w w w. s b f g n o s i a . o r g . b r / r e v i s t a
Original
Article
Evaluation
of
mutagenicity
and
metabolism-mediated
cytotoxicity
of
the
naphthoquinone
5-methoxy-3,4-dehydroxanthomegnin
from
Paepalanthus
latipes
Rodrigo
R.
Kitagawa
a,∗,
Wagner
Vilegas
b,
Eliana
A.
Varanda
c,
Maria
S.G.
Raddi
d aDepartamentodeCiênciasFarmaceuticas,UniversidadeFederaldoEspíritoSanto,Vitória,ES,BrazilbInstitutodeQuímicadeAraraquara,UniversidadeEstadualPaulista“JúliodeMesquitaFilho”,Araraquara,SP,Brazil
cDepartamentodeCiênciasBiologicas,FaculdadedeCiênciasFarmaceuticasdeAraraquara,UniversidadeEstadualPaulista“JúliodeMesquitaFilho”,Araraquara,SP,Brazil dDepartamentodeAnálisesClinicas,FaculdadedeCiênciasFarmaceuticaldeAraraquara,UniversidadeEstadualPaulista“JúliodeMesquitaFilho”,Araraquara,SP,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received22April2014 Accepted19December2014 Availableonline12February2015
Keywords: Cytotoxicity
5-Methoxy-3,4-dehydroxanthomegnin Mutagenicity
a
b
s
t
r
a
c
t
Alargenumberofquinoneshavebeenassociatedwithantitumor,antibacterial,antimalarial,and anti-fungalactivities.Resultsofpreviousstudiesof5-methoxy-3,4-dehydroxanthomegnin,anaphthoquinone isolatedfromPaepalanthuslatipesSilveira,Eriocaulaceae,revealedantitumor,antibacterial, immunomod-ulatory,andantioxidantactivities.Inthisstudy,weassessedthemutagenicityandmetabolism-mediated cytotoxicityof5-methoxy-3,4-dehydroxanthomegninbyusingtheAmestestandamicroculture neu-tralredassayincorporatinganS9fraction(hepaticmicrosomalfractionandcofactors),respectively. WealsoevaluatedthemutagenicactivityinSalmonellatyphimuriumstrainsTA100,TA98,TA102,and TA97a,aswellasthecytotoxiceffectonMcCoycellswithandwithoutmetabolicactivationinboth tests.Resultsindicatedthatnaphthoquinonedoesnotcausemutationsbysubstitutionorbyaddition anddeletionofbasesinthedeoxyribonucleicacidsequencewithandwithoutmetabolicactivation.As previouslydemonstrated,theinvitrocytotoxicityof5-methoxy-3,4-dehydroxanthomegnintoMcCoy cellsshowedasignificantcytotoxicindex(CI50)of11.9g/ml.Thisindexwasnotalteredbyadditionof theS9fraction,indicatingthattheS9mixturefailedtometabolicallymodifythecompound.Ourresults, alliedwithmorespecificbiologicalassaysinthefuture,wouldcontributetothesafeuseof 5-methoxy-3,4-dehydroxanthomegnin,compoundthathasshowedinpreviousstudiesbeneficialpropertiesasa potentialanticancerdrug.
©2014SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.
Introduction
“For many centuries, plants have provided a rich source of
therapeutic agents and bases for synthetic drugs. Despite the
developmentof organic synthesis, 25% of thedrugs prescribed
worldwide are derived from plant sources, showing that plant
speciesarestillanimportantsourceofnewdrugs”(Sacomanetal.,
2008).Muchresearchhasbeenconducted onplants inpopular
use,withtheobjectiveofidentifyingnaturalproductswith
thera-peuticpotential(BalunasandKinghorn,2005;Gurib-Fakim,2006;
NewmanandCragg,2012).
∗ Correspondingauthorat:DepartmentofPharmaceuticalSciences,Federal
Uni-versityofEspíritoSanto–UFES,AvenidaMarechalCampos1468,29043-900Vitória, ES,Brazil.
E-mail:rodrigo.kitagawa@ufes.br(R.R.Kitagawa).
