Journal
of
Coloproctology
w w w . j c o l . o r g . b r
Original
Article
Comparative
study
of
1,2-dimethylhydrazine
and
azoxymethane
on
the
induction
of
colorectal
cancer
in
rats
Mario
Jorge
Jucá
a,∗,
Bruno
Carneiro
Bandeira
a,
Davi
Silva
Carvalho
a,
Antenor
Teixeira
Leal
baMedicineSchool,UniversidadeFederaldeAlagoas(UFAL),Maceió,AL,Brazil bUniversityStudent,HealthSciences,Maceió,AL,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received12February2014 Accepted13March2014 Availableonline2July2014
Keywords: Colorectalcancer Experimentalmodel Carcinogenesis Azoxymethane 1,2-Dimethylhydrazine
a
b
s
t
r
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c
t
Theinducedcolorectalcarcinogenesisinrodentshasalonghistoryandcurrentlyusesthe substances1,2-dimethylhydrazineandazoxymethane.
Objective:The aimofthisstudy wastocomparethe inductiveeffect ofthesubstances azoxymethaneand1,2-dimethylhydrazineincolorectalcarcinogenesis.
Method:30randomlychosenmaleWistarratsweredividedintofourgroups.G1groupwas treated with1,2-dimethylhydrazine andC1wasitscontrolgroup;G2 groupwastreated azoxymethane andC2 was its control group.The animals wereweekly weighed until euthanasia,whentheirintestineswereremoved,processedandanalyzedbyanexperienced pathologist.
Results:Amongthecontrolgroups(C1andC2)nohistologicchangeswereobserved; mod-eratedysplasiawasdetectedinG2group;hyperplasia,milddysplasia,severedysplasiaand carcinomawereobservedinG1group.Whenthisstudycomparedthecostofthesubstances, 1,2-dimethylhydrazinewasmorethan50timeslessexpensivethanazoxymethane. Conclusion: Azoxymethaneisabletopromotehistologicalchangesconsistentwithcolorectal carcinogenesis.1,2-Dimethylhydrazineproducedneoplasiaanddysplasia,and,compared totheazoxymethane,wasmoreefficientintheinductionofcolorectalcancer.
©2014SociedadeBrasileiradeColoproctologia.PublishedbyElsevierEditoraLtda.All rightsreserved.
∗ Correspondingauthor.
E-mail:mario.juca@uol.com.br(M.J.Jucá).
http://dx.doi.org/10.1016/j.jcol.2014.06.003
Estudo
comparativo
das
substâncias
1,2-dimetil-hidrazina
e
azoximetano
na
induc¸ão
de
câncer
colorretal
em
ratos
Palavras-chave: Câncercolorretal Modeloexperimental Carcinogênese Azoximetano 1,2-Dimetil-hidrazina
r
e
s
u
m
o
Acarcinogênesecolorretalinduzidaemroedorestemlongahistóriaeutiliza,atualmente, assubstâncias1,2dimetil-hidrazina(DMH)eazoximetano(AOM).
Objetivo: CompararoefeitoindutivodassubstânciasAOMeDMHparaocâncercolorretal (CCR).
Método: 30ratosWistarmachosforamrandomizadosemquatrogrupos.OgrupoG1foi inoculadocomDMH,ogrupoC1foiseucontrole;G2recebeuoAOMeC2foiseucontrole. Osanimaisforampesadossemanalmenteatéaeutanásia,quandotiveramseusintestinos retirados,processadoseanalisadosporumpatologistaexperiente.
Resultados: Osanimaisdosgruposdecontroleapresentaramtecidocolorretalnormaleos animaisdogrupoG2apresentaramumpadrãodedisplasiamoderada.Naslâminasdogrupo G1,foramencontradasregiõesdehiperplasia,displasialeve,displasiagrave,ecarcinoma. ComparadoocustodassubstânciasAOMeDMH,esteúltimoteveumprec¸omaisde50 vezesmenoraodoAOM.
Conclusão: AOMécapazdepromoveralterac¸õeshistológicascompatíveiscoma carcinogê-nesecolorretal.DMHproduziuneoplasiaedisplasiagravee,comparadaaoAOM,foimais eficientenainduc¸ãodocâncercolorretal.
©2014SociedadeBrasileiradeColoproctologia.PublicadoporElsevierEditoraLtda. Todososdireitosreservados.
