Inhibitory effect of dibenzoylmethane on mutagenicityof food-derived heterocyclic amine mutagens

Shishu, A.K. Singla, I.P. Kaur

University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India

Summary

Dibenzoylmethane (DBM), a structural analogue of curcumin (a bioactive phytochemical present in awidely used spice turmeric) was screened for its inhibitory effect against seven cooked food mutagens(heterocyclic amines): 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimida-zo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-6-methyldipyrido[1,2-a:3’,2’-d]imidazole (Glu-P-1), in both TA98 and TA100 strains of Salmonella typhimurium using AmesSalmonella/reversion assay in the presence of Aroclor1254-induced rat liver S9 homogenate. DBMhas been reported to antagonize the mutagenicity of several chemical carcinogens in vitro and has re-cently been shown to be even more effective than curcumin in suppressing the 7,12-dimethyl-benz[a]anthracene (DMBA)-induced mammary tumors in rats. But there are no reports regarding itsantimutagenic properties against cooked food mutagens. Results of the present investigations clearlyindicate that dibenzoylmethane is a very potent antimutagenic agent, that could effectively inhibitmutagenicity induced by all the tested cooked food mutagens in both the frame shift (TA98) as well asthe base pair mutation sensitive (TA100) strains of S. typhimurium. These highly potent inhibitory ef-fects of dibenzoylmethane against heterocyclic amines observed in our preliminary investigationsstrongly warrant further studies of its efficacy as a cancer chemopreventive agent.

Key words: Antimutagenicity, dibenzoylmethane, cooked food mutagens, Ames assay

Abbreviations:2-AF – 2-aminofluorene; B(a)P – benzo[a]pyrene; DBM – di-benzoylmethane; DMBA – 7,12-dimethylbenz[a]anthracene;DMSO – dimethylsulphoxide; Glu-P-1 – 2-amino-6-methyl-dipyrido[1,2-a:3’,2’-d]imidazole; IQ – 2-amino-3-methylim-idazo[4,5-f]quinoline; MeIQ – 2-amino-3,4-dimethylimida-zo[4,5-f]quinoline; MeIQx – 2-amino-3,8-dimethylimida-zo[4,5-f]quinoxaline; PhIP – 2-amino-1-methyl-6-phenylimi-dazo[4,5-b]pyridine; TPA – 12-O-tetradecanoylphorbol-13-acetate; Trp-P-1 – 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]in-dole; Trp-P-2 – 3-amino-1-methyl-5H-pyrido[4,3-b]indole.

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� Introduction

Cooking of food is a process unique to humans. It en-hances the taste and the digestibility of food so muchso that its beneficial nature is taken for granted; howev-er, it induces profound changes in all types of food. Ithas been well established that these changes may be ofconcern to human health (Felton et al. 1997). Generalcooking procedures such as broiling, frying, barbe-quing, heat processing and pyrolysis of protein richfoods like beef, chicken and fish induce the formationof potent mutagenic and carcinogenic compoundscalled heterocyclic amines (Felton and Knize, 1991;Sugimura and Sato, 1983). These are potent mutagensand carcinogens in rodents, inducing tumors of severalorgans (Ohgaki et al. 1991; Wakabayashi et al. 1992)and in limited studies in monkeys (Adamson et al.

1994). There are reports in the literature indicating thepresence of heterocyclic amines in the urine of humanseating a normal non-vegetarian diet, thus, illustrating

Phytomedicine 10: 575–582, 2003© Urban & Fischer Verlaghttp://www.urbanfischer.de/journals/phytomed Phytomedicine

that a certain population eating animal protein is con-tinuously exposed to these carcinogens through diet(Reistad et al. 1997). Epidemiological studies, al-though not definitive, are also supportive of an associa-tion of heterocyclic amines intake to the etiology ofhuman cancer (deMeester and Gerber, 1995). Taken to-gether evidence from mutagenicity data, activation byvarious species including humans, carcinogenicity inanimals, human consumption data, epidemiologicalstudies and risk assessment supports the conclusionthat heterocyclic amines are probable human carcino-gens (Adamson et al. 1996).

