Experimental and Molecular Pathology 96 (2014) 15–26

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Experimental and Molecular Pathology

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Modulatory effect of troxerutin on biotransforming enzymes andpreneoplasic lesions induced by 1,2-dimethylhydrazine in ratcolon carcinogenesis

Rajamanickam Vinothkumar a, Rajenderan Vinoth Kumar a, Mani Sudha a, Periyaswamy Viswanathan b,Thangavel Balasubramanian c, Namasivayam Nalini a,⁎a Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar 608 002, Tamil Nadu, Indiab Department of Pathology, Rajah Muthiah Medical College, Annamalai University, Annamalainagar 608 002, Tamil Nadu, Indiac Faculty of Marine Sciences, Centre for Advanced Study in Marine Biology, Annamalai University, Parangipettai 608 502, Tamil Nadu, India

⁎ Corresponding author. Fax: +91 4144 238343.E-mail address: nalininam@yahoo.com (N. Nalini).

0014-4800/$ – see front matter © 2013 Elsevier Inc. All rihttp://dx.doi.org/10.1016/j.yexmp.2013.10.009

a b s t r a c t

a r t i c l e i n f o

Article history:Received 11 March 2013and in revised form 10 October 2013Available online 25 October 2013

Keywords:Colon cancer1, 2-dimethylhydrazineTroxerutinXenobiotic enzymesBacterial enzymesAberrant crypt foci

Colon cancer is the thirdmost global oncologic problem faced bymedical fraternity. Troxerutin, a flavonoid pres-ent in tea, coffee, cereal grains, and a variety of fruits and vegetables, exhibits various pharmacological and bio-logical activities. This study was carried out to investigate the effect of troxerutin on xenobiotic metabolizingenzymes, colonic bacterial enzymes and the development of aberrant crypt foci (ACF) during 1,2-dimethylhydra-zine (DMH) induced experimental rat colon carcinogenesis. Male albinoWistar rats were randomly divided intosix groups. Group 1 served as control. Group 2 received troxerutin (50mg/kg b.w., p.o. every day) for 16weeks.Groups 3–6 received subcutaneous injections of DMH (20mg/kg b.w.) once a week, for the first four weeks. Inaddition, groups 4–6 received different doses of troxerutin (12.5, 25, 50mg/kg b.w., p.o. every day respectively)along with DMH injections. Our results reveal that DMH treated rats exhibited elevated activities of phase I en-zymes such as cytochrome P450, cytochrome b5, cytochrome P4502E1, NADPH-cytochrome P450 reductaseand NADH-cytochrome b5 reductase and reduced activities of phase II enzymes such as glutathione-S-transferase (GST), DT-diaphorase (DTD) and uridine diphospho glucuronyl transferase (UDPGT) in the liverand colonicmucosa of control and experimental rats. Furthermore, the activities of fecal and colonicmucosal bac-terial enzymes, such as β-glucronidase, β-glucosidase, β-galactosidase and mucinase were found to be signifi-cantly higher in DMH alone treated rats than those of the control rats. On supplementation with troxerutin toDMH treated rats, the alterations in the activities of the biotransforming enzymes, bacterial enzymes and thepathological changeswere significantly reversed, the effect beingmore pronouncedwhen troxerutinwas supple-mented at the dose of 25 mg/kg b.w. Thus troxerutin could be considered as a good chemopreventive agentagainst the formation of preneoplastic lesions in a rat model of colon carcinogenesis.

© 2013 Elsevier Inc. All rights reserved.

Introduction

Colorectal cancer is amajor cause ofmorbidity andmortality aroundthe world (Gellad and Provenzale, 2010). As of 2008, it is ranked as thethirdmost commonly diagnosed cancer and is the third leading cause ofcancer death in both men and women in the Western world (Siegelet al., 2011). The incidence rate of colorectal cancer is expected to in-crease substantially in economically transitioning countries includ-ing most parts of Asia where the overall risk was formerly low. Thisincrease may reflect the adoption of Western lifestyle and behavior(Center et al., 2009). The incidence of new colon cancer patients inIndia is increasing gradually (Sinha et al., 2003) there are about 3.5million cases of cancer of which about 35,000 are found to sufferfrom colorectal cancer (Srikhande et al., 2007). Colon cancer arises

ghts reserved.

due to various environmental factors, including diet, genetic predisposi-tion and epigenetic alterations in the colonic epithelium (Giovannucci,2002). Accumulating evidence from epidemiological studies reveal aninterplay between diet and the prevalence of gastrointestinal tract tu-mors, especially colorectal cancer, which can be promoted by a dietrich in fat and meat (Aggarwal, 2008). Several studies from our labora-tory as well as others have shown that a high fat diet promotes tumor-igenesis in the chemically induced experimental model of coloncarcinogenesis (Aranganathan et al., 2009; Bansal et al., 1978; Reddyet al., 1977; Sangeetha et al., 2010). It may be related to the increasedconcentrations of secondary bile acids within the colon lumen, whichmay enhance cell proliferation in the colonic mucosa, and have beenfound to be carcinogenic in animal models (Baijal et al., 1998).

1, 2-dimethylhydrazine (DMH) induced rat colon carcinogenesis isone of the widely studied experimental models in several chemopre-vention studies. Repetitive treatment with this methylating agent wasreported to produce colon tumors in rodents that exhibit many of the

Fig. 1. A. Chemical structure of troxerutin. B. Diagrammatic representation of the experi-mental design.

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cell kinetics,molecular characteristics and pathological features associat-ed with the human colorectal cancer (Ma et al., 1996; Shamsuddin andPhillips, 1981). DMH is metabolized to form azoxymethane (AOM),which is further metabolized to methylazoxymethanol (MAM) by cyto-chrome P4502E1 in the liver (Sohn et al., 1991). MAM gets conjugatedin the liver by glucuronic acid and is released into the colon via bile asMAM glucuronide where it is deconjugated by the actions of gut micro-bial enzymes especially β-glucuronidase to yieldMAM (Rowland, 1988).Thus, from a chemical perspective, the intestinal flora tends to reversethe metabolites produced in the liver. The regenerated MAM is furthermetabolized to produce electrophilic methyldiazonium ion, whichinturn generates carbonium ion that is responsible for the methyla-tion of nucleic acids and acts as a trigger for colon carcinogenesis(Fiala, 1977; Rosenberg et al., 2009).