Introduction
Thenumberofnewcasesofcolonandrectalcancerestimated forBrazilin2012is30,140,with14,180inmenand15,960in women.1
Theetiologyofcolorectalcancer(CRC)isknowntobe multi-factorial,includingfamily,environmentalanddietaryagents. Despite many advances in our understanding of the pro-cessesofcarcinogenesis,todate,therapiesincludingsurgery, radiation andchemotherapydrugs are stilllimited totreat advancedstagesofCRC.2–4 Theonlysatisfactoryanswer to theproblemofmalignancyisitsprevention.Thisinvolvesan extensivesearchforacquiringknowledgeofthebasicaspects ofcarcinogenesis.5,6
Thecarcinogenesis and development ofCRC are multi-stepprocesses, characterizedbyprogressivechanges inthe amountoractivityofproteinsthatregulatetheproliferation, differentiation, andcell survival, andthat are mediatedby geneticmechanisms. An ordered sequence ofnon-random events leadstothe development ofcolorectalcancer, with theepitheliumundergoinganinvasivetransformation,with progressionfromnormalintestinalepitheliumtothe devel-opmentofinvasivecarcinoma.5,7–12
Animalmodelsaregoodchancestostudythebiologyof dis-easedevelopment.Inaddition,thesemodelsallowfortesting hypothesesrelatingenvironmentalfactorstotheetiologyand preventionofcancer.7
The study of colorectal carcinogenesis in rodents has a long history, dating back approximately 80 years. Cur-rently, experimental models use colorectal carcinogens 1,2-dimethylhydrazine(DMH)andazoxymethane(AOM).13–16 DMHfallsinthecategoryofanindirectinducerdrug.This drug has the ability to promote DNA hypermethylation of
colorectalepithelialcellsinthesegment.AOMisaderivative ofdimethylhydrazine.However,unlikeDMH,AOMfallsunder thecategoryofadirectinducer,withoutrelyingonconversion invivo.17
Thisstudyaimstocomparetheinductiveeffectofthe sub-stancesAOMandDMHforcolorectalcarcinomainanattempt toidentifyamoreefficientanimalmodelfortheinductionof CRCinrats.
Method
Animals
30 Wistarrats fromthe CentralAnimalLaboratory, Univer-sidadeFederaldeAlagoas(UFAL),submittedtoalight–dark cycle of12h,andfedwithstandarddietand waterad libi-tum, were used. The study was approvedby the Ethics in ResearchCommittee(ERC),UniversidadeFederaldeAlagoas, and all experimental steps were performed in accordance withthe principlesestablishedbythe ColégioBrasileiro de Experimentac¸ãoAnimal(COBEA).
Experimentalgroupsandtechnique
Theanimalswererandomizedintofourgroups:twogroupsof tenanimals(G1andG2)andtwooffive(C1andC2).G1was submittedtoinductionbyDMH,andC1wasitscontrolgroup. G2receivedAOMandC2wasitscontrolgroup.
AOMwasadministereddissolvedin0.9%NaCl,resultingin AOM20mg/mLandappliedsubcutaneouslyfortwoweeksat adoseof20mg/kg/wk.3
InC1andC2,onlysaline(sodiumchloride0.9%)wasapplied inaproportionalvolumeandinthesametimeschemeofG1 andG2,respectively.
AfterthesecondinoculationofAOM,wewaitedtwoweeks fortheactionofthissubstance,andtenweeksfortheaction ofDMH,afterits fifthinoculation.Theanimals were prop-erlyidentifiedandsubmittedtotheadministrationofsodium thiopental 150mg/kg, intraperitoneally, whose lethal dose causedaquickandpainlessdeathbycentralnervousaction withcardiopulmonaryarrest.3,18
Immediatelyaftereuthanasia,theintestinewasremoved enblocfromthececumtotheanusandopenedwithscissorsin theantimesentericborder.ThegutwasstretchedinStyrofoam platesforcleaningwith0.9%NaCl.
Histopathology
Tissueswerefixedin10%bufferedformaldehydefor24hand thereafterdehydratedinincreasingconcentrationsofethanol. Afterdehydration,thesampleswere embeddedinparaffin, andfromthesematerialstissuesectionswereobtainedand subsequently mountedon glassslides, whichwere stained withhematoxylin–eosin(HE).Theslideswereanalyzedbyan experiencedpathologist.