The carcinogenic risk imposed by these probablehuman carcinogens depends not only on the level ofexposure, but is also modulated by other dietary factorsthat influence their uptake and biotransformation.Presently, there is enough evidence to show that chem-ical mutagenesis and carcinogenesis can be inhibitedby a large number of naturally occurring compounds ofplant origin. These inhibitors are minor constituents ofsome commonly consumed vegetables, fruits, bever-ages and spices (Block et al. 1992; Ramel et al. 1986;Steinmetz and Potter, 1996). Therefore, it will be of in-terest to screen and identify the novel chemical con-stituents from plants that could play role in restrictingthe onset of mutagenesis/carcinogenesis.

Curcumin is a major yellow constituent of turmeric(Curcuma longa), a commonly used spice and coloringagent in food preparations (Nadkarni, 1976; Marmion,1979) and has been extensively investigated for its po-tential antioxidant, anti-inflammatoy and chemopre-ventive effects (Ammon and Wahl, 1991; Anto et al.1996 and 1998; Huang et al. 1998; Kawamori et al.1999). Like curcumin, DBM that structurally resem-bles curcumin (diferuloylmethane) in having a centralβ-diketone group and conjugated double bonds (Fig. 1)has also been screened for its various pharmacologicalactivities. DBM* and its synthetic derivatives havebeen reported to inhibit the mutagenicity and nucleicacid binding of chemical carcinogens in vitro (Choshiet al. 1992; Wang et al. 1991). Topical application ofDBM to the mouse skin has been reported to inhibitboth 12-O-tetradecanoylphorbol-13-acetate (TPA)-in-duced skin inflammation and skin tumor promotion ina dose-dependent manner (Conney et al., 1991). Di-etary DBM has been observed to reduce 7,12-di-methylbenz[a]anthracene (DMBA)-induced mammarytumors in rodents and these inhibitory effects are morepotent than those of curcumin (Huang et al. 1998; Linet al. 2001b; Singletary et al. 1998). It has also been re-ported to inhibit the formation of DNA-adducts follow-ing exposure to benzo[a]pyrene (B[a]P) and 1,6-dini-tropyrene in human mammary epithelial cell lineMCF-10F (Singletary and MacDonald, 2000). Recentstudies in several human prostate carcinoma cell lines

have also indicated its potential in the prevention andtreatment of prostrate cancer (Jackson et al. 2002).

Cooked food-derived heterocyclic amines are pro-mutagens and have been reported to be activated by cy-tochrome P450 isozymes particularly cytochromeP4501A2 (Boobis et al. 1994; Snyderwine et al. 1997).Further more, mechanistic studies have indicated thatchemopreventive effects of DBM are due to inhibitionof metabolism of chemical carcinogens to their proxi-mate carcinogenic forms by cytochrome P450 1A1,1A2 and 1B1 isozymes (Lin et al. 2001a; MacDonaldet al. 2001). Therefore, based upon these literature re-ports it is proposed to evaluate the antimutagenic po-tential of DBM against these probable food generatedcarcinogens/mutagens.

In the present investigation we evaluated the antimu-tagenic properties of DBM using Ames Salmonella/re-version assay in two strains of S. typhimurium namelyTA98 and TA100 against various classes of hetero-cyclic amines found in human diet namely: amino imi-dazoazaarenes; IQ, MeIQ, MeIQx and PhIP; pyridoin-dole derivatives; Trp-P-1 and Trp-P-2; and dipyridoim-idazole derivative; Glu-P-1.

� Materials and Methods

Bacterial strains A set of histidine requiring TA98 and TA100 strains ofSalmonella typhimurium were obtained as a kind giftfrom Dr Bruce N. Ames (University of California,Berkley, USA).

Chemicals 2-Amino-3-methylimidazo[4,5-f]quinoline(IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ),2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline

576 Shishu et al.

Fig. 1. Structures of dibenzoylmethane and curcumin.

