Impact of Novel Prebiotic Galacto-Oligosaccharides on Various Biomarkers of Colorectal Cancer in ...

12 Pages·2017·0.77 MB·English

International Journal o f
Molecular Sciences
Article
Impact of Novel Prebiotic Galacto-Oligosaccharides
on Various Biomarkers of Colorectal Cancer in
Wister Rats
TahirRasoolQamar1,SanaullahIqbal1,*,FatimaSyed1,MuhammadNasir1 ID,
HabibRehman2,MuhammadAamirIqbal1andRuiHaiLiu3
1 DepartmentofFoodScienceandHumanNutrition,UniversityofVeterinary&AnimalSciences,
Punjab54000,Pakistan;[email protected](T.R.Q.);[email protected](F.S.);
[email protected](M.N.);[email protected](M.A.I.)
2 DepartmentofPhysiology,UniversityofVeterinary&AnimalSciences,Punjab54000,Pakistan;
[email protected]
3 DepartmentofFoodScience,CornellUniversity,Ithaca,NY14850,USA;[email protected]
* Correspondence:[email protected];Tel.:[phone](ext.283);Fax:[phone]
Received:21July2017;Accepted:10August2017;Published:31August2017
Abstract: Colorectalcancer(CRC)isoneoftheleadingcausesofcancerdeathsaroundtheglobe.
Bioactive food ingredients such as prebiotics have protective potential in colon cancer. Data on
galacto-oligosaccharides(GalOS)againstCRCareverylimitedandGalOSusedinthisstudyhave
β-1,6andβ-1,3asmajorglycosidiclinkagesand, toourbestknowledge, wereneverusedbefore
againstanycancertreatment.ThisstudyaimstoinvestigatetheprotectiveroleofnovelGalOSagainst
variousbiomarkersofCRCincludingaberrantcryptfoci(ACF),bacterialenzymesandshortchain
fattyacids(SCFA)inarodentmodelinducedwith1,2-dimethylhydrazinedihydrochloride(DMH).
InulingroupwastakenaspositivecontrolinpresentstudytocomparenovelGalOSprotectiveeffects.
GalOSdosesof76–151mgandinulindosesof114mgweregiventodifferentgroupstreatedwith
DMH.ResultsshowedthatACFformationwassignificantly(p≤0.05)lessinhighdoseGalOSgroup
(27.3%). GalOS also had protective effects against DMH-induced body weight loss and showed
higherlevelofcecalandfecalSCFA(acetate,propionateandbutyrate). HighdosesofGalOSalso
resultedinsignificant(p≤0.05)reductionofbacterialenzymaticactivities. Increasedpopulationsof
beneficialbacteria(bifidobacteriaandlactobacilli)anddecreasedconcentrationsofharmfulbacteria
wereobservedinallprebioticstreatmentgroups. ItcanbeconcludedthatnovelGalOSexhibitrobust
protectiveactivityagainstACFformationinvivo.
Keywords: galacto-oligosaccharides;colorectalcancer;aberrantcryptfoci;bacterialenzymes
1. Introduction
Foodplaysimportantroleinimprovinghealthstatusandtomanagedifferentdiseases.Inmodern
times,muchimportancehasbeengiventoinvestigateimpactoffoodcomponentsonhealth. Inrecent
years,functionalfoodshavegainedsignificantimportancebyscientistsandconsumersandaredefined
as“foodingredientsthatprovidehealthbenefitsbeyondtraditionalnutrientsitcontains”[1]. Inmany
countries,herbalmedicinesarebeingcombinedwithtraditionalfoodstomakefunctionalfoodsand
arealsousedindietarysupplementsforvarioushealthbenefits. Prebioticsarefunctionalingredients
thatarenotdigestedbymammalianenzymesandreachincolon,fermentedbyselectivemicrobesto
improvegrowthofgastrointestinalmicrobiotaforhostwell-beingandhealth[2]. Prebioticshelpin
selectivestimulationofbeneficialbacteriaespeciallylactobacilliandbifidobacteriaaswellastheir
saccharolyticmetabolitessuchasshortchainfattyacids(SCFA),particularlybutyrate[3].
Int.J.Mol.Sci.2017,18,1785;doi:10.3390/ijms18091785 www.mdpi.com/journal/ijms
Int.J.Mol.Sci.2017,18,1785 2of12
Therearedifferenttypesofprebioticsandareindigestiblecarbohydratessuchasoligofructose,
lactulose, isomalto-oligosaccharides, xylo-oligosaccharides, soybean oligosaccharides, inulin-type
fructans,galacto-oligosaccharides(GalOS)andarabinoxylan-oligosaccharides[4]. PrebioticGalOS,
also called trans-galactosylated oligosaccharides (TOS), are composed of galactose and glucose
moleculeshavingaβ-linkageandareproducedbyaprocessknownastransgalactosylationoflactose
catalyzedbyβ-galactosidase(β-gal)[5]. GalOSareprincipallycomposedof2–8saccharideunits,with
glucosebeingtheterminalunitandotherunitsaregalactose[6].
Colorectal cancer (CRC) is a complex multistage disease and is considered a major cause of
mortalityinhumans,irrespectiveofgender,amongallcancer-relateddeathsaroundtheglobe[7,8].
