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2005 24: 149Hum Exp ToxicolTajdar Husain Khan, Lakshmi Prasad, Anuradha Sultana and Sarwat Sultana

Soy isoflavones inhibits the genotoxicity of benzo(a)pyrene in Swiss albino mice  

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Human & Experimental Toxicology (2005) 24: 149-155www.hetjournal.com

Soy isoflavones inhibits the genotoxicity ofbenzo(a)pyrene in Swiss albino miceTajdar Husain Khan, Lakshmi Prasad, Anuradha and Sarwat Sultana*

Section of Chemoprevention and Nutrition Toxicology, Department ofMedical Elementology and Toxicology,Faculty of Science, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi, India

Dietary factors are considered important environmentalrisk determinants for various diseases. Isoflavones areone of the biologically active polyphenolic plant consti-tuents that possess potential chemopreventive propertiesagainst a wide variety of chronic diseases. In the presentstudy we have evaluated the antimutagenic potential ofsoy isoflavones against benzo(a)pyrene (B[a]P) (125 mg/kg) induced genotoxicity in Swiss albino mice. The effectof soy isoflavones was studied by in vivo bone marrowchromosomal aberration and micronuclei induction test.Using an alkaline unwinding assay we monitored theDNA strand breaks. Two doses of soy isoflavones (20 and40 mg/kg b.wt) were given orally for seven days prior tothe administration of B[a]P. Soy isoflavone inhibited thegenotoxicity of B[a]P in terms of chromosomal aberrationand micronucleus formation. DNA strand break levels inonly B[aiP treated group remained significantly high

from the control values (P <0.001). The pretreatmentof soy isoflavone showed gradual reduction in strandbreaks significantly (P <0.001) and dose dependently.Soy isoflavone pretreatment also decreased cytochromeP450 (CYP) content. The activity of CYP was alsodecreased dose dependently by pretreatment withsoy isoflavone. The chemopreventive effect of soy iso-flavone on the inhibition of CYP activity and DNAintegrity mediate the possible mechanism of inhibitionof genotoxicity. Human & Experimental Toxicology (2005)24, 149-155

Key words: benzo(a)pyrene; chromosomal aberration; cyto-chrome P450; DNA strand breaks; micronuclei tests; soy isofla-vones

Introduction

Natural products are gaining more and more atten-tion due to less toxicity and high efficacy againstvarious types of diseases. Epidemiological studieshave shown that fruits, vegetables, spices, tea, andmedicinal herbs rich in antioxidants and othermicronutrients protect against diverse forms ofchemically induced carcinogenesis, inhibit DNA-damage, mutagenesis and lipid peroxidation.1 Con-sumption of soybean has been shown to producebeneficial effect on glucose and lipid metabolism.There are some reports that show that the prophy-lactic treatment of soybean in different forms havethe potential to decrease the risk of prostratecancer,2 breast cancer3 and colon cancer develop-ment.4 From some other reports we get the idea thatthe chemopreventive effects of soybean and soycontaining food may be related to their isoflavone

*Correspondence: Dr. Sarwat Sultana, Section of Chemopreven-tion and Nutrition Toxicology, Department of Medical Elementol-ogy and Toxicology, Faculty of Science, Jamia Hamdard (HamdardUniversity), Hamdard Nagar, New Delhi 110062, IndiaE-mail: sarwat786@rediffmail.com

Received 10 May 2004; revised 26 November 2004; accepted16 December 2004

© 2005 Edward Arnold (Publishers) Ltd

content.5 In spite of the estrogenic activity, isofla-vones show DNA topoisomerase activity, synthesisand release of TGF3, modulation of apoptosis, anti-oxidant activity and potent inhibitory activity oftyrosine specific protein kinases.6 It has been re-ported that populations having high intake of iso-flavones show lower incidence of cardiovasculardiseases, osteoporosis, kidney diseases and cancerrisk.7-9 The active constituents of soy includeisoflavones, genistein and daidzein. Genistein anddaidzein have direct free radical quenching abil-ity.1011 They have also been shown to prevent8-hydroxy-2'-deoxyguanosine formation in toxicantexposed cells.12Exposure to genotoxicants has been associated

with carcinogenecity and embryotoxicity.13 Geno-toxicity may cause a long-term effect on the sustain-ability of particular populations.14 Genotoxicsubstances may bring about changes in normalDNA integrity leading to pathological condi-tions.15'16 Damage to the structure of affected DNAis unequivocally the main cause of mutagenesis.17 Afrequently occurring form of DNA damage is theDNA strand breaks, divided into single strand