(MeIQx), 3-amino-1-methyl-5H-pyrido[4,3-b]indole(Trp-P-2) acetate and 2-amino-1-methyl-6-phenylimi-dazo[4,5-b]pyridine (PhIP) were purchased fromToronto Research Chemicals Inc., Canada. 2-Amino-6-methyldipyrido[1,2-a:3’,2’-d]imidazole (Glu-P-1) hy-drochloride (monohydrate) was purchased from WakoPure Chemicals, Japan. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) acetate was kindly giftedby Dr T. Nohmi, National Institute of Hygenic Sci-ences, Tokyo, Japan. Dibenzoylmethane 98% (DBM)was purchased from Aldrich Chemical Company, Inc.USA. Albumin, bovine and nicotinamide adenine dinu-cleotide phosphate (NADP) sodium salt, were pur-chased from Sisco Research Laboratories, Bombay,India. D-Glucose-6-phosphate monosodium salt and d-biotin were purchased from Sigma Chemical Compa-ny, USA. Oxoid, nutrient broth no.2 was purchasedfrom Oxoid Ltd., Basingstoke, Hampshire, England.Nutrient agar was purchased from Hi media Lab. Pvt.Ltd., India. All other reagents used were of AR grade.

Preparation of liver homogenate S9 fractionThe S9 fraction was prepared from the pooled liver ho-mogenate of 2 male Sprague-Dawley rats previouslyinduced with Aroclor 1254, by the method of Garner et al. (1972).

Determination of protein concentration of S9 Protein concentration of induced rat liver S9 was deter-mined by biuret method (Gornall et al. 1949) and wasfound to be 54 mg/ml.

Antimutagenicity testingThe plate incorporation procedure given by Maron andAmes, 1983 was used for antimutagenicity testing withthe inclusion of pre-incubation step (Yahagi et al. 1977)

Sterile test tubes each containing 500 µl of S9 mix(50 λ; the optimum concentration when tested against100 µg/plate of 2- aminofluorene), 100 µl of bacterialsuspension (16 hour bacterial culture, 1–2 × 109

cells/ml), 50 µl of DBM solution in DMSO and 50 µl ofthe respective mutagen solution in DMSO, were gentlyvortexed and incubated at 37 °C for 20 minutes. Then 2 ml of molten top agar, maintained at 45 °C, was addedto each of the above tubes. The tubes were vortexedthoroughly, and the mixture was immediately poured onto minimal glucose agar plates, prepared previously.The plates were allowed to harden horizontally forabout half an hour and were incubated at 37 °C for 48hours and the number of induced revertants was scored.

Simultaneously, each assay included a set of negativecontrol (500 µl of S9 mix, 100 µl of bacterial cultureand 100 µl of DMSO), and a positive control [500 µl ofS9 mix, 100 µl of bacterial culture and 100 µl of stan-dard mutagen 2-aminofluorene (100 µg/ml in DMSO)solution].

DBM in the concentration range of 0.25–100µg/plate was also checked for possible toxic or muta-genic effects in both TA98 and TA100 strains and nochange in spontaneous revertant count indicated ab-sence of any mutagenic/toxic effects of DBM in thetested dose range (see footnote of Tables 1–3 for rever-tant counts).

Inhibitory effect of dibenzoylmethane 577

Table 1. Inhibition of Aroclor induced S9-mediated mutagenicity of cooked food mutagens by dibenzoylmethane in TA98strain of Salmonella typhimurium.

No. of His+ revertants/plate*

Cooked food mutagen IQ MeIQ MeIQx Trp-P-1 Trp-P-2 PhIP Glu-P-1(µg/plate)…. (0.010) (0.005) (0.026) (0.225) (0.021) (0.897) (0.050)

Dibenzoylmethane(µg/plate)

0 502 ± 8 1200 ± 42 864 ± 32 390 ± 51 459 ± 128 160 ± 5 959 ± 26 0.25 316 ± 15 732 ± 44 479 ± 26 341 ± 8 451 ± 129 116 ± 3 428 ± 250.5 357 ± 26 649 ± 18 271 ± 34 272 ± 64 335 ± 56 51 ± 2 429 ± 191.0 254 ± 9 487 ± 45 197 ± 16 234 ± 53 263 ± 68 57 ± 5 400 ± 142.0 191 ± 12 520 ± 7 149 ± 7 213 ± 82 203 ± 71 19 ± 2 404 ± 114.0 – – – 70 ± 12 235 ± 8 – –

*A ll values are expressed as mean ± S.D. (n = 6) and include spontaneous revertant count (negative control) of 30 ± 4 (n = 15).With 2-AF (positive control) revertant count is 5326 ± 187 (n = 15).The spontaneous revertant count in presence of various concentrations of DBM alone is: 35 ± 2 (0.25 µg/plate); 29 ± 3 (0.50µg/plate); 30 ± 3 (1.0 µg/plate); 26 ± 6 (2.0 µg/plate); 28 ± 5 (4.0 µg/plate), using three plates per point.