Thereareseveralriskfactorsforcoloncancersuchasage,gender,physicalactivity,geneticfactors,
food habits, inflammatory bowel diseases and pro-carcinogens present in food supply chain and
environment [9]. Diet has major influence on gut related cancers; therefore those foods which
positivelyaffectcolonichealthbymodifyinggutmicrofloraareconsideredusefulstrategyagainst
CRC protection [10]. CRC has been induced chemically in various animal models successfully to
studytumordevelopment,riskfactorsofCRCanditsprevention[11,12]. CRCdevelopmentinboth
humanandrodentsisfollowedbyasequenceofpathologicalchanges,rangingfromaberrantcrypt
foci(ACF),whicharedistinctmicroscopiclesionsinmucosa,tomalignanttumors[8,13]. ACFare
paraneoplasticlesionsfoundincolonmucosaofpatientsathighriskofCRCdevelopmentandin
animalstreatedwithchemicalcarcinogens[14,15]. ACFcanbeinducedchemicallyinrodentcolon
bysingleormultipledosesof1,2-dimethylhydrazinedihydrochloride(DMH)orazoxymethane[8].
DetectionandquantificationofACFhasbeenproposedtoidentifychemo-preventiveagentspresent
inshortaswellasmediumtermbioassaysofrodentcoloncarcinogenesis[11,14].
Inthelastfewdecades,foodscientistsandnutritionistshavefocusedonprebioticsasnatural
dietary ingredients to improve smooth functioning of gastrointestinal tract and to protect colon
cancer[16]. Mostcommonlyinvestigatedoligosaccharidesforcancerprotectionareinulinandseveral
invivostudieshaveproventheirprotectiveeffectsagainstCRC[17–19]. However,verylimiteddata
areavailableforefficacyofGalOSagainstCRC.Furthermore,tothebestofourknowledge,nostudy
hasbeenconductedsofarusingGalOSwithβ-1,6andβ-1,3asmajorglycosidiclinkages. Onlytwo
studieshavebeenconductedbyoneresearchgroupusingGalOSagainstCRChavingβ-1,4asmain
linkage[20,21]. Itisthereasonthatinpresentstudy,prebioticinulingroupwasaddedaspositive
controltocomparetheprotectiveeffectsofnovelGalOShavingβ-1,6andβ-1,3asmainglycosidic
linkage. ThisstudyaimstoinvestigateprotectiveeffectofGalOSagainstvariousbiomarkersofCRC
including ACF, enzymatic activities of microbiota and short chain fatty acids (SCFAs) in a DMH
induced rodent model. The main finding of our study is that GalOS have more protective effects
againstCRCathigherdosesascomparedtolowdose.
2. Results
2.1. BodyWeightChanges
DataofweeklybodyweightchangesareshowninFigure1. Allgroupswerefedonbasaldietad
libitumandnosignificantdifferencewasobservedinfoodintakeamongallgroups. GroupG1(basal
dietgroup)gainedmaximumbodyweight(p≤0.05)ascomparedtoallothergroups. DMHshowed
highimpactonloweringbodyweightsofDMHtreatedanimalsandthenegativecontrolGroupG2
(basaldiet+DMH)gainedthelowestbodyweightascomparedtoallothergroups. Interestingly,
thoseanimals(GroupsG3–G6)receivingprebioticsastreatmentsshowedresistanceagainstDMH
effectonhinderingbodyweightgain. AmongGalOSgroups,G5groupshowedsignificant(p≤0.05)
increaseinbodyweightgainascomparedtoGroupG2. GroupG6,whichwasreceivinginulinas
treatment,gainedmaximumbodyweightamongallprebioticsgroupsandwassignificantly(p≤0.05)
higherascomparedtoGroupG2,howeverthisbodyweightgainwasstatisticallysimilartoGalOS
groups(G4andG5).
Int.J.Mol.Sci.2017,18,1785 3of12
Int. J. Mol. Sci. 2017, 18, 1785 3 of 12
G1 (Basal Diet Control Group) G2 (DMH Control Group)
Int. J. Mol. Sci. 2017, 18, 1785 3 of 12
G3 (DMH + GalOS 76 mg) G4 (DMH + GalOS 114 mg)
G5 (DMH + GalOS 151 mg) G6 (DMH + Inulin 114 mg)
35
G1 (Basal Diet Control Group) G2 (DMH Control Group)
g) 30 G3 (DMH + GalOS 76 mg) G4 (DMH + GalOS 114 mg)
s ( G5 (DMH + GalOS 151 mg) G6 (DMH + Inulin 114 mg)
e3255
g
n
g)ha3200
hanges (weight c22110505
ht cdy 155
go
eiB100
w
dy -55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
o Weeks
B -100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
-5
FigureFig1u.re B1o. dByodwye iwgehitghcth achnagnegsesi nind dififfeferreennttW ggerrooeuukppsss ooff aannimimalasl sthtrhoruoguhgohuot uthtet heexpeexripmeernimt einn t in
-110,2-dimethylhydrazine dihydrochloride (DMH) treated and non-treated groups (n = 15).
1,2-dimethylhydrazinedihydrochloride(DMH)treatedandnon-treatedgroups(n=15).
2.2. AFibgeurrrea n1t. CBroydpty Fwoceii g(AhtC Fch) anges in different groups of animals throughout the experiment in
2.2. AberrantCryptFoci(ACF)
1,2-dimethylhydrazine dihydrochloride (DMH) treated and non-treated groups (n = 15).