ACF has been widely used as an early biomarker for colon carcino-genesis and is considered to be a surrogate preneoplastic lesion sinceit is found in the colon of carcinogen treated rodents and humans.ACF are easily identified in formalin fixed methylene blue stainedwhole-mount colon preparations under a light microscope. Biochemi-cal, genetic and morphological studies have shown that ACF and colontumors share similar alterations, further criteria supporting the hypoth-esis that ACF are precursors of colorectal cancer (Bird, 1995; Bird andGood, 2000; Mori et al., 2005; Rudolph et al., 2005). Therefore, it is im-portant to identify chemopreventive compounds which can block thepotential biochemical factors responsible for ACF formation in DMH in-duced colon carcinogenesis.

Drug development from natural products is currently emergingas a highly promising strategy to identify novel anticancer agents.During the past decade, a large number of phytochemicals from thediet and medicinal plants have been evaluated for anticancer activitydue to their ability to interfere with multiple pathways controllinggrowth and apoptosis of cancer cells (Khan et al., 2008). Many epide-miological studies have reported that high consumption of wholegrains, fruits and vegetables is associatedwith a low risk of colorectalcancer. In addition, much of the attention given to flavonoid com-pounds comes from the results of epidemiological studies that sug-gest high fruit and vegetable consumption is associated with thedecreased risk of several types of cancer, including breast, colon,lung, larynx, pancreas, oral and prostate cancer (Ross and Kasum,2002).

Troxerutin (Fig. 1A), a trihydroxyethylated derivative of rutin,known as vitamin P4, is a flavonoid present in tea, coffee, cerealgrains and a variety of fruits and vegetables. Troxerutin is an effec-tive scavenger of reactive oxygen species (ROS) and may also functionindirectly as antioxidant through its effects on enzyme activities(Blasig et al., 1987, 1988; Fan et al., 2009). Troxerutin in recent timeshave gained great importance by virtue of its numerous pharmacologi-cal and biological properties such as antifibrinolytic (Boisseau et al.,1995), antiinflammatory (Fan et al., 2009), anti γ-radiation (Mauryaet al., 2005) and antidiabetic (Chung et al., 2005) effects. Chemopreven-tion aims to halt or reverse the development and progression ofcancerous cells through use of non-cytotoxic nutrients and/orpharmacological agents during the period between tumor initia-tion and malignancy. Modulation of enzymes involved in metabolicactivation and excretion of carcinogens is one of the best-investigatedmechanisms for chemopreventive agents. The objective of the presentstudy was to investigate the chemopreventive efficacy of troxerutinemploying DMH induced colon carcinogenesis in male albino Wistarrats as an experimental model.

Materials and methods

Chemicals

Troxerutin and 1,2-dimethylhydrazine (DMH), were purchasedfrom Sigma Chemicals Co., St. Louis, MO, USA. All other chemicals

and solvents utilized were of analytical grade and obtained fromHi-Media Laboratories Ltd., Mumbai, India.

Animals and diet

The experiment was carried out using male albino Wistar rats withbodyweight ranging from 130­150g (5weeks old). Thuswere procuredand maintained at the Central Animal House, Rajah Muthiah MedicalCollege & Hospital, Annamalai University, Tamil Nadu, India. The ani-mals were housed in solid-bottomed polypropylene cages with a wiremesh top and a hygienic bed of husk in a specific-pathogen-free animalroom under controlled conditions of a 12-h light/12-h dark cycle, with atemperature of 25±2°C and relative humidity of 50±10% till the end ofthe experimental period. Commercial pellet diet containing 4.2% fat(Hindustan Lever Ltd., Mumbai, India) was powdered and mixed with15.8% peanut oil, making a total of 20% fat in the diet (Table 1). Thismodified diet and water was fed ad libitum throughout the experimen-tal period of 16weeks. The animals were cared as per the principles and

Table 1Composition of the experimental diet.

Nutrient Commercial diet (84.2%) Peanut oil (15.8%) Total (100%)

Protein 17.7 – 17.7Fat 4.2 15.8 20.0Carbohydrate 50.5 – 50.5Fiber 3.4 – 3.4Minerals 6.7 – 6.7Vitamins 1.7 – 1.7

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guidelines of the Ethical Committee for Animal Care of Annamalai Uni-versity in accordance with the Indian National Law on animal care anduse (Reg.No.160/1999/CPCSEA:789).

Experimental design

Rats were randomly divided into six experimental groups of twelverats in each group. Group 1: rats received modified diet for 16 weeksand served as control, Group 2: rats received modified diet withtroxerutin (50mg/kg b.w.) everyday orally throughout the experimen-tal period of 16 weeks, Groups 3–6: rats received modified diet withsubcutaneous injections of DMH (20mg/kg b.w.) once a week, for thefirst four weeks of the experimental period. In addition, Groups 4–6: rats received different doses of troxerutin everyday (12.5, 25 and50mg/kg b.w., respectively) along with modified diet and DMH in-jections. The experimental design is clearly shown in Fig. 1B.

Preparation of troxerutin

Troxerutinwas solubilized in double distilledwater just before treat-ment and was administered everyday orally at the doses of 12.5, 25 or50mg/kg b.w for 16weeks.

Induction of colon cancer

For induction of colon cancer, DMH was dissolved in 1 mM EDTA,just prior to use and the pH was adjusted to 6.5 with 1mM NaOH andadministered subcutaneously in the right thigh at a dose of 20mg/kgb.w., once a week for the first 4weeks of the experiment.

