stress by the treatment of DMH as evidenced by enhanced MDA and decreased GSH and ac tiviti es of GSH-Px , catalase and SOD in rat li ver. The concomitant feeding of AL

30

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Fig.2- Effec t o f AL wit h o r w ithout the trea tme nt of DMH o n (A) GGT activ ity (nmo le p-nitroanlide re leased/min/mg pro te in) o f kid ney; and (B) femur bone marrow mi cronucle i of rats IValues bearing diffe rent supe rscripts in the fi gure are

s ign ificantly different (P<0.05); Val ues are Mean±SD for 6 rats].

and administration of DMH reversed the DMH­induced elevation of MDA and the DMH-induced reduction of GSH in li ver. The reduced levels of GSH might allow perox ides to accumulate in erythrocytes and haemolysis can occur due to thei r oxidati ve effect on lipids of the red cell membrane. Formation of MDA might result in oxidative s tress in vivo. Oxidative stress is a state of imbalance between generation of reactive oxygen species like hydroxy l and superoxide radicals and the level of antioxidant defence sys tem29

.

The increased GSH levels in rats fed wi th amaranth leaves might be helpful in reducing the DMH -induced elevated levels of oxidative radicals. Peroxidation in vivo is influenced by the activity of SOD, catalase and GSH-Px30

. The interactive effects of AL and DMH treatments, which led to decrease in lipid peroxidation in liver, may be ascribed to the protective phytochemicals. Carotenoids are thought to scavenge free radical s and o ther ox idants in volved in d isease process3t

. Decrease in levels of GSH-Px and SOD in li ver after feeding of amaranth leaves as observed in the present study warrant further studies.

Gamma glutamy ltranspeptidase is important in transport of amino ac ids required for the synthesis of GSH in cells. The elevated levels of GGT in kidney of rats treated with DMH might reduce the stress on GSH in the cell and enable the cells to respond to proliferative and other stimuli. GGT has been show n by WHO to be a very sensi tive and reliable index of pre-cancerous changes in ti ssues32

. Amaranth leaves produced remarkable change in the DMH-i nduced increase in GGT activity .

Amaranth leaves modulated the signifi cant reduction produced by DMH in colonic GSH-Px. At the same time GSSGR activity was significantly increased by 20% o f AL. The increase.d GSH-Px and

Table 3--EfTec t of AL wi th or without DMH on colonic ant ioxidant Idetox ifying enzymes

(Values are mean ± SD of 6 rats)

Rat group GSH ~l11o l es/g MDA Catalase GSH- x GSSGR G-6-PDH GST SOD nlllole/g *** x 10 -.1 X 10 -.1 @ * $

** **

Cont ro l 62.1 ±7.1 9" 1.39 ± 0.21" 0. 14±0.0 18" 0.21 ±0.033" 0.41 ± 0 .056 " 11 .28 ±1.39 " 46.2 ± 4 .90" 3.7±0.39" DMH 40.2 ± 5.29 h 1.40 ± 0.24 " 0.25 ± 0.024 h 0. 1 I ±0.02 1 b 0.39 ± 0.042 " I I. I I ± I .48 ' 37. 1 ±4.23" 1.6±0. 18" ALlO % 87. 1 ±9.1 2 c 2.40 ± 0.23 be O. 12 ± 0.020 " 0 .32 ±0.042 c 0.45 ± 0.059" 12.39 ± 1.34 ' 46.9 ± 5.29" 1.8 ± 0.4 1 h

AL 10% +DMH 58.2 ± 6 .22" 2.07 ± 0. 19 h!J 0.25 ± 0 .031 h 0.25 ±0.028" 0.38 ± 0.043 " 12.02 ± I .49" 34.1± 3.91" 1.8 ± 0.23 " AL20 % 85.2 ± 9 .56 c 2.39±0.14 c 0. 12 ± 0.0 19" 0.30 ±0.042 c 0 .52 ± 0.063 b 12.01 ±1.38 " 45.2 ± 4.68" 1.6 ± 0.24 " AL 20%+ DMH 59 . 1 ±6.8 1" 2.0 1 ±0.16 d 0.22 ± 0.038 b 0.24 ±O. 32" 0.40 ± 0 .051 ' I 1.29± 1.51 , 36.8 ± 3.91" 1.9 ± 0.2 1 "

Values bearing differenl superscript s in the same column are significantl y d ifferent at p<0.05 *mnlOle conjuga te forl11ed/l11in/l11g protein; ** mmole NADP formcd/lllin/ lllg protein ; ***t!,.A of O.l/llli Img protein ; $ un its/lllin/lllg protein ; @ ~Illole ADP' reduced/min/mg protein

ANILA KUMAR 1'1 al.: EFFECT OF AMARANTH LEAVES ON DlMETHYLHYDRAZINE INDUCED CHANGES 599

GSSGR activiti es seen in 20% of group were associated with a s ignificant enhancement in colonic GSH level, because the proper GSH status is achieved by the activities of GSH-Px and GSSGR activities. The dehydrated amaranth leaves did not affect the colonic detoxifying enzymes like GST and catalase, but did produce increase in GSH levels .