Microscopic examination of ACF in colon of DMH treated and non-treated animals is shown in
MFiigcurores c2o. pDiicstreixbaumtioinn aotfi oAnCFo finA dCifFfeirnencto plaorntso off DcoMloHn intr ealal tgerdouapnsd isn sounm-tmreaartizeedd ainn iTmaballes 1is. Nshoo wn
2.2. Aberrant Crypt Foci (ACF)
inFigAuCreF 2w.aDs disettreicbtuedti oinn GorfouApC GF1i n(badsiaflf edrieent tgrpoaurpt)s. oMfacxoimlounmi nACalFl ignr opuropxsimisals (u2m7.4m ±a 2r.i1z0e)d, minidTdaleb le1.
No A(C44F.7Mw ±ia c2sr.9od8s)ce oatepncidct ededixsatimanl i(nG1a1rt9oio.1un p± o 5fG. 5A16C)( Fcbo ailnso anclo wldoeinre eot ofg bDrsoMeurHvpe )dt.r eiMnat Geadxro iamunpdu Gmn2o n(Ab-taCrseFaalt iedndie pat nr+oi mDxMiamlsH ai)sl. sP(h2ro7ebw.4ion±t iincs 2 .10),
middFltiergeu(a4rtm4e .27e.n ±Dt gi2srt.or9iu8bp)usat iwnondit hod fih sAitgCahlF (d i1no1s 9de.si1f fse±hroe5wn.t5e pd6a )brctesot tloeofr n croewlsouenrl teisn oi nabl slh eginrrvdoeuedrpisni ngis tGshuremo oumcpcaurGrirz2eend(cb eian so Taf laAbdClieeF t1 i.+n N Daol lM H).
ApCarFt sw oafs dcoetleocnt.e dA imn oGnrgo uGpa GlO1S ( btarseaalt mdieent tg rgoruopu)p. sM, aGx4i maunmd AGC5 Fr einsu pltreodx imina ls (i2g7n.i4f i±ca 2n.1t 0()p, m≤ id0d.0l5e)
PrebioticstreatmentgroupswithhighdosesshowedbetterresultsinhinderingtheoccurrenceofACF
(r4e4d.7u ±c t2io.9n8 )o af nAdC dFi/sctoallo (n1 1a9s. 1c o±m 5.p5a6r)e cdo ltoon G wroeurep o Gbs2e. rGvreodu ipn GG6ro ruepce Giv2in (gb aisnaul ldinie ats + t DreMatHm)e.n Ptr webaiso tailcsso
inallpartsofcolon. AmongGalOStreatmentgroups, G4andG5resultedinsignificant(p ≤ 0.05)
tsreigantmifiecnatn tg r(po u≤p 0s. 0w5)it ihn hriegdhu cdtoiosens o sfh AowCFe/dc obleotnte ars r ceosumltpsa irne dh itnod Gerrionugp t Ghe2 .o Mccauxrirmenucme opfe ArcCenFt aing ea lolf
reductionofACF/colonascomparedtoGroupG2. GroupG6receivinginulinastreatmentwasalso
ptaorttasl AofC Fco ilnohni.b iAtiomno wnga sG sahloOwSn tbreya Gtmroeunpt gGr6o u(2p9s.3, %G)4 a nadnd G 5G (52 7r.e3s%u)l taesd coinm spiagrneidfi ctaon Gt 2( pa s≤ w 0e.l0l 5a)s
signifiroectdahunecrtt ip(oprne≤ boifo0 At.i0cC5sF )g/ircnooulropensd a(uGsc c3toi aomnndpo aGfre4Ad).C toF /Gcrooulopn Ga2s. cGormouppa Gre6d retoceGivrinogu pinuGl2in. aMs atrxeiamtmuemntp wearcs eanlstoa geof
totalsAigCnFifiicnahnti b(pit i≤o 0n.0w5)a isn srhedouwcntiobny oGf AroCuFp/cGol6on( 2a9s. 3co%m)paanrdedG to5 G(2r7o.u3p% G)2a.s Mcoaxmimpuamre dpetroceGn2taagse wofe llas
othertoptrael bAioCtFic isnhgirboiutiopns w(Ga3s sahnodwGn 4b)y. Group G6 (29.3%) and G5 (27.3%) as compared to G2 as well as
other prebiotics groups (G3 and G4).

Figure 2. Microscopic view of the colons showing: (a) normal crypts of control animals; and (b–f)
1,2-dimethylhydrazine dihydrochloride (DMH) treated animals showing aberrant crypt foci stained

with methylene blue (40×). Arrows indicate ACF: singlet (S), doublet (D), triplet (T), and cluster (C).
Figure 2. Microscopic view of the colons showing: (a) normal crypts of control animals; and (b–f)
Figure2. Microscopicviewofthecolonsshowing: (a)normalcryptsofcontrolanimals; and(b–f)
1,2-dimethylhydrazine dihydrochloride (DMH) treated animals showing aberrant crypt foci stained
1,2-dimethylhydrazinedihydrochloride(DMH)treatedanimalsshowingaberrantcryptfocistained
with methylene blue (40×). Arrows indicate ACF: singlet (S), doublet (D), triplet (T), and cluster (C).
withmethyleneblue(40×).ArrowsindicateACF:singlet(S),doublet(D),triplet(T),andcluster(C).