Homogenate preparation

Preparation of cytosolic and microsomal fractions

At the end of the experimental period all the animals were sacrificedunder anesthesia (i.p. administration of ketamine hydrochloride30mg/kg b.w.) by cervical dislocation after an overnight fast. Cyto-solic and microsomal fractionation was carried out by the methodof Schladt et al. (1986). Briefly the liver and colon mucosal scrapingswere homogenized in 10 mM Tris–HCl buffer (pH 7.4) containing0.25 M sucrose, centrifuged at 9000 ×g for 20min and the superna-tant was collected. The collected supernatant was centrifuged at100,000 ×g for 20 min, and the clear cytosolic fractions obtainedwere promptly assayed for the activities of phase II enzymes. Thepellet after centrifugation at 100,000 ×g was resuspended in ice-cold 0.15M Tris–KCl buffer (pH 7.4) and recentrifuged for 60min at100,000×g. The microsomal pellet was resuspended in homogeniza-tion buffer equivalent to half of the original buffer and was used forthe assay of phase I enzymes.

Fecal and colonic mucosal tissue processing

Fresh fecal pellets were collected for the assay of bacterial en-zymes. The mucosa from the colon was collected by scraping with aslide. The fecal pellets and colonic mucosa were homogenizedusing phosphate-buffered saline (pH 7.2), centrifuged at 2000 ×g

for 10min at 4 °C, and the supernatant collected for the assay of theactivity of fecal and colonic mucosal bacterial enzymes.

Biochemical estimations

Assay of phase I enzymesCytochrome P450 (EC. 1.14.14.1) and cytochrome b5 content were

measured by the method of Omura and Sato (1964). Cytochrome P450was determined using carbon monoxide (CO) difference spectra. Theabsorbance of CO adducts formed by the reaction of reduced cyto-chrome P450 with CO were measured at 450 nm. The rapid reductionof cytochrome b5 was characterized by an increase in absorbance at427 nm. Cytochrome P450 and b5 were determined using the ab-sorption coefficient of 91 and 185 cm2 M−1 m−1 respectively. Thevalues are expressed as μmoles/mg protein.

Cytochrome P4502E1 (EC. 1.14.13.n7) activity was measured bythe method of Watt et al. (1997). The assay mixture contained100 μg microsomal protein, 40 mM p-nitrophenol and 0.1 M phos-phate buffer. The reaction was initiated by the addition of 10mM ofNADPH and incubated at 37 °C for 60min. The reaction was stoppedwith 20% TCA and centrifuged at 1000 rpm for 5 min, 10 mM NaOHwas added to the supernatant and the absorbance was measured at450nm. The values are expressed as mmoles of p-nitrocatechol liber-ated/min/mg protein.

NADPH-cytochrome P450 reductase (EC. 1.6.2.4) activity was mea-sured by Omura and Takesue (1970), bymeasuring the rate of oxidationof NADPH at 340 nm. The reaction mixture contained 0.3M potassiumphosphate buffer (pH7.5), 0.1mMNADPH, 0.2mM potassium ferricya-nide and the microsomal preparation in a final volume of 1mL. Thereaction was started at 25 °C with the addition of NADPH. The en-zyme activity was calculated using the extinction coefficient of6.33mM−1cm−1. One unit of enzyme activity is defined as that caus-ing the oxidation of one mole of NADPH oxidized/min/mg protein.

NADH-cytochrome b5 reductase (EC. 1.6.2.2) was measured by themethod of Mihara and Sato (1972). The reaction mixture contained0.1M potassium phosphate buffer (pH7.5), 0.1mMNADH, 1mMpotas-sium ferricyanide andmicrosomal preparation in a final volume of 1mL.The reaction was started at 25 °C by the addition of NADH, and the rateof reduction of potassium ferricyanide by NADH was measured at420nm. The enzyme activity was calculated using the extinction coeffi-cient of 1.02mM−1cm−1. One unit of enzyme activity is defined as thatcausing the reduction of one mole of ferricyanide reduced/min/mgprotein.

Assay of phase II enzymes

Glutathione-S-transferase (EC. 2.5.1.18) was measured by the meth-od of Habig et al. (1974). The reaction mixture contained 100mM phos-phate buffer (pH6.5), 30mM 1-choloro 2,4-dinitrobenzoic acid (CDNB)and30mMreduced glutathione. The reactionwas started by the additionof cytosolic sample and the absorbancemeasured at 340nm. The specificactivity of GST is expressed as μmoles of 1-chloro-2,4-dinitrobenzene(CDNB)-GSH conjugate formed/min/mg protein using the extinction co-efficient 9.6mM−1 cm−1.

DT-diaphorase (EC. 1.6.99.2) activity was measured by the methodof Ernster et al. (1962)with NADHas the electron donor and 2,6-dichlo-rophenol indophenol (DCPIP) as the electron acceptor at 600 nm. Thereactionmixture contained 25mMTris–HCl (pH7.4), fewmg of crystal-line bovine mixture, 0.01% Tween 20, 0.1 mM NADPH, 10 μM cyto-chrome c, dicumarol and an appropriate amount of cytosolic sample ina final volume of 3mL. The electron acceptor, 40 μM DCPIP, was addedto initiate the reaction. The reduction of DCPIP was measured at600 nm and the activity of DTD is expressed as μmoles of DCPIP re-duced/min/mg protein using the extinction coefficient 21mM−1 cm−1.

UDP-glucuronyl transferase (EC. 2.4.1.17) was measured by themethod of Isselbacher et al. (1962). The incubation mixture containing

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0.5 mL buffer, 0.2 mL TritonX-100, 50 μL MgCl2, 50 μL p-nitrophenol,0.18 mL water and 0.1 mL enzyme were incubated at 37 °C for 2 min.Then 0.1 mL of UDP-glucuronic acid was added. The reaction wasarrested at 0, 10 and 15minwith TCA and centrifuged. To 1mLof the su-pernatant 0.25mL of NaOHwas added and measured at 450nm using aspectrophotometer. The activity of UDP-glucuronyl transferase isexpressed as nmoles/min/mg protein.