It is interesting to note the onset of oxidative stress by AL as evidenced by increase in MDA with an assoc iated decrease in SOD activity in the colon. At the same time liver was found to be effecti ve in mitigating the DMH-induced peroxidative stress by reducing MDA and increas ing the antioxidant/detoxifying enzy mes. Thi s dramatic contrast might depend on the pro-oxidant action of carotenoids in AL, which is present in large amount. The antioxidant activity of carotenoids may shift into pro-oxidant actIVity , depending on the redox po tential o f the carotenoid molecules as we ll as on the biological environment in which they act33

. The ox idative s tress induced by DMH in conjunction with hi gh carotenoids concentration in AL might have contributed towards MDA formati on in the co-admini stered group.

Eva luat ion of preventive effec t of AL using the technique of bone marrow mi cronucleus assay was also carri ed o ut. The decrease in mi cro nuclei by AL was inte res ting because, studies reporting the preventive role of AL aga inst the che mica lly induced genotox icity are limited and are of considerable importance with respect to nutrition and cance r.

From the foregoing di scuss io n, it was concluded that the supplementati o n with AL at 20% leve l produced sig nifi cant changes in DMH impaired antioxidant status . Measurement of cytotoxicity was carri ed out by assay ing li pid pe rox idatio n product viz., MDA. Pos iti ve effects o f AL may be ascribed to the high amount of caroteno ids present in them or the sy nerg isti c effects of po ly pheno lic compo unds and

other phytochcmical s . Effect of [3-carotene and o ther carotenoids on human chronic di seases is kno w n3

.J .

Reports are the re that people who ingest more di e tary carotenoids ex hibit less ri sk for several di seases and some cancers. Thus evaluati o n of antioxidants as suppressors of chemically induced lipid peroxidati on provides a scope to se lect natural free radi cal scavengers which on co-admini stration ill vivo, may reduce tox ic e ffects o f chemicals. At the same time, the amaranth leaves affect the hepatic GSH-Px and SOD activItIes adversely. Further studies are warranted to e lucidate thi s pheno menon.

Acknowledgement Authors thank Dr K Jeevaratnam , Project

Coordinator, Bioche mi stry and Nutrition Di scipline and Dr A S Bawa, Directo r, Defence Food Research Laboratory , Mysore for the ir keen interest. The secretarial assistance rendered by Mrs S Sukanya is gratefully acknowledged.

References I Baneljee S, Prashar R, Kumar A & Rao A R. Modulatory

influence of extract of Ocillllllll leaves on carcinogen­metaboli zing enzyme acti vities and reduced glutathione levels in mouse, NlIIr. Callce r , 25 ( 1996) 205.

2 Coldin G A, Branch L G, Lipnick R J. Willet W C. Rosner B 1'1 ai, Increased green and ye llow vegetable intake and lowered cancer death in elderl y population. Alii J Ciill Nll lr. 4 1 ( 1985) 32.

3 Koshimizur K. Ohigashi H. Tokuda H. Kondo A & Yamaguchi K. Screening of ed ible plants against possible antitumour promoting acti vity, Callcer Lell, 39( 1988) 2--1 7.

4 Bidlack W R. Brown R C & Mohan C. Nutrit ional parameters that alter hepatic drug metaboli sm conjugation and toxic ity. Fed Proc. 45( 1986) 142.

5 National Research Council. Dil'l N lIIrilioll alld Callcer

(National Academy Press, Washington DC) 1982. 6 Vi swanathan K R, A nilakumar K R, Farhath Khanum &

Sudarshanakrishna K R. M odulatory effect of dehydrated cabbage on the detox ify ing cnzymes of rats w ith a carcinogen trca tment, Nlllr Res. 18 ( 1998) 1733.

7 Khanum F. Ani lakumar K R. Sudarshanakrishna K R & Viswanathan K R. Effect of feeding fresh garl ic and garlic oil on detox ify ing enzymes and micronuclei forillation in rats treated with azox Ylllethane. 1111 J Vita lllill N ll l r Res. 68 ( 1998) 208.

8 A nilakulllar K R. Khanum F. Sudarshana Kri shna K R .. Vi swanathan K R & Santhanam K. Effect of dehyd rated carro t on the antiox idant enzy mes and micronuclei format ion in rats challenged w ith dimethy l hydrazine. NlIIr Res. 20 (2000) 93.

9 Khanulll F. A nil akumar K R. Sudarshanakri shna K R. Vi swanathan K R & Santhanalll K. A nticarcinogenic potential of curry leaves. Plow Food HlllliWi NII/r. 55 (2000) 3--1 7.