TheEriocaulaceaefamily,commonlyfoundintheStatesofBahia
andMinasGerais,Brazil,hasbeenthesourceofseveral
biolog-ically active compounds. Paepalanthus,with about 500species,
isoneof itsprincipalgenus.Anumberof studieshave
demon-stratedthatalmostallspeciesofPaepalanthussubgenusPlatycaulon
possessnaphthopyranonederivatives,includingpaepalantineand
8,8′-paepalantinedimer isolatedfromPaepalanthusbromelioides
(Vilegas et al., 1990; Coelho et al., 2000), planifolin isolated fromPaepalanthusplanifolius(Santosetal.,2001;Varandaetal.,
2006), and 5-methoxy-3,4-dehydroxanthomegnin isolated from
PaepalanthuslatipesSilveira,Eriocaulaceae,(Kitagawaetal.,2004, 2008).
The compound 5-methoxy-3,4-dehydroxanthomegnin (1) is
a naphthoquinone with potentialtherapeutic applications.
Pre-vious studies have shown that this compound has
antitu-mor and immunomodulatory effects (Kitagawa et al., 2011),
as well as anti-Helicobacter pylori and antioxidant properties
http://dx.doi.org/10.1016/j.bjp.2014.12.001
(Kitagawa et al.,2012).Theantitumoreffect of
5-methoxy-3,4-dehydroxanthomegnin may be enhanced by association with
ascorbic acid as demonstrated by a significant cytotoxic index
(CI) for McCoy cells. The enhanced effect is probably due to
hydrogenperoxidegeneratedbyascorbate-driven
5-methoxy-3,4-dehydroxanthomegninredoxcycling(Kitagawaetal.,2008).
Currently, interest is focused on cytotoxic compounds that
appeartoexerta beneficialeffectonkey mechanismsinvolved
inthepathogenesisofcancerandinfectiondiseases.Manyactive
compoundsthatworkbyinterferingwiththefunctionofDNAseem
toplayadecisiveroleinantitumoractivity(Harvey,2008;Maand
Wang,2009;SudanandRupasinghe,2014).
Short-termteststhatdetectgeneticdamagehaveallowed
eval-uationofthecarcinogenicrisksofchemicalstohumans.TheAmes
assay, which is recommended for testing the mutagenicity of
chemicalcompoundswithpotentialpharmacologicalapplications
(Varandaetal.,2006;Resendeetal.,2012;Aardema,2013),was
usedinthepresentstudytoevaluateinvitrothemutageniceffect
of5-methoxy-3,4-dehydroxanthomegnin(1).
In vitro cytotoxicity tests simulate injury to cells from the
testedsubstances,whichmaybecausedbyanumberof
incom-pletemechanisms,duringperiodsofexposurethatarerealisticfor
acutetoxicity(Benbowetal.,2010).Thecentralpointregarding
in vitro/in vivo comparisons refers to xenobiotic-metabolizing
capacity. Bioactivationis an importantconsideration in invitro
cytotoxicity assays, since in vivo the test agent may be
bio-transformed. The incorporation of the S9 microsomal fraction
has been used in the study of metabolic activation of
chem-icals in a variety of cell culture models (Soares et al., 2006;
Ponsoni et al., 2013).The test is applicable to the analysis of
toxic ranges, for the detection of biotransformation of parent
compounds, and for the evaluation of the cytotoxic effects of
chemotherapeuticagents(BorenfreundandPuerner,1987).Inthis
study,weinvestigatedinvitrometabolism-mediatedcytotoxicity
of5-methoxy-3,4-dehydroxanthomegninusingtheS9system.For
comparison,weevaluatedcyclophosphamide,anindirect-acting
cytotoxicantemployedinantineoplastictherapy,asthecontrolof
biotransformationinductionbythehepaticS9fraction(Hilland
Hill,1984).
Materialandmethods
Plantmaterial
Paepalanthus latipes Silveira, Eriocaulaceae, was collected at
Serrado Cipóin theCadeiado Espinhac¸o, MinasGerais, Brazil,
andauthenticatedbyProfessorPauloTakeoSanofromthe
Insti-tuteofBiosciences,UniversityofSãoPaulo.Thevoucherspecimen
(CFSC, 13846) is on file at the Herbarium in the Department
of Botany, Institute of Biosciences, University of São Paulo,
Brazil.
Chemicalsandculturemedia
Eagle medium was purchased from Adolfo Lutz (São Paulo,
Brazil),andfetal bovineserumfromCultilab(Campinas,Brazil).