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breaks (ssBs) and double-strand breaks (dsBs). SsBsoccur during different processes including directscission of the sugar-phosphate backbone by che-mical or radical attack, hydrolysis of alkali-labilesite, enzymatic incision during base and nucleotideexcision repair, post replicational or recombina-tional repair, enzymatic scissions, due to the actionof topoisomerase or lysosomic hydrolases.18 DNAstrand breaks which result following excisionrepair, 19,20 are capable of inducing structural da-mage to the chromosomes, which is analysed asstructural aberrations to the morphology of chromo-somes and also chromosome breaking effects in theform of micronuclei induction.21'22Most of the chemicals are not reactive themselves

and require metabolic activation by a variety ofenzymes responsible for their metabolism to exerttheir genotoxicity. CYP is one of the major group ofenzymes mainly involved in the activation of carci-nogens, especially polycyclic aromtic hydrocarbon(PAHs).23'24 Thus, we designed the present study toassess the activity of CYP in order to find out howmuch the carcinogen alters the level of theseenzymes and if the soy isoflavones exert effect onthe whole content of CYP. PAHs used in this studywas B[a]P, which is a well known genotoxicant andenvironmental pollutant that requires metabolicactivation to exert genotoxicity.25'26 It is widespreadin environment, food, ambient and indoor air. Someof its metabolites produced are highly reactive toDNA and may lead to covalent binding causing DNAadduct formation. It has been reported to causemutations, chromosome aberrations, sister chroma-tid exchanges and cancer in animals.27 Some of theother B[a]P reactive intermediates form alkali-labilesites on DNA.Soybeans and soyfoods potentially have multi-

faceted health-promoting effects. Keeping in viewthe potential health promoting effects of soybeansand soyfoods we have hypothesized that the soyisoflavones may inhibit genotoxicity and attenuateDNA integrity in animals.

Materials and methods

ChemicalsNovasoy® 40% soy isoflavone concentrate wasprocured from Archer Daniels Midland Co., Decatur,Illinois, USA. The product yields 33 mg of genisteinand 67 mg of daidzein in 100 mg product, bovineserum albumin (BSA), B[a]P, sodium dithionate,colchicine, bisbenzamide, EDTA, SDS, Trisma, Phe-nol, chloroform, isoamyl alcohol, RNase were pur-chased from Sigma St. Louis. Stains were purchasedfrom Hi-Media Labs, Mumbai, India and other

reagents and solvents were of a high analyticalgrade.

AnimalsEight-week-old adult male Swiss albino mice(20-25 g) were obtained from the Central AnimalHouse Facility of Hamdard University, New Delhiand were housed in a ventilated room at 25+2°Cunder a 12-h light/dark cycle. The animals wereacclimatized for one week before the study and hadfree access to standard laboratory feed (HindustanLever Ltd., Mumbai, India) and water ad libitum.

Treatment scheduleSoy isoflavones were suspended in normal salineand B[a]P in corn oil. B[a]P and soy isoflavoneswere administered orally. Each group consisted offive animals. In group I (vehicle control) animalswere given normal saline (0.9%) orally. Theanimals of group II served as positive controland were administered single oral dose of B[a]P(125 mg/kg b.wt). Animals of group III and V werepretreated with 40 mg/kg b.wt of soy isoflavoneswhile group IV were pretreated with 20 mg/kgb.wt of soy isoflavones for seven consecutive days.The above-mentioned doses of soy isoflavoneswere selected based on the preliminary studiesby the investigator. On day 8, the animals of groupII, IV and V were administered a single oral doseof B[a]P (125 mg/kg).

Assays

Chromosomal aberration test Mice were sacri-ficed by light ether anesthesia 30 hours after treat-ment with B[a]P. About 90 min prior to killing, asingle ip dose of colchicine (4 mg/kg) was adminis-tered to the animals. The time of sacrifice wasdecided based on preliminary experiments as opti-mal for scoring of aberrations. The slides of bonemarrow cells were prepared and stained accordingto the routine schedule for metaphase plate analysis,i.e., hypotonic-acetic acid-methanol-flame-drying-Giemsa.28 At least 100 well-spread intact meta-phases were scored per animal under 100 x oilimmersion using a light microscope (Olympus BX50). The type of chromosomal aberration (CA)included chromatid and chromosome breaks andchromosomal rearrangements. All aberrations wereconsidered as equal regardless of the number ofbreaks involved, gaps were not included.29 A singleobserver carried out blind scoring.