Int.J.Mol.Sci.2017,18,1785 4of12
Table1.Effectofprebiotictreatmentsonaberrantcryptfoci(ACF)inproximal,middleanddistalcolon
in1,2-dimethylhydrazinedihydrochloride(DMH)-initiatedandnon-initiatedanimals(n=15).
G1(BasalDiet G2(DMH G3(DMH+ G4(DMH+ G5(DMH+ G6(DMH+
ACF
ControlGroup) ControlGroup) GalOS76mg) GalOS114mg) GalOS151mg) Inulin114mg)
ACF/proximalcolon ND 27.4±2.10a 24.7±3.11a,b 18.7±2.56b,c 14.3±1.78c 16.4±2.83c
ACF/middlecolon ND 44.7±2.98a 42.3±2.86a,b 34.3±3.8b,c 22.9±3.45d 28.3±3.23c,d
ACF/distalcolon ND 119.1±5.56a 115.3±7.22a,b 110.4±4.63a,b 101.7±3.32b,c 90.3±3.31c
TotalACF/colon ND 191.2±6.46a 182.4±7.92a 163.4±4.72b 139.0±3.24c 135.1±3.73c
Percentinhibition
- - 4.6 14.53 27.3 29.3
oftotalACF
ValuesareexpressedasMean±SE.Meansinthesamerowwithdifferentsuperscriptaresignificantlydifferent
(p≤0.05).DMH,1,2-dimethylhydrazinedihydrochloride(4×40mg/kgb.wt.,s.c.);ND,Notdetected.
2.3. ShortChainFattyAcids(SCFAs)
ResultsofSCFAs(acetate,butyrateandpropionate)incecalandfecalcontentsaregiveninTable2.
ThoseanimalsreceivingprebioticsastreatmentshowedhigherconcentrationofSCFAs,particularly
highdosegroups. GroupsG5andG6showedsignificantly(p≤0.05)higherconcentrationofacetate
andbutyrateincecalcontentsascomparedtoGroupG2(basaldiet+DMH).OnlyGroupG6had
significantly(p≤0.05)higherconcentrationsofpropionateincecumascomparedtoGroupG2.Infecal
contents,GroupsG5andG6resultedinsignificant(p≤0.05)higherconcentrationsofacetateand
butyrate as compared to Group G2 While Groups G4–G6 were significantly (p ≤ 0.05) higher in
concentrationsofpropionateinfecalcontentsascomparedtoGroupG2.
Table2.Effectofprebiotictreatmentsonshortchainfattyacids(SCFAs)concentrationsofcecaland
fecalcontentsin1,2-dimethylhydrazinedihydrochloride(DMH)initiatedandnon-initiatedanimals
(n=15).
G1(BasalDiet G2(DMH G3(DMH+ G4(DMH+ G5(DMH+ G6(DMH+
Parameters
ControlGroup) ControlGroup) GalOS76mg) GalOS114mg) GalOS151mg) Inulin114mg)
Cecum
Acetate 80.8±3.41c 79.2±3.28c 82.8±3.59b,c 86.2±3.85a,b,c 92.4±3.49a,b 94.9±3.83a
Propionate 21.9±2.03a,b 20.6±1.85b 22.1±2.11a,b 23.4±1.78a,b 26.1±1.54a,b 26.6±1.44a
Butyrate 14.1±1.11c 13.9±1.03c 14.8±0.93b,c 16.4±1.21a,b,c 18.5±1.36a 17.8±1.09a,b
Fecal
Acetate 51.7±2.58b 50.6±2.71b 53.2±3.11b 56.8±2.35a,b 62.1±3.13a 64.8±2.67a
Propionate 14.3±0.99c,d 14.1±0.79d 15.5±0.68c,d 17.4±1.05b,c 20.3±1.26a,b 21.2±1.34a
Butyrate 5.2±0.51b 4.92±0.48b 5.5±0.53b 6.1±0.53a,b 7.3±0.57a 7.5±0.67a
ValuesareexpressedasMean±SE.Meansinthesamerowwithdifferentsuperscriptaresignificantlydifferent
(p≤0.05).DMH,1,2-dimethylhydrazinedihydrochloride(4×40mg/kgb.wt.,s.c.);SCFAs,µmol/g.
2.4. BacterialEnzyme
Table 3 summarizes bacterial enzyme activities in fresh cecal and fecal samples. Group G2
(basal diet + DMH) showed overall high bacterial enzymes activities in cecal and fecal contents.
Amongprebioticstreatmentgroups,GroupsG5andG6showedlowestenzymeactivities(formost
enzymes) in both cecal and fecal contents. Groups G5 and G6 resulted in significant (p ≤ 0.05)
lowercontentsofnitroreductasewhileonlyinGroupG5β-glucoronidasewassignificantlyreduced
in cecum as compared to Group G2. However, in the case of β-glucosidase, only Group G6 had
significantly(p≤0.05)lowercontentsincecumcomparedtoG2group. Infecalsamples,GroupG6
couldsignificantly(p≤0.05)decreaseβ-glucosidase,nitroreductaseandazoreductaseconcentrations,
whileGroupG5significantly(p≤0.05)reducedactivitiesofonlyβ-glucoronidaseandnitroreductase
ascomparedtoDMHcontrolgroup(G2).