Dimethyl sulfoxide (DMSO), nicotinamide adeninedinucleotide
phosphatesodiumsalt(NADP),d-glucose-6-phosphatedisodium
salt, magnesium chloride (MgCl2), l-histidine monohydrate, d
-biotin, sodiumazide, 2-anthramine, and 2-aminofluorene were
purchasedfromSigmaChemicalCo.(St.Louis,MO,USA).Oxoid
NutrientBrothNo.2(Oxoid;Basingstoke,UK)andBactoAgar(BD
BactoTM;Sparks,MD,USA)wereusedasbacterialmedia.d-glucose,
magnesium sulfate, citric acidmonohydrate, anhydrousdibasic
potassiumphosphate,sodiumammoniumphosphate,monobasic
sodiumphosphate,dibasicsodiumphosphate,andsodium
chlo-ridewerepurchasedfromMerck(WhitehouseStation, NJ,USA).
Neutralred(NR)wasobtainedfromRiedel-de-HaënAG(Seelze,
Hannover,Germany).The5-methoxy-3,4-dehydroxanthomegnin,
isolated and characterized as previously described (Kitagawa
et al., 2004), was stored as stock solution at 10mg/ml in
DMSO.
Metabolicactivationsystem(S9mixture)
The S9 fraction, prepared from livers of Sprague-Dawley
ratstreated with thepolychlorinatedbiphenyl mixtureAroclor
1254(500mg/kg),waspurchasedfromMolecularToxicology,Inc.
(Boone,NC,USA).ThemetabolicactivationsystemconsistedofS9
fraction(4%),0.4MMgCl2 (1%),1.65MKCl (1%),1Md
-glucose-6-phosphatedisodium(0.5%),0.1MNADP(4%),0.2Mphosphate
buffer(50%),andsteriledistilledwater(39.5%)(MaronandAmes,
1983).
Salmonellamutagenicassay
MutagenicactivitywastestedbySalmonella/microsomeassay,
using the Salmonella typhimurium tester strains TA97a, TA98,
TA100, and TA102 (kindly provided by B.N. Ames; Berkeley,
CA, USA), with and without metabolization by the
preincuba-tion method(Maronand Ames, 1983).The strainsfrom frozen
cultures were grown overnight for 12–14h in Oxoid Nutrient
Broth No.2. The metabolicactivation mixture(S9) wasfreshly
preparedbeforeeachtest.Variousconcentrationsof
5-methoxy-3,4-dehydroxanthomegnindissolvedinDMSOweretested:62.5,
125.0,250.0,500.0,and750.0g/plateforstrainsTA98,TA97a,and
TA100;6.25,12.5,25.0,50.0,and75.0g/plateforstrainTA102.
Theseconcentrationswereselectedbasedonapreliminary
toxic-itytest.Inallsubsequentassays,theupperlimitofthedoserange
testedwaseitherthehighestnontoxic doseorthelowesttoxic
dosedeterminedinthispreliminaryassay.Toxicitywasapparent
asareductioninthenumberofHis+revertantsorasanalteration
intheauxotrophicbackground(i.e.,backgroundlawn).The
vari-ousconcentrationsof5-methoxy-3,4-dehydroxanthomegninwere
addedto0.5mlof0.2Mphosphatebuffer(pH7.4)orto0.5mlof
4%S9mixturecombinedwith0.1mlofbacterialcultureandthen
incubatedat37◦Cfor20–30min.Afterthistime,2mloftopagar
wasadded tothemixtureandpoured ontoaplate containing
minimalagar.Theplateswereincubatedat37◦Cfor48handthe
revertantcolonieswerecountedmanually.Allexperimentswere
analyzedintriplicate.TheresultswereanalyzedwiththeSalanal
statisticalsoftwarepackage,adoptingtheBernsteinetal.(1982)
model. Thedata(revertants/plate)were assessedbyanalysisof
variance(ANOVA),followedbylinearregression.Themutagenic
index(MI)wasalsocalculatedforeachconcentrationtested,this
beingtheaveragenumberof revertantsperplatewiththetest
compounddividedbytheaveragenumberofrevertantsperplate
withthenegative(solvent)control.Asamplewasconsidered
muta-genicwhenadose–responserelationshipwasdetectedanda2-fold
increaseinthenumberofmutants(MI≥2)wasobservedwithat
least1concentration(Santosetal.,2006).Thestandardmutagens
used as positive controls in experiments without the S9
Table1
Mutagenicactivityandmutagenicindex(MI)of5-methoxy-3,4-dehydroxanthomegnin(naphthoquinone)atvariousconcentrationsinstrainsTA100,TA98,andTA97aof Salmonellatyphimuriuminthepresence(+S9)andabsence(−S9)ofmetabolicactivation.