Micronucleus test For this test, mice were sacri-ficed 28 h after treatment with B[a]P. Bone marrow

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was collected from the femur bone of the mice formicronucleus assay while the liver tissue wasobtained for the estimation of CYP and DNA strandbreaks. Preliminary studies were conducted at dif-ferent time intervals and the time at which there wasmaximum induction of micronucleus formation wasselected for sacrificing the animals. The informationof kinetics of erythropoiesis and the recommenda-tions and observations made by Salamone andHeddle30 and Henderson et al.31 were also takeninto consideration. The mouse bone marrow micro-nucleus test was carried out according to the methodof Schimd.32 The air-dried slides were stained withMay-Grunwald and Giemsa as described bySchimd,32 made permanent and coded. A total of2500 polychromatic erythrocytes (PCEs) were scoredper animal by the same observer for determining thefrequencies of micronucleated polychromatic ery-throcytes (MnPCEs).

DNA isolation DNA was extracted from approxi-mately 500 mg of liver tissue by homogenizing thetissue in 5 mL TNE buffer (50 mM Trisma, 100 mMEDTA, 0.5% SDS, pH 8.0) in a 2 mL ground glasshomogenizer. Each sample was homogenized with10 standardized strokes of the pestle so as tominimize any potential effect on DNA integrityintroduced by the homogenization procedure. Anequal volume of buffered phenol/chloroform/isoa-myl alcohol (PCI) (25:24:1, v/v/v, pH 8.0) was thenadded to the sample. The sample was gently mixedand allowed to settle for 5 min. It was thencentrifuged for 5 min at 13000 rpm at 40C. Theaqueous layer was transferred to a new microcentrifuge tube and PCI extraction was repeated.The aqueous layer was then digested by 5 pL ofRNase (10 mg/mL) for 30 min at 370C and thedigestate was extracted once by PCI and once by500 iL of chloroform. DNA was precipitated fromthe resulting aqueous layer by adding 2 volumes ofabsolute ethanol and 1/10 volume of 3 M sodiumacetate, pH 5.2. The sample was then centrifuged(13 000 rpm, 15 min), and the resulting pellet rinsedwith 500 pL of 70% ethanol and air-dried. Theamount of DNA was quantitated spectrophotometri-cally at 260 and 280 nm.33 2 pg/pL of DNA samplewas dissolved in 1 mL of TE buffer (10 mM Trisma, 1mM EDTA) and subsequently used in the DNAalkaline unwinding assay.

Alkaline unwinding assay The procedure usedfor alkaline unwinding was essentially the same asthat outlined in Shugart20 3 with slight modifica-tions.35 In the alkaline unwinding assay, the rate oftransition of double stranded DNA (dsDNA) to

single stranded DNA (ssDNA) under pre-definedalkaline denaturing condition was proportional tothe number of breaks in the phosphodiester back-bone and thus was used as a measure of DNAintegrity. Bisbenzamide was used as a DNA-bindingdye from the fluorescence of which various types ofDNA were quantitated. For the fluorescence deter-mination of dsDNA, ssDNA and partially unwoundDNA (au-DNA), three equal portions of diluted DNAsample were prepared. The amount of dsDNA wasobtained from the fluorescence of a sample withoutany treatment, while ssDNA was determined fromthe sample that had been boiled for 30 min.Fluorescence of the DNA sample which had beensubjected to alkaline treatment (pH 12.2) on ice for30 min provided an estimate of the amount of auDNA.The fluorescence of initial or double stranded

DNA was determined by placing 100 pmol DNAsample, 100 ptL NaCl (25 mM) and 2 pL SDS (0.5%)in a prechilled test tube, followed by an addition of3 mL 0.2 M potassium phosphate pH 9, and 3 pLbisbenzamide (1 mg/mL). The contents were mixedand allowed to react in darkness for 15 min to allowfluorescence to stabilize. The fluorescence of thesample was the measured using a spectrofluorimeter(Ex: 360 nm, Em: 450 nm).36 The fluorescence ofsingle stranded DNA was determined as above butusing the DNA sample that had already been boiledfor 30 min to completely unwind the DNA.

50 [tL NaOH (0.05 N) was rapidly mixed with100 pL ofDNA sample in a prechilled test tube. Themixture was incubated on ice in darkness for 30 min(Rao et al.35) followed by rapid addition and mixingof 50 ptL HCI (0.05 N). This was followed immedi-ately by an addition of 2 ItL SDS (0.5%) and themixture was forcefully passed through a 21 G needlesix times. Fluorescence of alkaline unwoundedDNA sample was measured as described above.Measurement of the alkaline unwounded samplewas performed in triplicate and the average wasreported.The ratio between double stranded DNA to total

DNA (F value) was determined as follows:

F value = (auDNA - ssDNA)/(dsDNA - ssDNA).

where auDNA, ssDNA and dsDNA are the degreesof fluorescence from the partially unwound, singlestranded and double stranded determinations,respectively. The F value was inversely proportionalto the number of strand breaks present and thuscould be used as an indicator of DNA integrity.