Int.J.Mol.Sci.2017,18,1785 5of12
Table3.Effectofprebiotictreatmentsoncecalandfecalenzymeactivitiesin1,2-dimethylhydrazine
dihydrochloride(DMH)initiatedandnon-initiatedanimals(n=15).
G1(BasalDiet G2(DMH G3(DMH+ G4(DMH+ G5(DMH+ G6(DMH+
Enzymes
ControlGroup) ControlGroup) GalOS76mg) GalOS114mg) GalOS151mg) Inulin114mg)
Cecum
β-Glucosidase 0.82±0.11a,b 0.96±0.11a 0.94±0.09a 0.80±0.10a,b 0.77±0.11a,b 0.59±0.09b
β-Glucoronidase 3.43±0.18a,b 3.52±0.19a 3.25±0.12a,b 3.12±0.18a,b 2.96±0.15b 2.99±0.19a,b
Nitroreductase 4.38±0.29a 4.47±0.28a 4.27±0.25a 3.83±0.25a,b 3.44±0.19b 3.26±0.17b
Azoreductase 11.67±1.01a 11.42±0.56a,b 10.08±0.67a,b 10.17±0.81a,b 9.16±0.79b 9.25±0.72a,b
Fecal
β-Glucosidase 0.72±0.11a,b 0.91±0.10a 0.76±0.08a,b 0.69±0.09a,b 0.64±0.11a,b 0.53±0.09b
β-Glucoronidase 2.97±0.25a 3.07±0.16a 2.93±0.10a,b 2.72±0.14a,b 2.43±0.15b 2.55±0.16a,b
Nitroreductase 3.13±0.18a 3.18±0.19a 2.86±0.15a,b 2.76±0.15a,b 2.49±0.17b 2.55±0.18b
Azoreductase 8.17±0.51a,b 8.42±0.42a 7.92±0.47a,b 7.41±0.59a,b 6.91±0.45a,b 6.67±0.57b
ValuesareexpressedasMean±SE.Meansinthesamerowwithdifferentsuperscriptaresignificantlydifferent
(p≤0.05).DMH,1,2-dimethylhydrazinedihydrochloride(4×40mg/kgb.wt.,s.c.);β-Glucosidase,µg/min/mg
cecalorfecalprotein; β-Glucoronidase,µg/min/mgcecalorfecalprotein; NitroreductaseandAzoreductase,
µg/h/mgcecalorfecalprotein.
2.5. BacterialPopulations
FecalbacterialconcentrationdataareshowninTable4. Microbialconcentrationsofbeneficial
bacteria(bifidobacteriaandlactobacilli)wereincreasedinallprebioticstreatmentgroupsanddecreased
concentrations of harmful bacteria were observed in these treatment groups. Among all prebiotic
treatmentgroups,onlyGroupG5showedsignificantly(p≤0.05)higherconcentrationsoflactobacilli
ascomparedtoGroupsG2(basaldiet+DMH)andG1(BasalDietControlGroup). GroupsG5and
G6 had significantly (p ≤ 0.05) higher population of bifidobacteria as compared to Group G2. No
significantdifferencewasobservedinconcentrationofenterococciamongallgroups;however,Group
G6hadsignificantly(p≤0.05)lowerconcentrationsofclostridiaascomparedtoGroupG2.
Table4. Effectofprebiotictreatmentsonfecalmicrobialconcentrationsin1,2-dimethylhydrazine
dihydrochloride(DMH)initiatedandnon-initiatedanimals(n=15).
G1(BasalDiet G2(DMH G3(DMH+ G4(DMH+ G5(DMH+ G6(DMH+
Microbes
ControlGroup) ControlGroup) GalOS76mg) GalOS114mg) GalOS151mg) Inulin114mg)
Lactobacillus 7.18±0.24b 7.14±0.17b 7.39±0.18b 7.64±0.09a,b 7.93±0.16a 7.52±0.18a,b
Bifidobacteria 8.11±0.14c,d 8.07±0.15d 8.18±0.14c,d 8.24±0.13b,c,d 8.51±0.15b,c 9.12±0.11a
Clostridia 8.82±0.12a,b 8.92±0.11a 8.77±0.15a,b 8.59±0.20a,b,c 8.51±0.14a,b,c 8.45±0.11b,c
Enterococci 7.42±0.11 7.48±0.10 7.53±0.18 7.37±0.17 7.22±0.15 7.27±0.09
ValuesareexpressedasMean±SE.Meansinthesamerowwithdifferentsupersciptsaresignificantlydifferent
(p≤0.05). DMH,1,2-dimethylhydrazinedihydrochloride(4×40mg/kgb.wt.,s.c.) log10 CFU/gwetweight
offecal.