Measurement of bacterial enzymes

β-glucuronidase (EC. 3.2.1.31) activity wasmeasured by themethodof Freeman (1986). β-glucuronidase assay mixture contained knownvolume of 0.02 M phosphate buffered saline (pH 7.0), 0.1 mM EDTA,0.01M p-nitrophenyl-β-D-glucuronide and the enzyme supernatant ina final volume of 1 mL, and the mixture was incubated at 37 °C for15min in a shaking water bath. The reaction was arrested with 0.2Mglycine buffer (pH 10.4) and the amount of p-nitrophenol releasedwas measured at 540nm. All reactions were linear with respect to con-centration and incubation time to 45min. The amount of p-nitrophenolliberated was determined by comparison with a standard nitrophenolcurve. Values are expressed asmgof p-nitrophenol liberated/min/g pro-tein for fecal samples and μg of p-nitrophenol liberated/h/g protein formucosal tissue.

β-glucosidase (EC. 3.2.1.21) activity wasmeasured by themethod ofFreeman (1986). β-glucosidase assay mixture contained 2 mM p-nitrophenyl-β-D-glucopyranoside, 50 mM potassium phosphatebuffer (pH7.2), a suitable amount of the fecal or mucosal suspensionin a final volume of 1mL, and the mixture was incubated at 37 °C for60min in a shaking water bath. The reaction was arrested with 0.2MNa2CO3 and the amount of p-nitrophenol liberated was determined(450 nm) by comparison with a standard nitrophenol curve. Valuesare expressed as mg of p-nitrophenol liberated/min/g protein forfecal samples and μg of p-nitrophenol liberated/h/g protein for mu-cosal tissue.

β-galactosidase (EC. 3.2.1.23) activity was measured by the methodof Freeman (1986). The mixture contained 3 mM p-nitrophenyl-β-D-galactopyranoside, a known volume of sample and incubated at 37 °Cfor 15 min. Then the reaction was terminated by the addition of0.25M Na2CO3. Release of p-nitrophenol was measured spectrophoto-metrically at 405nm. The amount of p-nitrophenol liberated was deter-mined by comparison with a standard nitrophenol curve. Values areexpressed asmg of p-nitrophenol liberated/min/g protein for fecal sam-ples and μg of p-nitrophenol liberated/h/g protein for mucosal tissue.

Mucinase (EC. 4.2.2.1) activitywasmeasured by themethodof Shiauand Chang (1983). The assay mixture contained 0.2M porcine gastricmucin with a known amount of fecal or mucosal suspension. The reac-tionmixture was incubated at 37°C for 25min. The amount of reducingsugar wasmeasured by themethod of Nelson (1944) at 620nm. Valuesare expressed as mg of glucose liberated/min/mg protein.

Estimation of protein

The protein content was determined by the method of Lowry et al.(1951) using bovine serum albumin (BSA) as standard, at 660nm.

Determination of aberrant crypt foci (ACF)

Topographical analysis of the colonic ACF was performed accord-ing to method of Bird (1987). After laparotomy, colons were excised,flushed with saline and opened longitudinally from anus to caecum.Each colon was cut into three segments (proximal, middle, and dis-tal) of equal length and fixed flat between filter papers in 10% buff-ered formalin for at least 24 h. Formalin fixed colons were stainedwith 0.2% methylene blue for 2 min, washed with distilled waterfor 5 sec to remove excess stain, placed on microscopic slides withthe mucosal side facing up and viewed under a light microscope

(Carl Zeiss, Germany) at low magnification and the morphologicalchanges within colon were photographed with a digital camera.ACF were identified by their increased size, intense methylene bluestaining, irregular and dilated luminal opening, thicken epitheliallining and increased pericryptal zone. The total number of ACF percolon, the total number of aberrant crypts (ACs) observed in eachfocus, and the location of each focus was recorded.

Combined Alcain blue-periodic acid schiff (AB-PAS) analysis of colonicmucin

Histochemical analysis of mucin was carried out by combined AB-PAStechnique (Mowry, 1958). Briefly, 5 μm thickness paraffin-embeddedcolon sections were heated for 60min at 60 °C, deparaffinized in xylene,and rehydrated through graded alcohols at room temperature. Thesections were stained with Alcian blue for 25min and washed withdistilled water followed by staining with Schiff's reagent for 15 min,washed in running tap water and counterstained with haematoxylin.The over stained sections are washed in running tap water, dehydratedin graded alcohol andmounted using DPX. To determine whether DMHcaused morphological changes in the mucus cell number and integrity,Alcian blue-positive cells were quantified for each category of controland experimental groups of rats (Carl Zeiss, Germany).

Histopathological study

Histological analysis was performed to observe the pathologicalalterations if any. A portion of the liver and colonic tissues from dif-ferent groups were fixed in 10% neutral buffered formalin for a week,dehydrated by passing successively in different concentrations ofethanol, cleaned in xylene, embedded in paraffin, sectioned andprocessed by routine histological methods with haematoxylin andeosin (H and E) staining.

Statistical analysis

The data are presented as means±S.D. The results were analyzedby one way analysis of variance (ANOVA) and any significant differ-ence among treatment groups was evaluated by Duncan's MultipleRange Test (DMRT). The data were considered statistically signifi-cant at p b 0.05. All statistical analyses were performed using SPSSversion 11.0 software package (SPSS, Tokyo, Japan).

Results

Tolerability of treatment

Troxerutin was well tolerated by the rats throughout the experi-mental period of 16 weeks as evidenced by the absence of clinical/pathological signs in group 2 animals.

Effect of DMH and troxerutin on phase I xenobiotic metabolizing enzymes(microsomes)

Figs. 2 and 3 shows the influence of treatment with troxerutin on theactivities of phase I xenobioticmetabolizing enzymes such as cytochromeP450, cytochrome b5, cytochrome P4502E1, NADPH-cytochrome P450reductase, and NADH-cytochrome b5 reductase in the liver and colonicmucosa of control and experimental rats. The activities of phase I en-zymeswere significantly (pb0.05) elevated in the liver and colonicmu-cosa of DMH treated rats (group 3) relative to the control at the end of16weeks. Supplementation with different doses of troxerutin (groups4–6) significantly (p b 0.05) diminished the activities of all the abovephase I xenobiotic metabolizing enzymes, a more pronounced effectbeing observed in the rats supplemented with troxerutin 25 mg/kgb.w., (group 5). However, the troxerutin alone supplemented rats

Fig. 2. Effect of troxerutin on phase I enzymes in the liver of control and experimental rats. Values are given as means±S.D. for six rats in each group. Values not sharing a common su-perscript symbol (a,b,c) differ significantly at pb0.05 (DMRT). A— μmoles/mg protein; B—mmoles of p-nitrocatechol liberated/min/mg protein; C— onemole of NADPH oxidized/min/mgprotein; and D — one mole of ferricyanide reduced/min/mg protein.