10 DeGroot A P. Willems M I & De Vos R H, Effects of high levels of Brussels sprouts in the diets of rats. Food Chelll Toxicol, 29 ( 199 1) 828.

II Dwyer J. Dietary fiber and colorectal cancer ri sk. NII/r ReI'. 51 ( 1993) 147.

12 Lupton J R. Steinbach G. Chang W C. O'Brien 13 C. Wiese S. Stoltzfus C L. GlobeI' G A , Wargov itch M J, McPherson R S & Winn R J, Calcium supplementation modifies the relati ve amounts of bile ac ids in bile and affects key aspects of human colon physiology, J NII/r, 126 ( 1996) 142 1.

13 Cravo M L, Mason J B. Dayal Y . Hutchinson M . Smith Advances, Selhub J & Rosenberg I H. Folate deficiency enhances the development of colonic neoplasia in dimethylhyd razine- treated rats. Callcer Res, 52 ( 1992) 5002.

14 Gopalan C. Ramasastry 13 V & Balasubramanian S G. in Nutriti ve value of Indian foods. (National Institute of NutrilOn. Indian Council of Medical Research. Hyderabad. India) 1989. 67.

600 INDIAN J EXP BIOL, JUNE 2004

15 Laughton M J, Evans P J & Moroney M A, Inhibition o f mammalian 5-lipoxygenase and cyclo-oxygenase by fl avonoids and phenolic and dietary additi ves, Biochelll Phanllacol. 42 ( 199 1) 1673 .

16 Bronzetti G, Antimutagens in food, Trellds Food Sci Tech, S (1994) 390.

17 Dudeja P D & Brasitus T A. 1,2-Dimethylhydrazine-induced alterations in lipid peroxidation in pre-neoplastic and neoplastic colonic, ti ssues. Biochelll Biophys Acta. 1046 ( 1990) 267. ,

18 Girotti A W & Deziel M, Photodynamic action of protoporphyrin on resealed eryth rocyte membranes: Mechanism of trapped markers, Porphyrill PllOtosellsitizatioll, edited by D Kessel and T J Doughenty, (Plenum Press, New York) (1983) 213.

19 Ellman G L, Li ver glutathione-A colorimetric method for determining low concentrations of mercaptans, Arch Biochelll Biophys, 74 ( 1958) 443.

20 Cohen G, Dembiec C & Marens J, Measurement of catalase acti vity in ti ssue extracts, Allal Biochelll . 34 ( 1970) 30.

2 1 Weiss C, Maker H S & Lehrer G M, Sensiti ve fluorometric assays for glutathione perox idase and reductase, Allal Biochelll . I 06( 1980) 51 2.

22 Habig W B, Pabst M J & Jakoby W, Glutathione S­transferases: the first enzymatic step in mercapturic ac id formation, J BioI G elll , 249(1974) 7130.

23 Flohe L & Otting F, Superox ide assays, Methods Ell zYlllol, ( 1984) 105.

24 Balinsky D & Bernstein R E, The purificat ion and properties of glucose-6-phosphate dehydrogenase from human eryth rocy tes, Biochelll Biophys Acta, 67 ( 1963) 13.

25 Meister A, Tate S S & Griffith 0 W. y-g lutamyl transpeptidase, Methods EII ZYlllol. 77 (198 1) 237.

26 Countryman P I & Heddle J A, The production of micronuclc i fro m chromosome aberrations in irradiated cultures of human lymphocytes, Mlllat Res. 4 1 (1976) 32 1

27 Lowry 0 H, Rosebrough N J, Faro A L & Randa ll R J, Protein measurement with the Folin phenol reagent J Bioi Chelll . 193 ( 1951 ) 265.

28 Fisher R A & Yates F. Statistical allalysis fo r biological. agricultural alld medical research, 6th edition, edited by Oli ves and Boyd, (Edinburgh 1963)

29 Halliwell B & Gutteridge J M C, in Free radicals ill biology alld medicille, 2nd edition (C larendon Press, Oxford ,) 1989.

30 Machlin L J & Bendich A, Free radical ti ssue damage: Protective ro le o f antiox idant nutrients, FASEB. I( 1987) 44 1.

3 1 Sies H & Stahe W, Vitamins E and C, f)-carotene, and other carotenoids as antiox idants, Alii J Ciill Nutr. 62 (suppl ) (1995) 13 15s.

32 WHO, International programme on chemical safety­Environmental health criteria (WHO, Geneva) 1990, 109.

. . 33 Palozza P, Prooxidant actions of carotenoids in biological

systems. Nlltr Rev, 56( 1998) 257.

34 Mayne S T, f)-Carotene, caroteno ids, and di sease prevention in humans, FASEB J, 10(1 996) 690.