3. Discussion
CRC is prevalent worldwide and annually 500,000 people die. Western developed countries
have higher incidence of CRC [22]. Diet is considered a major factor as it can play either good or
badroleforCRCdevelopment. Dietrichinsweets,alcohol,refinedgrains,redmeatandprocessed
meatincreasesCRCriskanddietrichindairyproducts,fruits,vegetables,fish,oliveoilandfiberis
consideredbeneficialforcancerprotection[23]. Thecurrentstudywasperformedtoassessprotective
roleofprebioticsGalOSandinulin(aspositivecontrol)againstvariousbiomarkersofCRC.Prebiotics
are bioactive ingredients of recent past and have several confirmed and postulated impacts on
gastrointestinalmicrobiotaandtheirmetabolismespeciallyincolon[24]. Theprebiotics,probiotics[25]
andsynbiotics[26]haveappliedinvariousCRCinducedratmodelsandhaveshownprotectiveaswell
aspreventiverolesagainsttumordevelopmentandvariousotherearlybiomarkers[17,27]. Todate,
studiestofindprotectiverolesofGalOSinCRCareverylimited. Theprimaryglycosidiclinkagein
GalOSusedinpreviousstudiesrelatedtocancerwasβ-1,4andnoproductwithβ-1,6and/orβ-1,3
Int.J.Mol.Sci.2017,18,1785 6of12
linkage have been used yet. Furthermore, purity of GalOS in these studies was 58.8% with other
mono-anddisaccharides[20,21]. Toourbestknowledge,prebioticGalOSthatcontainsβ-1,6and/or
β-1,3glycosidiclinkageisusedforthefirsttimeinthepresentstudywithpurityof30%. Ourdata
demonstratethathighdoseofGalOSprovideasignificantprotectiveeffectagainstdevelopmentof
coloncancer.ItwasrevealedthattheinhibitionofACFformationwassignificantlyhigherininulinand
highdoseGalOSgroups. TheresultsinthisstudysuggestthatGalOShaddose-dependentresistance
againstbodyweightlossinDMH-inducedgroupsandhighdosegroupsshowedhigherlevelsofcecal
andfecalSCFAs.
ACFareconsideredverygoodbiomarkersforinitiationstageofcoloncancerandcanbeeasily
detectedincolonmucosaofpatientsathighriskofCRCdevelopment[13]. NoACFformationwas
detectedinGroupG1(controlgroupfedonbasaldiet). DataofACFincidenceindicatethatDMH
successfullyinducedACFinallDMHtreatedanimals. HighestnumberofACFwasobservedinGroup
G2(basaldiet+DMH)inallpartsofcolon. Theseresultsdemonstratethatprebioticshavesignificant
effectonreductionofDMH-inducedACF,particularlyGroupsG5andG6. ThereductionofACFin
ratsintheprebioticsgroupsmaybeduetoinductionofapoptosisincoloniccrypts,modulationof
microbiota,increasedenzymaticactivityandimprovedconcentrationsofSCFAs,asallthesefactorsare
verymuchimportantfornormaldevelopmentandsmoothfunctioningofcolonicepithelialcells[17,28].
Becauseofprebioticfermentationincolon,SCFAsareproducedanditisbelievedthat,amongthese
SCFAs, butyrate plays an important role in inhibiting tumor cells [29,30]. These results of ACF
inhibitionsupportpreviousinvestigationbyVermaandShukla[18]inwhichtheystudiedtheeffects
ofinulinandlactuloseondifferentbiomarkersofCRCin-vivo. Theyalsoconcludedthatprebiotic
inulinsignificantlyreducedACFcountascomparedtoonlyDMHtreatedanimals. Findingsofanother
studyconductedbyWijnands,Schoterman,Bruijntjes,HollandersandWoutersen[21]usingGalOSas
treatmentssupportourfindingsofprotectiveeffectofGalOSonACFinhibition.
Elevated levels of SCFAs in prebiotic groups of present study are in accordance with
previous studies in which higher concentration of SCFAs were observed in rats after inulin
supplementations[31–33].AnotherstudyconductedbyWijnands,Schoterman,Bruijntjes,Hollanders
andWoutersen[21]usingcommercialGalOShavingβ-1,4linkagealsoshowedhigherlevelsofSCFAs
inhighdoseGalOSgroupswhichstrengthenourfindings. Wealsoobservedthatgroupswhichhad
higherlevelsoftheseSCFAsthetotalnumberofACFcountwaslowinthosegroups,itmightbedue
totheincreasedlevelsofbutyrate,whichisconsideredimportantfortumorsuppressionasitplays
selectiverolesinnormalandcolonictumorcells. Itinducesapoptosisincancerouscellsandprotects
colonfromgenotoxiccarcinogens[30,34]. Infusionofhighdoseofbutyrateindistalcolonhasshown
morethan60%reductionintheformationoftumorsinazoxymethaneinducedratsascomparedto
controlgroups[35].
Itiswellestablishedthatmicrobialenzymes(β-glucuronidase,β-glucosidase,azoreductaseand
nitroreductase)caninfluencecarcinogenesisdevelopment[18,36];increasedactivityofβ-glucuronidase
isassociatedwithincreasedincidenceofneoplasmsincolon[37]. Oneoftheconditionsforprebiotics
istoescapethedigestioninuppergastrointestinaltractsothatbeneficialmicrobiota(bifidobacteria
andlactobacilli)inlowertractutilizeundigestedprebioticsincolon[38]. Decreasedactivityofthese
enzymes in prebiotics treatment groups in present study may be due to the increased population
ofhealthbeneficialmicrobiotaaspreviousstudieshavealsodocumentedtheincreasedgrowthof
probioticsanddecreasedgrowthofpathogensandlowactivityofpathogenbacterialenzymesafter
supplementationofprebiotics[26,28,33,38].