A suitable dose of the test mutagens was selectedfrom the linear portion of the dose-response curve of therespective mutagen (Alldrick et al. 1986) for both TA98and TA100. Further, mutagens were applied to the test insuch doses which resulted in a maximum of about 2000his+ revertants/plate (9–12 fold increase over sponta-neous count), so as to ensure accurate counting, since at

this count overlapping of bacterial colonies is avoidedand inhibition or enhancement by modulators can be de-tected with a minimum statistical variation.

All assays were carried out in duplicate/triplicate onseparate occasions. Results are expressed as mean ± SDof his+ revertants per plate (uncorrected for spontaneouscount) for each dose.

578 Shishu et al.

Fig. 3. Effect of varying concentrations of dibenzoylmethane on S9-mediated mutagenicity of various cooked food mutagens– IQ (1.0 µg/plate), MeIQ (0.11 µg/plate), MeIQx (2.56 µg/plate), Trp-P-1 (22.50 µg/plate), Trp-P-2 (2.14 µg/plate) and PhIP(89.7 µg/plate) in TA100 strain of S. typhimurium.*Mean value is not significantly different from the preceding mean value at P < 0.05.

Fig. 2. Effect of varying concentrations of dibenzoylmethane on S9-mediated mutagenicity of various cooked food mutagens– IQ (0.010 µg/plate), MeIQ (0.005 µg/plate), MeIQx (0.026 mg/plate), Trp-P-1 (0.225 µg/plate), Trp-P-2 (0.021 µg/plate),PhIP (0.897 µg/plate) and Glu-P-1 (0.050 µg/plate) in TA98 strain of S. typhimurium. *Mean value is not significantly different from the preceding mean value at P < 0.05.

and Figs. 2–5) clearly indicate that DBM is a very po-tent antimutagenic agent, that could effectively inhibitmutagenicity induced by all the tested cooked food mu-tagens at very low doses (ID50 values lie between 0.16to 2.29 µg/plate) except Glu-P-1 in TA100 (ID50 = 16 µg/plate). In TA98 maximum inhibition of~60–90% was observed against imidazoazaarenes i.e.IQ, MeIQ, MeIQx and PhIP, 46–54% inhibition againstpyridoindole derivatives Trp-P-1 and Trp-P-2 and 58%inhibition against dipyridoimidazole derivative Glu-P-1 at the dose level of 2 µg/plate (Fig. 2).

In base pair mutation sensitive strain TA100, DBMshowed stronger antimutagenic effects against cookedfood mutagens. More than 80% inhibition was ob-served against imidazoazaarenes i.e. IQ, MeIQ, MeIQxand PhIP, and upto 73% inhibition against pyridoindolederivatives Trp-P-1 and Trp-P-2 at the dose level of 2 mg/plate (Fig. 3). At lower doses of DBM, an in-crease in mutagenicity of Glu-P-1 was observed (datanot shown), where as a potent inhibitory effect (morethan 80% inhibition at 25 µg/plate) was observed in thedose range of 12.5–100 µg/plate (Fig. 4).