19R. Vinothkumar et al. / Experimental and Molecular Pathology 96 (2014) 15–26

(group 2) did not show any significant change in the activities of phase Ienzymes as compared to the control rats (group 1).

Effect of DMH and troxerutin on phase II xenobiotic metabolizing enzymes(cytosol)

Fig. 4 illustrates the effect of troxerutin on the activities of phase II xe-nobiotic metabolizing enzymes viz., GST, DTD and UDPGT in the liver andthe colonic mucosa of control and experimental rats. The activities ofphase II enzymes were significantly (p b 0.05) decreased in the liver

Fig. 3. Effect of troxerutin on phase I enzymes in the colonic mucosa of control and experimencommon superscript symbol (a,b,c,d,e) differ significantly at p b 0.05 (DMRT). A — μmoles/mNADPH oxidized/min/mg protein; and D — one mole of ferricyanide reduced/min/mg protein.

and colonic mucosa of DMH treated rats (group 3) as compared to thecontrol rats at the end of 16 weeks. Supplementation with differentdoses of troxerutin (groups 4–6) significantly (pb0.05) elevated the ac-tivities of all the above phase II enzymes. However, troxerutin supple-mentation at the dose of 25 mg/kg b.w., (group 5) showed greatermodulatory effects on phase II xenobiotic metabolizing enzymes ascompared to the other treatment groups (group 4 and 6). Moreoversupplementation with troxerutin alone (group 2) did not induce anysignificant change in phase II enzymes as compared to the control rats(group 1).

tal rats. Values are given as means± S.D. for six rats in each group. Values not sharing ag protein; B — mmoles of p-nitrocatechol liberated/min/mg protein; C — one mole of

Fig. 4. Effect of troxerutin on phase II enzymes in the liver and colonic mucosa of control and experimental rats. Values are given as means± S.D. for six rats in each group. Values notsharing a common superscript symbol (a,b,c,d) differ significantly at p b 0.05 (DMRT). A — μmoles of 1-chloro-2,4-dinitrobenzene (CDNB)-GSH conjugate formed/min/mg protein; B —

μmoles of 2,6-dichlorophenolindophenol reduced/min/mg protein; and C — nmoles/min/mg protein.

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Effect of DMH and troxerutin on fecal and colonic mucosal bacterialenzymes

Tables 2 and 3 shows the activity of the bacterial enzymes such asβ-glucuronidase, β-glucosidase and mucinase obtained from freshfeces and colonic mucosa of control and experimental rats. The spe-cific activities of these bacterial enzymes was significantly(p b 0.05)increased in the DMH treated rats (group 3) as compared to the con-trol and the rats treated with troxerutin alone (group 1 and 2). How-ever, supplementation with troxerutin at different doses (12.5, 25and 50 mg/kg b.w.) along with DMH significantly (p b 0.05) de-creased the activities of these bacterial enzymes, the effect beingmore pronounced in rats supplemented with 25 mg/kg b.w., oftroxerutin (group 5).

Effect of DMH and troxerutin on ACF and crypt multiplicity

ACF analysis was carried out at the end of 16weeks in each experi-mental group. The effect of troxerutin on theACF occurrence, cryptmul-tiplicity and distribution of ACF are shown in Fig. 5 and Tables 4 and 5,respectively. The incidence of ACF as well as the crypt multiplicity was100% in DMH alone treated rats (group 3) whereas control andtroxerutin alone supplementation did not show ACF formation in thecolon of rats (group 1 and 2). Troxerutin supplementation (groups 4–6) for 16 weeks significantly reduced ACF formation and crypt

Table 2Effect of troxerutin and DMH on the fecal bacterial enzymes of control and experimental rats.

Groups Control Control+troxerutin(50mg/kg b.w.)

DMH

β-Glucuronidase A 19.23±1.46a 18.29±1.05a 24.56± 1β-Glucosidase A 73.28±5.43a 76.86±5.65ab 128.07± 9β-Galactosidase A 26.34±1.04a 29.79±2.50b 51.49± 3Mucinase B 2.59±0.23a 2.47±0.23a 5.10± 0

Data are presented as the means± S.D. of six rats in each group. Values not sharing a commonA mg of p-nitrophenol liberated/min/g protein.B mg of glucose liberated/min/mg protein.

multiplicity in a dose dependent manner as compared to the DMHalone treated rats (group 3). Most of the ACF with 3 or more cryptswas observed in middle and distal colon than in proximal colon.77.64% inhibition of ACF incidence was observed in rats supplementedwith troxerutin at the dose of 25mg/kg b.w. (group 5), whereas the in-hibition rate was significantly lower, that is 33.44% and 74.68% respec-tively, in groups 4 and 6.

Effect of DMH and troxerutin on mucin presenting cells

Fig. 6 shows the AB-PAS stained glycoprotein (mucin) in the mucussecreting cells of the control and experimental rats. In the control andtroxerutin alone supplemented rats the Alcian blue staining was strongand intense (group 1 and 2). On the other hand, the mucosa of DMHalone treated rats showed faint and weak Alcian blue staining and pro-gressive loss ofmucin. Themucosa of DMH treated ratswhichwere sup-plemented with troxerutin at different doses (groups 4–6) showed anincreased staining, as well as an obvious increase in the mucus cellsize and number as compared to those found in the mucosa of DMHalone treated rats (group 3).

Effect of DMH and troxerutin on liver and colon pathology

Figs. 7 and 8 represents the histopathological changes in the liverand colon of control and experimental rats. DMH alone treated rat

DMH+troxerutin(12.5mg/kg b.w.)