About100trillionmicrobialorganismsareresidentinadulthumangut;thesemicrobialorganisms
are collectively called microbiota [39]. Human health status directly and indirectly is dependent
on resident microbiota, as these microbes ferment various food substances in colon that are left
undigestedandunabsorbedinsmallintestine. Ingastrointestinaltract,carbohydrateandproteolytic
aretwomaintypesoffermentations. SCFAs(butyrate,acetateandpropionate)aremajorby-products
of carbohydrate fermentation and are considered beneficial for health. However, end products of
Int.J.Mol.Sci.2017,18,1785 7of12
proteolyticfermentation(phenoliccompounds,amines,ammonia,N-nitrosocompoundsandindoles)
can be toxic to the host [40]. We found increased concentrations of beneficial bacteria (lactobacilli
and bifidobacteria) in our study. As these microbes are known to ferment prebiotics in colon, we
alsoobservedelevatedlevelsofSCFAsinprebiotictreatmentgroups. Asincreasedpopulationsof
beneficialbacteriaissupposedtosuppresstheconcentrationsofpathogens,weobserveddecreasein
enterococciandclostridiapopulationsasthesemicrobesareefficientproducersofβ-glucoronidase
andβ-glucosidase[41],whichmightbeoneofthereasonsweobservedlowerconcentrationsofthese
enzymes in our all prebiotic treatment groups. Interestingly, growth of lactobacilli was highest
in Groups G4 and G5, which received prebiotics GalOS as compared to bifidobacterium, thus
strengtheningthepreviousfindingsbyTzortzisetal. thatprebioticsresultsinmaximumgrowthof
thosebacteriawhichhaveusedassourceofenzymefortheirproduction[42,43]. Inpresentstudy,
LactobacillusreuteriL103wasusedassourceofβ-galforGalOSproduction.
4. MaterialsandMethods
4.1. ChemicalsandReagents
The chemicals and reagents used in the study were of analytical grade, procured form
Merck Chemicals (Darmstadt, Germany) unless stated. The prebiotic inulin was purchased
from Cargill (Minneapolis, MN, USA). The ortho-nitrophenyl-β-galactopyranoside (oNPG), DMH,
phenolphthalein-β-D-glucuronide,m-nitrobenzoicacidandnitrophenyl-β-D-glucosidewerepurchased
fromSigma-Aldrich(St. Louis,MO,USA).
4.2. Animals,TreatmentsandDiets
Six-week-old male Wister rats (n = 90) were procured from Institute of Biochemistry and
Biotechnology, UVAS, Lahore and were housed in stainless steel cages under standard conditions
of temperature (23 ± 2 ◦C) and humidity (55 ± 10%) in a 12 h light–dark cycle. All experimental
protocolsusedhereinwereapprovedbythe“UniversityEthicalReviewCommittee(DASNo.: 5372
Date: 14-04-2017) for the use of Laboratory Animals”. Rats were fed on standard diet for 1-week
acclimatizationperiodbeforebeginningtheexperiment. Afteracclimatizationperiod,90ratswere
equallyandrandomlydividedintosixgroups. G1wascontrolgroup(CG)fedonAIN-93G/Mas
control basal diets [44]. Group G2 (basal diet + DMH) was DMH control group. Treatments with
prebioticsGalOSweregiventoGroupsG3–G5whileGroupG6waspositivecontrolandwasgiven
prebioticinulintreatment. Prebioticsdoseswerecalculatedbyusingthehumanequivalentdose(HED)
equation:HED(mg/kg)=animaldoseinmg/kg×(animalweightinkg/humanweightinkg)0.33[45].
GroupsG3, G4andG5received76mg(HED=4g), 114mg(HED=6g)and151mg(HED=8g)
GalOS,respectively,andGroupG6received114mg(HED=6g)ofinulin.
DMHtreatment(40mg/kgbodyweight)wasgiventoGroupsG2–G6throughfoursubcutaneous
injectionspreparedin1mMEthylenediaminetetraaceticacid(EDTA)solutionofpH6.0,twiceaweek
for2weeks[19,46]. ThecontrolgroupwastreatedwithsameconcentrationsofEDTAinasimilar
pattern. AftercompletingtheDMHdoses,prebioticstreatmentswerestartedtorespectivegroups
for16weeks. Bodyweightofeachratineachgroupwasrecordedweeklyanddosesofprebiotics
torespectivegroupsweregivenaccordingtomeanbodyweightofeachgroup. Tomakeconditions
similar to all groups, all prebiotics were given orally through tube feeding while control groups
(G1andG2)wereadministeredsameamountofwater. After16weeks,allanimalswerekilledusing
exsanguinationunderanesthesiawithsodiumpentobarbital(45mg/kgbodyweight).
4.3. PreparationofGalOS
The β-galactosidase (β-gal) was produced from Escherichia coli BL21 containing gene from
L. reuteri L103 following procedure explained by Iqbal et al. [47]. The enzyme activity was
measured for chromogenic substrate oNPG and natural substrate lactose following methods by
Int.J.Mol.Sci.2017,18,1785 8of12
Splechtnaetal.[48]. Thecrudeβ-galwasusedfortheproductionofGalOSthroughbioconversionof
lactose by following method of Splechtna, Nguyen, Steinböck, Kulbe, Lorenz and Haltrich [48].