A linear dose-response relationship was observedagainst all the tested mutagens (r = 0.8–0.9) exceptagainst MeIQ and Glu-P-1 in TA98 and against IQ andMeIQx in TA100. The order of antimutagenic poten-tial, quantified as ID50 values (the dose of DBM inmg/plate required to reduce the mutagenicity of a givenmutagen by 50%, calculated from corresponding doseresponse curves), observed against various cookedfood heterocyclic amines is as follows:

In TA98Glu-P-1 > MeIQx > PhIP > MeIQ > IQ > Trp-P-2 > Trp-P-1 (0.21) (0.31) (0.38) (0.67) (1.07) (1.60) (2.29)

In TA100IQ > PhIP > MeIQx > MeIQ > Trp-P-1 > Trp-P-2 > Glu-P-1(0.16) (0.20) (0.28) (0.43) (1.51) (1.68) (16)

Statistical AnalysisAll the data were statistically analysed by one wayanalysis of variance (ANOVA) followed by Student-Newman-Keuls method. The data which was not nor-mal or where variations, were not equal, was subjectedto Kruskal-Wallis one way analysis of variance(ANOVA) on ranks. Linear regression was used to testfor linearity of dose-response relationship.

� Results

Results of inhibitory studies, against S9-mediated mu-tagenicity induced by heterocyclic amines in S. ty-phimurium TA98 and TA100 (as shown in Tables 1–3

Inhibitory effect of dibenzoylmethane 579

Fig. 4. Effect of varying dose of DBM on S9-mediated mu-tagenicity of Glu-P-1 (5.05 µg/plate; 20 nmoles/plate) inTA100 strain of S. typhimurium.*Mean value is not significantly different from the precedingmean value at P < 0.05.

Fig. 5. Comparison of ID50 values observed against various cooked food mutagens in TA98 and TA100 strains of S. ty-phimurium.

� Discussion

Recently, much attention has been focused on the roleof diet in the etiology of cancer. Diet is a complex mix-ture of chemical entities and may contain substancesthat cause cancer as well as agents that can inhibit ormodulate the development of neoplasia. There is con-

siderable evidence to indicate that man is exposed toheterocyclic amines through diet and is susceptible tothe carcinogenic effects of these highly potent muta-gens and reported rodent and non-human primate car-cinogens. Antimutagens and anticarcinogens present inthe diet are well known to play a significant role incombating the actions of cancer causing agents.

In the present investigation, we have tried to evaluatethe antimutagenic potential of DBM, against highly po-tent heterocyclic amine mutagens that are generatedduring cooking of muscle meats such as beef, fish andchicken, using a short-term genotoxicity assay i.e.Ames Salmonella/microsome assay. The Ames assay iswell established and an excellent method for screeningof such cancer chemopreventive factors (Brockman etal. 1992; Edenharder et al. 1999).

Interestingly, dibenzoylmethane (structure given inFig. 1), possessing central β-diketone moiety (as pre-sent in the curcumin molecule) was found to be highlyactive against all the compounds investigated in thepresent study and these antimutagenic effects weremuch higher than those observed with curcumin (un-published results). Such a strong antimutagenic effectof DBM might be because of the relatively small sizeof the molecule compared to curcumin and absence ofhydroxyl groups on the aromatic rings. Dibenzoyl-methane is also reported to inhibit S9-mediated muta-genicity of DMBA (Huang et al. 1998; Singletary et al.1998) and very recently it has been established thatthese effects are due to inhibition of various cy-tochrome P450 enzymes namely 1A1, 1A2 and 1B1(Lin et al. 2001a; MacDonald et al. 2001). Based on

580 Shishu et al.

Table 3. Inhibition of Aroclor induced S9-mediated muta-genicity of Glu-P-1 by dibenzoylmethane in TA100 strain ofSalmonella typhimurium.

Cooked food mutagen Glu-P-1(µg/plate)…. (5.05)

No. of His+ revertants/plate*Dibenzoylmethane(µg/plate)

0 1710 ± 8512.5 1048 ± 4425 307 ± 1450 291 ± 13

100 110 ± 11

*All values are expressed as mean ± S.D. (n = 6) and includespontaneous revertant count (negative control) of 132 ± 10 (n= 15).With 2-AF (positive control) revertant count is 2758 ± 67 (n= 15).The spontaneous revertant count in presence of various con-centrations of DBM alone is: 131 ± 5 (12.5 µg/plate); 136 ± 3(25 µg/plate); 132 ± 6 (50 µg/plate); 134 ± 4 (100 µg/plate),using three plates per point.