Naphthoquinone (g/plate)
Numberofrevertants/plateand(MI)
TA100 TA98 TA97a
−S9 +S9 −S9 +S9 −S9 +S9
0 147±22 121±13 22±3 26±1 218±2 343±6
62.5 151±9(1.08) 199±33(1.32) 21±4(1.2) 28±2(1.27) 304±28(1.2) 332±27(1.05) 125 151±8(1.08) 176±9(1.17) 18±3(1.05) 25±2(1.13) 277±12(1.1) 399±10(1.26) 250 154±12(1.10) 185±30(1.23) 17±2(1.0) 26±2(1.18) 309±22(1.25) 476±16(1.51) 500 131±14(0.94) 185±19(1.23) 17±3(1.0) 27±3(1.22) 301±10(1.21) 398±26(1.26) 750 124±10(0.88) 216±53(1.44) 19±2(1.1) 27±4(1.22) 294±16(1.19) 431±25(1.36)
Control+ 957±24 973±10 1492±28 2002±60 1484±32 1521±31
0=negativecontrol:75.0lDMSO.
Positivecontrols:+S9→2-anthramine(2.5g/plate)forTA100,TA98andTA97astrains.−S9→sodiumazide(1.25g/plate)forTA100;4nitro-o-phenylenediamine (10.0g/plate)forTA98andTA97a.Theresultsarereportedasmeans±SD.
*p<0.05(ANOVA)5-methoxy-3,4-dehydroxanthomegninconcentrationscomparedwithnegativecontrol.
andTA97a,sodiumazide(1.25g/plate)for TA100,and dauno-mycin(3.0g/plate)forTA102.Intheexperimentswithmetabolic activation,2-anthramine(2.5g/plate)wasusedwithTA98,TA97a, and TA100, and 2-aminofluorene (10.0g/plate) with TA102. DMSOservedasthenegative(solvent)control(75.0l/plate).
Cytotoxicityassay
McCoy cells (CCL1–ATCC/USA, from the cell culture section oftheAdolfoLutzInstitute,São Paulo,Brazil)weremaintained inEaglemediumwith7.5%fetalbovineserum.After trypsiniza-tion,0.2mlofmediumcontainingapproximately104cells/mlwere
seeded into 96-well microtiter tissue culture plates and incu-batedat 37◦C. After24h, theEagle mediumwasremoved and the cells were placed in unmodified medium (control) or in mediummodifiedwithvariousconcentrationsof 5-methoxy-3,4-dehydroxanthomegnin(5,10, 20,40, 50,80, and 100g/ml)or cyclophosphamide(25,50,100,150g/ml)withandwithoutthe S9mixtureat10%.Afterincubatingforanother24h,themedium was removed and the plates were prepared for the NR assay (BorenfreundandPuerner,1985).Afterbriefagitation,theplates
weretransferred toa microplate reader (Spectra and Rainbow
[Shell]Readers;Tecan, Austria)and the opticaldensity ofeach
wellwasmeasuredusinga540nmfilteranda620nmreference
wavelength.Allexperimentswereperformedatleastfourtimes,
usingthreewellsforeachconcentrationofcompoundtested.The
cytotoxicitydatawerestandardized bydeterminingabsorbance
andcalculatingthecorrespondingchemicalconcentrations.Linear
regressionanalysiswitha95%confidencelimitwasusedtodefine
dose–responsecurvesandtocomputetheconcentrationsof
chem-icalagentsneededtoreduceabsorbanceoftheNRby50%(IC50),
calledcytotoxicmidpoint(Barile,1994).