Assay of cytochrome P450 content 10% (w/v)homogenate was prepared from the liver of mice

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and processed for the preparation of post mitochon-drial supernatant (PMS) and microsomes for CYPcontent.The method of Omura and Sato was followed.37 A

pinch of sodium dithionate was added to 2 mL ofsample. This was then divided equally between twomatched cuvettes. The contents of the test cuvettewere gently bubbled with carbon monoxide forabout one minute and then OD was taken simulta-neously at 450 and 490 nm.

Protein estimation Protein content in all sampleswas estimated by the method of Lowry et al.38 usingbovine serum albumin (BSA) as standard.

Statistical analysisThe level of significance between different groups isbased on Dunnett's t-test, followed by the analysis ofvariance test.

Results

B[a]P treatment alone produced gaps and DNAstrand breaks in the cells, However cells withmultiple chromosomal aberration and exchangeswere observed infrequently as they are not consid-ered as good indicator of chromosomal damage.29Table 1 shows the reduction in frequency of chro-mosomal aberrations induced in mouse bone mar-row cells following oral administration of the twodoses of soy isoflavones to the mice for 7 days beforeB[a]P treatment as compared with the group treatedwith clastogen, i.e., B[a]P alone. Significant reduc-tion in the frequency of chromosomal aberrationswas observed at 20 and 40 mg/kg b.wt doses of soyisoflavone treatment. Significantly higher incidenceof chromosomal aberrations was observed when wecompare control group with only B [a]P treatedgroup. In all cases, however, the aberration fre-Table 1 Effect of soy isoflavones on chromosomal aberrationsinduced by B[a]P in mice

Groups Chromosomal aberrations Mean + S.E.

quency was lower in the animals given the clastogenalone.The data presented in Table 2 shows the protec-

tive effect of soy isoflavone against B[a]P inducedmutagenicity as assessed by the bone marrowmicronucleus test. B[a]P produced significantmicronuclei formation when compared with thecontrol group (P <0.001). There was 37-57% in-hibition of the B[a]P-induced micronuclei by thepretreatment with soy isoflavones (P <0.001). B[a]Pproduces micronucleus formation and found signif-icant difference when compared to the control (P <0.001).

Figure 1 shows the mean F values in eachindividual treatment group. B[a]P caused reductionin DNA integrity as measured in terms of F valueand this F value showed significant differencewhen compared to the saline treated control group(P <0.001). Pretreatment with soy isoflavones(20 mg and 40 mg) causes recovery of DNA integritydose dependently and significantly (P <0.001).Figure 2 shows mean hepatic CYP content of each

individual group. Pretreatment with soy isoflavonessignificantly decrease the CYP content in a dosedependent manner as compared to the control group(P <0.001). In only B[a]P treated group CYP levelwas significantly elevated as compare to controlgroup (P < 0.001).

Discussion

Cancer and other chronic diseases may be relatedwith mutations produced by environmental agents;therefore, minimizing the exposure to harmfulagents has been recommended as a way to preventthese diseases. Unfortunately, it is not easy toeliminate the source of genotoxic agents completelyin modern society. Therefore, the identification andapplication of well-known antimutagens is essentialfor improving human health.39 Considerable empha-sis had been laid down on the use of dietaryconstituents to prevent mutagen induced cytogenicTable 2 Effect of soy isoflavones on micronuclei induced byB[a]P in mice

B' B" R Total'

1. Control2. Only B[a]P3. Only SIF40 mg/kg4. SIF20 mg/kg B[alP5. SIF40 mg/kg B[a]P

7508

2111

24133

I2152

1056102916

1.8 + 0.5911.09 + 1.67*

1.7 ± 0.565.6 ± 1.08#3.4 ± 0.60#

B': chromatid breaks; B": chromosomal breaks; R: rearrangements.a Total 500 metaphase plates were observed per group (n = 5animals) for scoring chromosomal aberrations; soy isoflavonealone did not induce chromosomal aberrations.* Significantly different from the control group (P < 0.001)." Significantly different from the only B[a]P treated group (P <0.05).