DMH+troxerutin(25mg/kg b.w.)

DMH+troxerutin(50mg/kg b.w.)

.84b 22.19±1.52c 17.69±1.32a 18.34±1.40a

.44c 98.64±3.86d 78.50±3.84ab 81.37±5.87b

.75c 37.68±1.92d 27.84±1.31ab 30.36±1.19b

.30b 4.07±0.17c 2.61±0.12a 2.73±0.19a

superscript letter (a–d) differ significantly at p b 0.05 (DMRT).

Table 3Effect of troxerutin and DMH on the colonic mucosal bacterial enzymes of control and experimental rats.

Groups Control Control+troxerutin(50mg/kg b.w.)

DMH DMH+troxerutin(12.5mg/kg b.w.)

DMH+troxerutin(25mg/kg b.w.)

DMH+troxerutin(50mg/kg b.w.)

β-Glucuronidase A 5.91±0.28ab 6.27±0.33b 11.73±0.67c 8.59± 0.40d 5.66±0.31a 5.75±0.32a

β-Glucosidase A 22.89±1.19a 23.46±2.32a 56.44±3.86b 38.27± 2.91c 24.84±1.26a 26.09±2.51a

β-Galactosidase A 19.39±1.57a 21.82±1.53ab 47.39±3.14c 41.62± 4.18d 22.59±2.02ab 24.07±2.02b

Mucinase B 04.86±0.32a 04.72±0.34a 08.41±0.53b 06.92± 0.39c 04.69±0.11a 04.97±0.27a

Data are presented as the means± S.D. of six rats in each group. Values not sharing a common superscript letter (a–d) differ significantly at p b 0.05 (DMRT).A μg of p-nitrophenol liberated/h/g protein.B mg of glucose liberated/min/mg protein.

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liver (group 3) showedmicro andmacro vesicular type of fatty changesinvolving all the zones of the liver, Kupffer cell hyperplasia and varyingdegrees of anaplasia (Fig. 7C). The liver section of DMH+ troxerutin(12.5 mg/kg b.w., i.e. group 4) rat showed micro and macro vesiculartype of fatty changes and Kupffer cell hyperplasia. The liver ofDMH+troxerutin supplemented rats (25 and 50mg/kg b.w., i.e groups5 and 6) showed near normal liver morphology (7E and F).

DMHalone treated rat colon (group 3) showed prominent lymphoidaggregates (circle mark), dispersed inflammatory cell infiltration inthemucosal and submucosal layers (arrowmark). In addition 6 aber-rant crypt foci contain 8–13 crypts with intense staining and in-creased pericryptal zone (dotted arrow mark) were noticed (8C).

Fig. 5. Topographical view of normal crypts and aberrant crypt foci (ACF). (A and B) Colons of(C) Topographical view of DMH alone treated rats colon showsmore than ten aberrant crypt foshows four crypts. (E and F) Colons of troxerutin (25 and 50mg/kg b.w., i.e groups 5 and 6) su

The colon section of DMH+ troxerutin (12.5 mg/kg b.w., i.e. group4) supplemented rat showed mild inflammatory cell infiltration inthe mucosal layer and occasionally lymphoid aggregates. The colonsof DMH+ troxerutin supplemented rats (25 and 50 mg/kg b.w., i.egroups 5 and 6) showed near normal appearing glands (8E and F).

Discussion

Nowadays chemoprevention is regarded as a promising avenuefor decreasing cancer burden. Dietary intervention is a convenientway for disease prevention or therapy. Due to their high tolerabilityand low toxicity phytochemicals are receiving more attention in

control and control+ troxerutin (50mg/kg b.w.) supplemented rats show normal crypts.ci. (D) Colon of troxerutin (12.5mg/kg b.w., i.e groups 4) supplemented DMH-treated ratpplemented DMH-treated rats show two crypts.

Table 4The effect of troxerutin and DMH on the number of ACF and crypt multiplicity in the colon of experimental rats.

Groups ACF Incidence Number of ACF Number of AC No. of foci containing

1 crypt 2 crypt N3 crypt

Control 6/0 Nil Nil Nil Nil NilControl+ troxerutin (50mg/kg b.w.) 6/0 Nil Nil Nil Nil NilDMH 6/6 61.83± 3.54a 141.00±4.47a 24.50±1.22a 20.83± 1.60a 16.50± 1.76a

DMH+ troxerutin (12.5mg/kg b.w.) 6/6 41.16± 2.13b 90.16± 2.31b 16.50±1.37b 14.33± 0.51b 10.00± 0.89b

DMH+ troxerutin (25mg/kg b.w.) 6/6 13.83± 0.40c 24.00± 1.26c 3.66±0.81c 10.16± 0.98c –

DMH+ troxerutin (50mg/kg b.w.) 6/6 15.66± 0.81c 26.66± 1.36c 4.66±0.51c 11.00± 0.63c –

Data are presented as the means± S.D. of six rats in each group. Values not sharing a common superscript letter (a–c) differ significantly at p b 0.05 (DMRT).

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chemoprevention studies (Gescher, 2004). Troxerutin is a flavonoid,which has undergone numerous clinical trials in human subjects;even with high doses, troxerutin had excellent safety and tolerabilityprofile (Maurya et al., 2005). In the present study, we have observedthat troxerutin supplementation modulates DMH induced deleteri-ous effects, by modifying the activities of biotransforming enzymes,bacterial enzymes and inhibiting the formation of preneoplastic le-sions, thereby minimizing the frequency of carcinogenicity in therat colon.