Results
Tables1and2showthemutagenicityresultsfor
5-methoxy-3,4-dehydroxanthomegninwhich demonstrate that this compound
doesnotpossessmutagenicactivityforthestrainsTA100,TA97a,
and TA98, with MI less than 2.0 at all tested concentrations
with or without metabolic activation. The TA102 strain was
foundto be more sensitiveto the toxic effects of
5-methoxy-3,4-dehydroxanthomegnin;thus,itwasnecessarytodecreasethe
concentrationsusedwithTA102relativetotheotherstrains. In
fact,thelowestconcentrationusedintheexperimentswithTA100,
TA97a,andTA98wasclosetothehighestconcentrationusedwith
Table2
Mutagenic activity and mutagenic index (MI) of 5-methoxy-3,4-dehydroxanthomegnin (naphthoquinone) at various concentrations in strain TA102ofSalmonellatyphimurium inthepresence(+S9)andabsence(−S9)of metabolicactivation.
Naphthoquinone(g/plate) Numberofrevertants/plateand(MI)
TA102
−S9 +S9
0 400±10 301±5
6.25 355±6(0,93) 292±17(0.92) 12.5 401±8(1.05) 295±38(0.93)
25 384±5(1.01) 307±8(0.96)
50 389±23(1.02) 288±15(0.90)
75 379±30(1.0) 282±22(0.88)
Control+ 1157±54 1348±66
0=negativecontrol:75.0lDMSO.
Positive controls: +S9→2-aminofluorene (10.0g/plate). −S9→daunomycin (3.0g/plate).Theresultsarereportedasmeans±SD.
* p<0.05 (ANOVA)5-methoxy-3,4-dehydroxanthomegnin concentrations com-paredwithnegativecontrol.
TA102.Nevertheless,theTA102straindidnotdisplaymutagenicity intheabsenceorpresenceofmetabolicactivation.
Theconcentration–responsecurveforcytotoxicitywas estab-lishedfor5-methoxy-3,4-dehydroxanthomegnininthepresence and absence of an S9 mixture as an external metabolizing system (Fig. 1). Table 3 shows the IC50 of
5-methoxy-3,4-dehydroxanthomegninandcyclophosphamide(control)forMcCoy
cellsinthepresenceandabsenceoftheS9system.TheIC50obtained
for5-methoxy-3,4-dehydroxanthomegnininthepresenceoftheS9
mixturedidnotdifferfromthatattainedwithoutthemetabolizing
system.ExposureoftheMcCoycellstocyclophosphamidewithand
withoutS9confirmstheefficacyofthehepaticmicrosomalfraction
forinvitrometabolicactivationassay.
Table3
Cytotoxic midpoint (g/ml) on McCoy cells for
5-methoxy-3,4-dehydroxanthomegninandcyclophosphamidewithoutandwiththeS9metabolic activation.
Compound WithoutS9a WithS9a
5-Methoxy-3,4-dehydroxanthomegnin 11.9±1.15 10.08±0.38 Cyclophosphamide >150 21.6±1.7
0 20 40 60 80 100 120 140 160 0.0
0.1 0.2 0.3 0.4
ABS
(540
/620
nm
)
Concentration (µg/ml)
0 5 10 15 20 25 30
0.0 0.1 0.2 0.3 0.4 0.5
ABS
(
540
/620
n
m)
Concentration (µg/ml)
A
B
Fig.1.Concentration-effectrelationshipof5-methoxy-3,4-dehydroxanthomegnin(A)andcyclophosphamide(B)onMcCoycellswith(redline)andwithouttheS9(black line)metabolicactivationsystem.Eachpointandbarrepresentsthemean±SDforatleastthreeindependentexperimentscarriedoutintriplicate.(Forinterpretationofthe referencestocolorinthistext,thereaderisreferredtothewebversionofthearticle.)
Discussion
During the past few decades, plant research has revealed
several chemical compounds with important pharmacological
activities. Some of these compounds have been incorporated
intodrugssuchasantineoplastics-vinblastineandvincristine
iso-latedfromCatharanthusroseus,camptothecinderivativesobtained
fromCamptothecaacuminata,derivativesofpodophyllotoxinfrom
the rhizomes of Podophyllum peltatum and P. hexandrum, and
taxolextractedfromTaxusbrevifolia(CraggandNewman,2005;
Srivastava et al., 2005; Basmadjian et al., 2014).However, the
literaturealsodescribesmanyplantscontainingmutagenic
com-pounds,suchasfurocoumarins,tannins,anthraquinones,alkaloids,
andflavonoids(Rietjensetal.,2005;Nesslanyetal.,2009;Guterres
etal.,2013;Minineletal.,2014).Thisevidencedrawsattentionto
theimportanceofstudyingthegeneticrisksofplantcompounds,
sincethepresenceofmutagensinmedicinescanbedangerousto
humanhealth.