The transgalactosylated mixture were analyzed for glucose using GOPOD assay kit (K-GLUC)
howevergalactoseandlactoseweremeasuredusingLactose/Galactoseassaykit(K-LACGAR)from
Megazyme (Wicklow, Ireland) by following protocol given in the provided documents. The main
glycosidiclinkageinpresentprebioticGalOSwasβ-1,6withmainproductsβ-D-Galp-(1–6)-D-Glc,β-
D-Galp-(1–6)-D-Galandβ-D-Galp-(1–6)-Lac,followedbyβ-1,3linkedproducts,i.e.,β-D-Galp-(1–3)-D
-Galandβ-D-Galp-(1–3)-Lac.Whilenoproductwithβ1–4linkagewasidentified[47,48]ascomposition
determined by High performance anion exchange chromatography with pulsed amperometric
detection(HPAEC-PAD)andcapillaryelectrophoresis.
4.4. MeasurementofBodyMass
Changesinbodyweightandfeedintakeweremeasuredonweeklybasisusingweighingscale
(SCL66110OlympiaPlusKentScientificCorporation,Torrington,WY,USA).
4.5. AberrantCryptFoci(ACF)Analysis
ACFanalysiswereperformedusingamethodpreviouslydescribedbyBird[49]. Briefly,after
sacrificinganimals,colonwasremovedstartingfromcecumtoanususingsharpbladeandcolonwas
openedalonglongitudinalaxis,remainingundigestedmaterialincolonwasgentlywashedawayby
salinesolution. Colonwasfixedin10%formalinatroomtemperaturefor24h. Methyleneblue(0.2%)
insalinewasusedforstainingofcolon. ACFwereidentifiedbymicroscopyatamagnificationof
×40. Colonwasdividedintothreepartsdistal(nearrectum),middleandproximalcolon(nearcecum).
ThenumberofACFinthreepartsofcolonwasaddedtogettotalnumberofACF.
4.6. SamplesCollectionandEnzymeAnalysis
Therectalregionofratswasgentlysqueezedtogetfreshfecalsamplewhilececumsamplewas
collectedduringopeningoflargeintestineaftersacrificingeachanimal. Bothtypesofsampleswere
processedimmediatelyaftercollection. Detailedprocedurestomeasureactivitiesofnitroreductase,
β-glucuronidaseandβ-glucosidaseincecumandfecalsamplesareexplainedinourpreviousstudy[50]
andazoreductaseactivitywasdeterminedfollowingGoldinandGorbach[36]method.
4.7. ShortChainFattyAcids(SCFAs)
TheamountsofSCFAsincecalandfecalcontentsweredeterminedusinggaschromatography
(Agilent6890Plus,SantaClara,CA,USA)followingmethoddescribedbyZhaoetal.[51].
4.8. MicrobialAnalysis
Bacterial groups from fecal samples were identified using plate count methods as previously
described by Van de Wiele et al. [52]. Specific media (Oxoid, Hampshire, UK) were used for each
bacterialgroup,includingRogosaagar(lactobacilli),raffinoseBifidobacteriumagar(bifidobacteria),
Enterococcusagar(enterococci),andtryptosesulfitecycloserinagar(clostridia).
4.9. StatisticalAnalysis
Allcollecteddatawereexpressedasmean±standarderror(SE).One-wayanalysisofvariance
inSPSSsoftware(ver. 18,IBMSPSSSoftwareCompany,Chicago,IL,USA)wasappliedtoanalyze
variationamonggroups. DuncanMultipleRange(DMR)testwasappliedforpost-hocanalysisin
significantlydifferentresults. Differenceswereconsideredsignificantatp<0.05.
Int.J.Mol.Sci.2017,18,1785 9of12
5. Conclusions
ItcanbeconcludedthatnovelprebioticGalOShavingβ-1,6andβ-1,3glycosidiclinkagehasdose
dependentprotectiveeffectsonvariousbiomarkersofCRCandaremoreprotectiveinhigherdoses.
Furtherstudiesarerequiredatgeneexpressionleveltounderstandthemechanismsofactionofthese
prebioticsintheprotectionofCRC.
Acknowledgments:TheauthorsarereallythankfultoDietmarHaltrich,FoodBiotechnologyLab.,Universityof
NaturalResourcesandLifeSciences,ViennaAustriaforprovidingbacterialstrainhavingβ-galactosidasegene.
AuthorContributions:TahirRasoolQamar,FatimaSyedandSanaullahIqbalDesignedthisstudy.TahirRasool
QamarandFatimaSyedconductedtheexperimentsandperformedthepracticalwork. HabibRehmanand
Muhammad Aamir Iqbal performed statistical analysis on the data. Tahir Rasool Qamar, Sanaullah Iqbal,
MuhammadNasirandRuiHaiLiuproofreadandfinalizedthemanuscript.
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.
Abbreviations
CRC Colorectalcancer
ACF Aberrantcryptfoci
GalOS Galacto-oligosaccharides
DMH 1,2-Dimethylhydrazinedihydrochloride
SCFAs Shortchainfattyacids
ITF Inulin-typefructans
DP Degreeofpolymerization
FOS Fructo-oligosaccharides
oNPG Ortho-nitrophenyl-β-galactopyranoside
HED Humanequivalentdose
EDTA Ethylenediaminetetraaceticacid
β-Gal β-Galactosidase
HPAEC-PAD Highperformanceanionexchangechromatographywithpulsedamperometricdetection
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