Cytochrome P450 is a family of phase I hemoproteins, representingmajor enzymes involved in the activation of various procarcinogensto carcinogens (Ding and Kaminsky, 2003). Accordingly cytochromeP450 catalyzes the oxidation of lipophilic chemicals via C- or N-hydroxylation to electrophilic carcinogens. The catalytic efficiencyof cytochrome P450 is enhanced by the ubiquitous electron trans-port protein cytochrome b5. Being a procarcinogen, DMH undergoesmetabolic activation by cytochrome P4502E1 to generate electrophilicmetabolites forming DNA adducts, inducing mutations that can initiateoncogenic transformation (Modugno et al., 2003;Williams and Phillips,2000). Therefore, the expression of carcinogen activating enzymes is akey component in chemically induced carcinogenesis, responsible fordriving normal cells to a malignant phenotype with very high prolif-erative potential (Ali et al., 2004; Begleiter and Fourie, 2004). In ourpresent study, we have observed increased activities of the cyto-chrome P450 family enzymes in the liver and colonic mucosa ofDMH-treated rats which could be due to the presence of DMHeliciting substrate induced activation of these enzymes. Supplemen-tation with troxerutin to DMH treated rats inhibited the activities ofthese phase I enzymes. This strategic inhibition of cytochrome P450enzymes could decrease the bioavailability of highly metabolizeddrugs. Thus, the inhibition of phase I enzymes are hypothetically im-portant in xenobiotic toxicity, because it could reduce the toxicity ofxenobiotics. Therefore the protective effect of troxerutin againstDMH induced colon carcinogenesis is driven by declining the activi-ties of the phase I enzymes.

Many of the toxic chemicals that enter the body are fat-soluble,difficult to excrete, and have a high affinity for fat tissues and cellmembranes. GST, the major phase II detoxification enzyme, favorselimination of polar end products of cytochrome P450 mediatedphase I reactions through conjugation with reduced glutathione toform nontoxic peptide derivatives (Coles and Kadlubar, 2003). DTDis a flavoprotein generally induced in coordination with GST, which

Table 5Distribution of ACF in the colon of experimental rats.

Groups Number of ACF Proximal colon M

DMH 61.83±3.54a 12.66±1.63a 1DMH+ troxerutin (12.5mg/kg b.w.) 41.16±2.13b 8.66±0.81b 1DMH+ troxerutin (25mg/kg b.w.) 13.83±0.40c 2.16±0.40c

DMH+ troxerutin (50mg/kg b.w.) 15.66±0.81c 2.83±0.40c

Data are presented as the means± S.D. of six rats in each group. Values not sharing a common

facilitates the metabolism of quinones, thereby protecting againstthe toxicity of quinones and their metabolic end precursors such aspolycyclic aromatic hydrocarbons (Karczewski et al., 1999). UDPGTis also a phase II detoxifying enzyme, which helps in the conjugationof glucuronic acid with numerous endobiotic and xenobiotic sub-strates, thereby increasing their water solubility and facilitatingrenal and biliary excretion. Importantly, glucuronidation facilitatedby UDPGT is increasingly recognized as a major phase II detoxifica-tion pathway in humans (Fisher et al., 2001). There is increasing ev-idence that human cancers can be prevented not only by avoidingexposure to carcinogens but also by favoring the intake of protectivefactors that potentiate the defensemechanisms of the host organism.The present data together with reports available from our laboratoryand others, suggest that flavonoids have immense chemopreventivepotential (Aranganathan et al., 2009; Siess et al., 1992; Yannai et al.,1998). In the present study, troxerutin supplementation to DMHtreated rats showed enhanced activities of phase II enzymes suchas GST, DTD and UDPGT. Dual acting agents are generally recog-nized to be more promising as chemopreventive agents becausethey inhibit metabolic activation of carcinogens while promotingdetoxification and excretion (Talalay, 1992). From the presentstudy, we observed that troxerutin acts as a dual acting agent byinhibiting phase I enzymes and inducing the activities of phase IIenzymes.

The intestinal microbiota are capable of hydrolyzing glycosidic link-ages by producing β-glucuronidase, β-glucosidase and β-galactosidase,leading to the release of mutagens, carcinogens and also tumor pro-moters in the gastrointestinal tract (Chadwick et al., 1992; Georgeet al., 2004; Rumney et al., 1993). These enzymes are responsible forthe deconjugation of complexes of toxins and carcinogens, which hadbeen previously detoxified by glucuronide conjugation in the liver andsubsequently delivered into the colon via the blood or bile. Moreover,β-glucosidase can hydrolyse plant glycosides, releasing aglycones withtoxic, mutagenic or carcinogenic properties and β-glucuronidase is akey enzyme in the final activation of DMHmetabolites to toxic carcino-gens (Nalini et al., 2004; Rowland, 1988). Thus, the compound DMH, acycasin derivative is carcinogenic in the presence of gut bacteria capableof producing the aglycone derivative. Previous findings in our laborato-ry showed that subcutaneous injection of DMH increased the activitiesof β-glucuronidase, β-glucosidase and β-galactosidase, in the rats(Aranganathan et al., 2008). Similarly in this study, there was a remark-able increase in the activities of bacterial enzymes in the feces and

iddle colon Distal colon Incidence of ACF % Inhibition of ACF %

9.66± 0.81a 29.50±1.87a 100 02.66± 0.81b 19.83±1.83b 66.56 33.443.16± 0.40c 8.50±0.83c 22.36 77.643.66± 0.51c 9.16±0.75c 25.32 74.68

superscript letter (a–c) differ significantly at p b 0.05 (DMRT).

Fig. 6. Cross section of rat colon stained with AB-PAS for mucin in the mucus secreting cells. (A and B) Control and control+ troxerutin (50mg/kg b.w.) supplemented rats show strongintense blue staining. (C)Mucosa of DMH alone treated rat shows faint andweak blue staining and progressive loss of mucin in the colonic architecture (group 3). (D–F) Mucosa of DMH-treated rats supplementedwith troxerutin at different doses showed (group 4–6) an increase in the staining for mucin and an increase in themucus cell size and number as compared tothose found in the mucosa of DMH alone treated rats (C).

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colonic mucosa of rats treated with DMH alone as compared totroxerutin supplemented DMH-treated rats. Moreover, various stud-ies indicated that the reduced activities of colonic bacterial enzymeshave been associated with the decreased incidence of colorectal tumors(Gorbach and Goldin, 1990; Lampe et al., 2002; Shiau and Chang, 1983).Thus the outcomeof this experiment reveals that supplementationwithtroxerutin decreased the activities of the retoxification enzymes(microbial enzymes), thereby reducing the toxins and mutagensin the rat colon, which could be attributed to the altered colonicmicroflora.