Previousstudieshaveshownthatspeciesbelongingto
Paepalan-thussubgenusPlatycaulonpossessnaphthopyranonederivatives.
Thenaphthopyranone paepalantine isolatedfrom P.vellozioides
exhibited strong mutagenicityand cytotoxicity (Varanda et al.,
1997).Thegenotoxicpotentialofpaepalantinewasalso
demon-stratedbyTavaresetal.(1999)ininvivoassayswithbonemarrow
cellsofWistarrats.Themutagenicactivityofplanifolinisolated
from P. planifolius was tested through Salmonella/microsome
assays,withresultsindicatingthatthisnaphthopyranonedimer
caused mutationsbysubstitution and by additionand deletion
ofbasesintheDNAsequence.Moreover,itsmutagenicpotential
increasedinthepresenceofmetabolization.Theseresults,alliedto
thechemicalstructure,suggestthatplanifolinmayactasan
inter-calatingagentintheDNAmolecule(Varandaetal.,2006).Inthe
presentstudy,weassessedthemutagenicactivityof
5-methoxy-3,4-dehydroxanthomegnin(1)usingSalmonella/microsomeassays.
The compound 5-methoxy-3,4-dehydroxanthomegnin was not
mutagenictostrainTA98,whichisusedtodetectframeshift
muta-tions.SubstitutionofDNAbasesinstrainTA100andoxidativeDNA
damageinstrainTA102werenotdetected.Nomutagenicitywas
observedintheTA97astrain,whichdetectsframeshiftmutations
that are sensitive to heavy metal mutagens. The results were
alsonegativeforalltheS.typhimuriumstrainstestedwiththeS9
mixture.
However, data reported in the literature reveal that some
quinones,includingnaphthoquinones,presentmutagenicityafter
metabolization. Chesis et al. (1984) concluded that the
muta-genicityof quinoneswasmainlydue toone-electron reduction
of quinones to semiquinones via the formation of
superox-ide anion radical (O2• −)and, subsequently, hydrogen peroxide
(H2O2). Tikkanen et al. (1983) observed that naphthoquinones
with1or2hydroxyl and/ormethylsubstituents aremutagenic
withmetabolicactivation.Nevertheless,it seemsthat the
posi-tion of substituents, as well as the number of substituents, is
important for mutagenicity. This point seems to explain the
absenceofmutagenicityof5-methoxy-3,4-dehydroxanthomegnin,
which has hydroxyl and methyl substituents and did not
present mutagenicity in tests with or without
metaboliza-tion.
Considerableinteresthasbeenfocusedonshort-terminvitro
cytotoxicityassayswithculturedcellsforevaluationofacute
tox-icitiesofchemicalagentsandpilotstudiesindrugdevelopment.
Suchassayswouldnotonlycurtailtheuseofanimalsformedian
lethaldose(LD50)andsimilartests,butwouldserveasan
econom-icalapproachtotherapidscreeningofxenobiotics(Greeneetal.,
2010).
Cytotoxicityduetodirect-actingchemicalsisreadily
demon-strable in vitro. However, the toxicity of many chemicals is
dependent upon metabolic activation, usually catalyzed by the
microsomal cytochrome P-450-dependent monooxygenase
sys-tem, and the majority of cell lines currently used in in vitro
cytotoxicitytestspossesslittle intrinsicdrug-metabolicactivity.
Consequently,problems arisewhenmetabolism-mediated
cyto-toxic events are studied in vitro. One possible answer to this
problemisthecoincubationofculturedcells withmetabolically
activerodentliverfractions,inamannersimilartotheirusein
invitromutagenesisassays(Gonzalez,2005;Lietal.,2012;Cole etal.,2014).