Histopathologicalchangeswereclassifiedasmild, moder-ateandseveredysplasias.Milddysplasiawascharacterized as havingelongated, crowded and pseudo-stratified nuclei with preserved polarity and a normal or slightly reduced number of goblet cells.Moderate dysplasia was character-ized as having hyperchromatic proprieties and deformity of the cell nuclei, increased number of mitoses, thicken-ing of the glandular epithelium and an increased number ofimmune (defense) cells in the connectivetissue. Severe dysplasiawascharacterizedashavingbroad,roundorovoid nucleiwithprominentnucleoli,andatypicalmitoticfigures. In severe dysplasia, the nuclear polarity was partially lost andthenumberofgobletcellswassignificantlyreducedor completelydisappeared. Colorectal carcinoma is character-izedbyacompletelossofthemorphologicalcharacteristics ofthe tissue of origin and by the presence of signet ring cells.19,20
Results
Weightgain
Theanimalswereweighedweekly,fromthefirstinoculation untileuthanasia.Table1showstheweightofeachG1animal overthe15weeksoftheexperiment,andTable2showsthe weightofeachC1animalinthesameperiodoftime.Fig.1
comparestheweightevolutionofG1versusC1animalsinthe weeksofevaluation.Table3showstheweightofeachG2 ani-maloverthefourweeksoftheexperiment,andTable4shows theweightofC2animalsinthesameperiodoftime.Fig.2
comparestheweightevolutionofG2versusC2animalsinthe
weeksofevaluation. T
T able 2 – W eight gain, in gr ams, of eac h animal of C1 gr oup (contr ol) dur ing the tr ial per iod. Animals W eek 1 W eek 2 W eek 3 W eek 4 W eek 5 W eek 6 W eek 7 W eek 8 W eek 9 W eek 10 W eek 11 W eek 12 W eek 13 W eek 14 W eek 15 1 105.7 144.2 183.1 228.3 250.0 263.0 273.9 272.6 278.9 294.8 308.1 315.5 322.8 326.0 340.9 2 102.3 143.9 180.1 215.5 231.2 239.5 251.3 250.8 257.0 272.9 285.1 290.2 294.4 301.9 315.4 3 100.8 142.1 177.3 212.7 222.9 235.4 243.2 249.2 254.8 271.8 281.3 287.7 290.5 298.6 313.3 4 96.9 132.4 167.2 199.8 212.2 226.9 230.6 242.9 249.9 260.1 276.4 284.5 287.5 294.5 304.9 5 93.4 132.4 165.1 194.6 203.1 214.7 222.3 230.5 235.8 245.3 259.8 264.9 264.1 273.2 286.4 Mean 99.8 139.0 174.6 210.2 223.9 235.9 244.3 249.2 255.3 269.0 282.1 288.6 291.9 298.8 312.2
Table3–Weightgain,ingrams,ofeachanimalofG2
groupduringthetrialperiod.
Animals Week1 Week2 Week3 Week4
1 174.5 128.0 193.6 218.8
2 174.8 145.3 164.3 196.6
3 168.8 168.6 196.2 212.1
4 155.1 164.7 198.8 215.4
5 154.5 158.4 160.5 187.3
6 156.8 135.5 165.5 186.5
7 152.5 146.3 161.6 188.8
8 180.0 183.0 194.4 236.3
9 209.0 216.0 220.2 253.6
10 197.0 157.0 191.6 236.1
Mean 172.3 160.3 184.7 213.2
Table4–Weightgain,ingrams,ofeachanimalofC2
controlgroupduringthetrialperiod.
Animals Week1 Week2 Week3 Week4
1 191.0 226.0 245.6 257.6
2 202.0 234.0 254.5 272.3
3 218.0 252.0 290.7 318.7
4 164.5 190.2 217.7 229.9
5 170.7 198.1 211.5 223.6
Mean 189.2 220.1 244.0 260.4
Histologicalanalysis
Nomacroscopiclesionincolorectaltissueofanyanimalwas found.
Theanimalsinthecontrolgroupsshowednormal colorec-taltissue.Intheslidesstudied,thehomogeneouspatternof staininginthenucleiwasmaintained,aswellasitsbasal loca-tion.Nomitotictissuechangeswereobserved,aswellasinthe sizeorshapeoftheglands,whichremaineduniform(Fig.3).
G2animalsshowedchangesconsistentwithmoderate dys-plasia(Fig.4).InG1slides,areasofhyperplasia(Fig.5),mild dysplasia(Fig.6),severedysplasia(Fig.7)andcarcinoma(Fig.8) wereobserved.
Discussion
Intheweightevolutioninthecontrolgroups(C1andC2),an increasingweightgainoccurred.