The colonic lumen is lined by a highly specialized epitheliumcomposed primarily of goblet cells, which secrete mucin (Thorntonand Sheehan, 2004). Mucin the principal glycoprotein of mucous se-cretion plays an important role in the protection against microbialinvasion due to its ability of glycosylation and gel formation.Mucinase is an enzyme present in the intestinal microflora, whichon activation may alter the protective layer of the colon (Shiau andChang, 1983). Many studies have demonstrated that analysis of theepithelial mucin and mucinase activity might be valuable markersto predict and monitor the progression of colon cancer (Aksoyet al., 2000; Salim et al., 2011). In our study, rats treated with DMHalone showed greater mucinase activity as compared to the controland moreover staining with AB-PAS we observed goblet cells devoid

of forming mucin. Furthermore in our earlier study (Manju andNalini, 2006) we reported that administration of the carcinogen,DMH to rats significantly increased the mucinase activity in thecolon and fecal contents. The enhanced mucinase activity in DMHalone treated rat can lead to hydrolyses of the protective mucinlayer of the colonic wall, thereby exposing the underlying epithelialcells to the carcinogens, that had been retoxified by β-glucuronidase(Bara et al., 2003; Miller and Hoskins, 1981; Reddy and Wynder,1973). Interestingly, our present findings show a significant decreasein the mucinase activity, and thus increased mucin content in thecolon of rats supplemented with troxerutin as compared to DMHalone treated rats. The significant reduction in the mucinase activityprotects the gel coating barrier of goblet cells against bacteria and toxinsin the intestinal lumen.

There are reports showing that specific enzymes of colonic bacte-ria in rats could effectively promote the ACF profile induced by DMH.DMH/AOM induced ACF own several biological aberrations includingincreased proliferative state and expansion of the proliferative zone.ACF is suggested to be a putative intermediate marker of colon can-cer and are thought to be good targets for assessing the activity ofchemopreventive agents (Bird, 1995; Corpet and Pierre, 2005;Rodrigues et al., 2002). In our present study increased crypt sizewith intense stinging was observed in DMH treated rats. The

Fig. 7. Liver histopathology of the control and experimental rats. (A andB) Control and control+troxerutin (50mg/kg b.w.) supplemented rats shownormal livermorphology. DMHaloneadministered rat liver (group3) showsmicro andmacro vesicular type of fatty changes, kupffer cell hyperplasia and varying degrees of anaplasia (C). The liver section of DMH+troxerutin(12.5mg/kg b.w., i.e. group 4) supplemented rat showmicro andmacrovesicular type of fatty changes. The livers of DMH+troxerutin supplemented rats (25 and 50mg/kg b.w., i.e groups5 and 6) show near normal appearing liver architecture (E and F).

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majority of ACF were perceived in the middle and distal parts of thecolon andmany of them containedmore than three crypts/foci in theDMH alone treated rats. These findings support the observation thatDMH induced ACF may reflect the initiation step of colorectal carci-nogenesis. Moreover the progressive increase in the number ofcrypts per ACF (i.e four or more aberrant crypts/focus) has beenshown to be a consistent predictor of colon tumorigenesis (Agneret al., 2005). Supplementation with different doses (12.5, 25, and50mg/kg b.w.) of troxerutin to DMH treated rats apparently reducedthe formation of ACF and its multiplicity. Besides, troxerutin supple-mentation at the dose of 25mg/kg b.w., showed themaximum inhib-itory effect (77.64%) on ACF incidence and multiplicity therebysuppressing the progression of preneoplasia to malignant neoplasia.Furthermore the reduction of ACF number/multipilicity by troxerutin isparalleled by the altered activities of biotransforming enzymes therebyplaying a significant role in growth inhibition.

Additionally DMH alone treated rat liver histology showed microand macro vesicular type of fatty changes, Kupffer cell hyperplasiaand varying degrees of anaplasia. On the other hand, supplementa-tion with troxerutin to DMH treated rat showed well preserved

liver morphology. In addition, troxerutin supplementation suppressedthe inflammatory responses in the colon by decreasing the prominentlymphoid aggregation and infiltration of the inflammatory cells in tothe mucosal and submucosal layers. Earlier, it was demonstratedthat troxerutin supplementaion protects the mouse kidney fromD-galactose induced inflammation (Fan et al., 2009). Similarly inthe present study we have observed well preserved liver andcolon morphology of rats when supplemented with 25 mg/kg b.w.,validating the antiinflammatory and anticarcinogenic efficacy oftroxerutin.

Conclusion

Overall, our biochemical and histological studies postulate thattroxerutin has definite chemopreventive efficacy in experimentalrats when supplemented at the dose of 25 mg/kg b.w., as evidentby its role in modulating the enzymes involved in the metabolic acti-vation of DMH and protecting against the development of colonicpreneoplastic lesions. Long term analysis is warranted in future tosubstantiate the colon cancer chemopreventive potential of troxerutin.

Fig. 8. Colon histopathology of the control and experimental rats. (A and B) Control and control+troxerutin (50mg/kg b.w.) supplemented rats showed normal colonic mucosa and nor-mal appearing glands. DMH alone administered rat colon (group 3) showed prominent lymphoid aggregates (circle mark), inflammatory cell infiltration in the mucosal and submucosallayers (arrow mark) and 6 aberrant crypt foci contain 8–13 crypts with intense staining and increased pericryptal zone (dotted arrow mark) were noticed (C). The colon section ofDMH+troxerutin (12.5mg/kg b.w., i.e. group 4) supplemented rat showedmild inflammatory cell infiltration and occasional lymphoid aggregates. The colons of DMH+troxerutin sup-plemented rats (25 and 50mg/kg b.w., i.e groups 5 and 6) showed near normal appearing glands (E and F).

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Conflict of interest statement

The authors declared that there are no conflicts of interest.

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