Groups

1. Control2. Only B[a]P3. Only SIF40 mg/kg4. SIF20 mg/kg B[a]P5. SIF40 mg/kg B[a]P

Mn PCEs0 PCEs/NCEs +PCEs

4.76 + 0.7628.67 + 2.8*4.23 + 0.5618.1 + 2.23#

12.23 + 1.9##

0.380.370.390.340.32

PCEs, polychromatic erythrocytes; NCEs, normochromatic ery-throcytes; MnPCEs, micronucleated polychromatic erythrocytes.a Mean+ S.E. of 2500 PCEs.* Significantly different from the control group (P <0.001).4 Significantly different from the B[a]P treated group (P <0.05).44 Significantly different from the B[a]P treated group (P < 0.001).

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0.9 -

0.8

0.7-

~,0.6-

0 54

0.1

Control Only Only D2 Dl +T D2+TToxicant

Groups

Figure 1 Effect of treatment of soy isoflavones on DNA integrity.* Significantly different from the control group (P <0.001).# Significantly different from the B[a]P treated group (P <0.001).

damage and DNA damage due to their nontoxiceffects. Many plants and their isolated compoundshave been tested to determine their antimutagenicpotential; like green and black teas are known to bestrong mutagenic and carcinogenic inhibitors.4043Catechins, theaflavins and thearubigins have beendetected as some of the main active components insuch inhibitors.4041 Studies on these agents havealso suggested that the whole mixture may bemore effective than its specific constituents.42'43This observation is congruent with the use of thesoy isoflavones in our study. In fact, favorable effectshave been suggested even with the consumption of a

mixture of isoflavones.10'44Our results show that soy isoflavones have the

efficacy to act as potent inhibitors of genotoxicity,the protection rendered by soy isoflavone may beattributed due to all three constituents genistein,daidzein and glycetin.As we know polycyclic aromatic hydrocarbons

(PAHs) in the environment are known or suspectprocarcinogens that require metabolic activation toexert their genotoxicity.21 CYP is one of the majorenzymes mainly involved in the activation of carci-nogens. CYP system is mainly localized in the liverin more abundant amounts than in any other organs

such as lung, kidney or intestine.45 B[a]P forms7-

.' 6

° 5-

4-

0

E 2 -

1-

Control OnlyToxicant

Only D2 D1+T D2+T

Groups

Figure 2 Effect of treatment of soy isoflavones on hepatic CytP450 content. * Significantly different from the control group

(P <0.001). # Significantly different from the B[a]P treated group

(P <0.001).

Genotoxicity of benzo(a)pyrene in miceTH Khan et al.

153

B[a]P-7,8-hydrodiol (7,8-diol), an immediate precur-sor to the highly-reactive ultimate carcinogen ofB[a]P, B [a]P-7,8-hydrodiol-9,10-epoxide (BPDE)which is an electrophile and binds with DNAmolecule.There are reports that nutrients and food additives

can modify CYP activities and consequently influ-ence toxicity and carcinogenicity of environmentalcarcinogens.46 This inhibition of hepatic CYP con-tent after repeated pretreatment with soy isoflavonesis a good indication of protection against genotoxi-city induced by B[a]P.The single strand DNA breaks assay detects sites

on the DNA where one of the strands have beennicked: (the more the nicks, more rapidly the DNAunbinds under alkaline conditions, and lower the Fvalue). Our results show that soy isoflavones arethe potent inhibitor of single strand DNA breaks,this protection of soy isoflavone may be attributeddue to all three constituents genistein, daidzein andglycetin.The chromosomal aberrations are produced by

error in DNA molecule. Chromosomal aberrationsare analysed in mitotic metaphases from prolifera-tive tissue such as bone marrow tissue. In micro-nuclei induction test, the clastogenic effects weremeasured indirectly by counting small nuclei ininterphase cells formed by acentric chromosomalfragment or whole chromosomes. Several studies ofDNA aberration have given a new insight to lookupon the antimutagenic properties of flavonoids,isoflavonoids that form the major part of diet. In ourstudy, soy isoflavones have been proven to reducethe micronuclei induction and chromosomal aberra-tion dose dependently. This mechanism can becompared as the protective action of cystein. Themechanisms by which the dietary agents inhibitgenetic damage induced by clastogens involve mod-ulation of intracellular and extracellular metabolicpathway.40 Also, the dietary constituents have theproperty of inhibiting free radical intermediatesgenerated by various pathways.47 Several vitamins,garlic eugenol, indole-3-carbinol and plant phenolshave been reported as antimutagenic agent in vivo.47The anticlastogenic effects of soy isoflavones can

be attributed to the combination mixture of allisoflavones. We conclude that soy isoflavones canbe used as a major chemopreventive agent againstB[a]P induced mutagenicity.

Acknowledgements

The authors would like to thank the Council ofScientific and Industrial Research (CSIR), NewDelhi, India, for providing the funds to carry outthis study.

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