InG2group,i.e.thoseanimalsthatreceivedAOM,therewas aweightlossinthefirstweekand,thereafter,weightgain. Probablythis wasduetothemetabolismofthis substance, thatactsdirectlyincarcinogenesis.15
AdifferenceinweightevolutionofG1andC1wasnoted, comparedtoG2andC2.InthegroupthatreceivedDMH(G1), althoughtherehasbeennoweightloss,weightgainwas con-sistentlylowerthaninthecontrolgroup.Thisweightbehavior wasnotanalyzedinsimilarstudies;howevertheresearch con-firmdifferentmetabolismsbetweenDMHandAOM,whichcan generatefurtherresearchtojustifysuchdevelopments.15,21
G1, 1, 92.8 G1, 2, 118.7
G1, 3, 135.5 G1, 4, 161.0
G1, 5, 182.3 G1, 6, 193.1
G1, 7, 206.2 G1, 8, 211.8 G1, 9, 215.7
G1, 10, 233.4 G1, 11, 246.4
G1, 12, 248.8 G1, 13, 255.1 G1, 14, 259.6 G1, 15, 272.5
C1, 1, 99.8
C1, 3, 174.6
C1, 5, 223.9 C1, 6, 235.9
C1, 7, 244.3 C1, 8, 249.2 C1, 9, 255.3
C1, 10, 269.0 C1, 11, 282.1
C1, 12, 288.6 C1, 13, 291.9 C1, 14, 298.8 C1, 15, 312.2
G1
C1
Weight (g)
Time (weeks) C1, 2, 39.0
C1, 4, 210.2
Fig.1–Comparativeweightevolution,ingrams,betweenG1andC1groupsinthefifteenweeksofevaluation.
Weight (g)
G2,1,172.3 C2,1,189.2
G2,2,160.3 C2,2,220.1
Time (weeks)
G2,3,184.7 C2,3,244.0
G2,4,213.2 C2,4,260.4
G2
C2
Fig.2–Comparativeweightevolution,ingrams,betweenG2andC2groupsinthefourweeksofevaluation.
Fig.3–PhotomicrographofcolonictissuestainedwithHE, representinganormalpatternofcontrolgroups.
inoculation andeuthanasia,sinceother studies have iden-tified carcinoma when the time elapsed was superior to ours.15,19,22,23
The analysis of G1 slides showed histological changes in differentstages, which confirmed the high carcinogenic capacityofDMH.7,13,15,18,21,24
Itisknownthatthefinalamountofcarcinogenfoundin the tissues isa functionofactivity of themetabolic path-waysleadingtoitsformation,oftheactivityofdetoxification pathways, aswell as thehalf-livesof all biologicalspecies involved.25ConsideringthatinthemethodologyofDMH car-cinogenesis 15 weeksshould elapse for the final analysis, therewasmoretimefortheevolutionoflesions,which con-firmstheimportanceofgeneticandenvironmentalfactorsin carcinogenesis.12
Fig.4–PhotomicrographofcolonictissuestainedwithHE, showingapatternofmoderatedysplasia(G2group).
Fig.5–PhotomicrographofcolonictissuestainedwithHE, showinghyperplasiaofglandularepithelium(G1group).
Fig.6–PhotomicrographofcolonictissuestainedwithHE, withmilddysplasia(G1group).
Fig.7–PhotomicrographofcolonictissuestainedwithHE, exhibitingseveredysplasia(G1group).
Fig.8–PhotomicrographofcolonictissuestainedwithHE, featuringcarcinoma–thearrowsignalsasignetringcell (G1group).
someauthorsareusingsubstancestoinhibitthecarcinogenic process.5,22,24,26,27However,thecostofsubstances–alimiting factorofthefeasibilityofanexperiment–isnotbeing ana-lyzednorvalued.
WheninthisstudywecomparedthecostofAOMversus DMH,thelatterwasmorethan50timeslessexpensivethan AOM.EvenrequiringmorecarcinogenesistimewithDMHand, consequently,anhighermaintenancecostofanimals,theuse ofAOMdidnotpayoff.
Themethodologyusedconfirmedthatthefiveweeks’time ofinoculationfollowedbytenweeksofobservationis suffi-cientforcompletionofexperimentalcarcinogenesisbyDMH.
Conclusion
thatleadstothedevelopmentofcolorectalcancer,being con-sideredagoodcolorectalcarcinogen.
Timeisapreponderantfactorforevidencingthe histologi-calchanges.Neoplasiaandseveredysplasiawereproducedby DMHwhich,comparedtoAOM,wasmoreefficientininducing colorectalcancer.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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