Table 2. Inhibition of Aroclor induced S9-mediated mutagenicity of cooked food mutagens by dibenzoylmethane in TA100strain of Salmonella typhimurium.

No. of His+ revertants/plate*

Cooked food mutagen IQ MeIQ MeIQx Trp-P-1 Trp-P-2 PhIP(µg/plate)…. (1.00) (0.11) (2.56) (22.5) (2.14) (89.7)

Dibenzoylmethane(µg/plate)

0 1048 ± 22 918 ± 18 1121 ± 12 560 ± 24 462 ± 10 1205 ± 180.25 203 ± 6 828 ± 16 592 ± 11 498 ± 5 375 ± 7 455 ± 70.5 185 ± 6 220 ± 10 233 ± 5 403 ± 11 364 ± 6 224 ± 71.0 171 ± 7 203 ± 4 218 ± 6 352 ± 10 289 ± 8 207 ± 132.0 172 ± 3 160 ± 5 152 ± 4 206 ± 6 202 ± 6 205 ± 15

*A ll values are expressed as mean ± S.D. (n = 6) and include spontaneous revertant count (negative control) of 132 ± 10 (n =15).With 2-AF (positive control) revertant count is 2758 ± 67 (n = 15).The spontaneous revertant count in presence of various concentrations of DBM alone is: 127 ± 8 (0.25 µg/plate); 136 ± 2 (0.50µg/plate); 132 ± 3 (1.0 µg/plate); 126 ± 7 (2.0 µg/plate), using three plates per point.

these literature reports and considering the fact thatcooked food mutagens are activated only in the pres-ence of liver microsomal enzymes, it can be concludedthat these strong antimutagenic effects of DBM,against cooked food mutagens could probably be dueto the inhibition of bioactivation of these promutagens.Since in the present study, DBM has shown strong in-hibitory effects against all the tested food-derived hete-rocyclic amines, it may thus be suggested that a com-mon mechanism of inhibition is involved i.e. alterationin metabolic activation.

In the strain TA100, treatment with DBM at lowerconcentrations (less than 12.5 µg/plate) resulted in anincrease in the mutagenicity of Glu-P-1, where as athigher doses (12.5–100 µg/plate) showed a strong in-hibitory effect. No change in the spectroscopic absorp-tion maxima of Glu-P-1 and DBM (results not shown)upon mixing the solutions of these two compoundsrules out any in vitro molecular interaction or complex-ation of DBM with Glu-P-1. Since the concentration ofGlu-P-1 required to induce mutagenicity in TA100 wasfound to be very high, i.e. 20 nmoles/plate; hence, onlyhigher amounts of DBM could inhibit the mutagenicityof Glu-P-1. The increase in mutagenicity of Glu-P-1 atlow concentrations of DBM could, however, be due tothe induction of Phase-1 activating enzymes by theseagents at low doses as there are reports in the literaturethat indicate that DBM behaves as a bifunctional induc-er of liver enzymes (Singletary et al. 1998). However,this behavior of DBM towards Glu-P-1 in TA100 isquite intriguing and more exhaustive investigations arenecessary and are suggested to explain these effects.

To summarize, these findings indicate that DBM is ahighly potent inhibitor of S9-mediated mutagenicity ofheterocyclic amines and may play a beneficial role inchemoprevention of human cancers induced by thesecooked food mutagens. Since these are only prelimi-nary investigations using in vitro bacterial system, it isvery hard to predict whether similar effects can be ex-pected in humans as complex mechanisms, namely, in-teractions with metabolic activation reactions are notadequately represented in in vitro assays with exoge-nous enzyme homogenates. However, this study doesprovide an impetus for the further evaluation of DBMas possible chemopreventive agent in human cancersinduced by these cooked food mutagens using the suit-able mammalian cell lines and by carrying out bio-chemical, enzymatic and in vivo investigations in ani-mal models as well as in humans.

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� Address

Shishu, University Institute of Pharmaceutical Sci-ences, Panjab University, Chandigarh-160014, India. Tel.: ++91-172-534112(O), ++91-172-782099(R),Fax: ++91-172-541142;e-mail: shishugoindi@yahoo.co.in, goindi@satyam.net.in

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