In previous studies, 5-methoxy-3,4-dehydroxanthomegnin
showedsignificantinvitrocytotoxicitytoMcCoycellsintheNR
microculturetest comparedwithcis-diamminedichloroplatinum
(cisplatin),oneofthemostwidelyusedchemotherapeuticdrugs
(Kitagawaetal.,2004).Ourresultsindicatedthatcombined
treat-mentwith5-methoxy-3,4-dehydroxanthomegninandhepaticS9
microsomalfractiondidnotalterthecytotoxicityofthis
One hypothesis explaining the same cytotoxic potential of
5-methoxy-3,4-dehydroxanthomegnin withand without theS9
microsomalsystemisthatthiscompounddoesnotactasa
sub-stratefortheenzymaticsystem.Previousstudiesexploredredox
cycling of 5-methoxy-3,4-dehydroxanthomegnin in a
nonenzy-matic system.In this study, we usedthe NRassay toevaluate
the ability of ascorbic acid associated with
5-methoxy-3,4-dehydroxanthomegnin to cause cell death in the same cell
line. The synergic effect of ascorbic acid on
5-methoxy-3,4-dehydroxanthomegnin(1)resultedinaCIthatwasseventimes
lowerthan the index for 5-methoxy-3,4-dehydroxanthomegnin
aloneaddedtotheMcCoycellline.Theobservedsynergiceffect
wasmostprobablydue toH2O2 generated byascorbate-driven
5-methoxy-3,4-dehydroxanthomegnin redox cycling (Kitagawa
et al., 2008), indicating that the association of
5-methoxy-3,4-dehydroxanthomegninwithascorbicacidmaybepromisinginthe
treatmentofsolidtumorsthataredeficientinantioxidantdefenses.
The resultsof this study investigating the mutagenic
activ-ity and metabolism-mediated cytotoxicity of
5-methoxy-3,4-dehydroxanthomegnin, allied in the future with more specific
biologicalassays, willcontribute tothesafeuseof
5-methoxy-3,4-dehydroxanthomegnin,signifyingitsbeneficialpropertiesas
apotentialanticancerdrug.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Authors’contributions
R.R.K.contributedtorunningthelaboratoryworkinisolation
and identification of the 5-methoxy-3,4-dehydroxanthomegnin,
biologicalstudies,analysisofthedata,anddraftedthepaper.W.V.
supervisedthelaboratoryworkintheisolationandidentification
ofthe5-methoxy-3,4-dehydroxanthomegnin andcontributedto
criticalreading ofthemanuscript. E.A.V. supervisedthe
labora-toryworkinAmesTestandcontributedtocriticalreadingofthe
manuscript.M.S.G.R.designedthestudy,supervisedthelaboratory
workinthecytotoxicitytest,andcontributedtocriticalreadingof
themanuscript.Alltheauthorshavereadthefinalmanuscriptand
approvedthesubmission.
Acknowledgements
ThisstudywassupportedbyagrantofCNPqtoR.R.K.,toFAPESP
forfinancialaidtoW.V.andfinancialassistancefromthe
PADC-UNESP.
References
Aardema,M.J.,2013.Theholygrailingenetictoxicology:follow-upapproachesfor positiveresultsintheAmesassay.Environ.Mol.Mutagen.54,617–620. Balunas,M.A.,Kinghorn,A.D.,2005.Drugdiscoveryfrommedicinalplants.LifeSci.
78,431–441.
Barile,F.A.,1994.IntroductiontoInVitroCytotoxicology:MechanismsandMethods. CRCPress,Inc.,BocaRaton.
Basmadjian,C.,Zhao,Q.,Bentouhami,E.,Djehal,A.,Nebigil,C.G.,Johnson,R.A., Serova,M.,Gramont,A.,Faivre,S.,Raymond,E.,Désaubry,L.G.,2014.Cancer wars:naturalproductsstrikeback.Front.Chem.2,1–18.
Benbow,J.W.,Aubrecht,J.,Banker,M.J.,Nettleton,D.,Aleo,M.D.,2010.Predicting safetytolerationofpharmaceuticalchemicalleads:cytotoxicitycorrelationsto exploratorytoxicitystudies.Toxicol.Lett.197,175–182.
Bernstein,L.,Kaldor,J.,Mccann,J.,Pike,M.C.,1982.Anempiricalapproachtothe statisticalanalysisofmutagenesisdatafromtheSalmonellatest.Mutat.Res.97, 267–281.
Borenfreund,E.,Puerner,J.A.,1985.Toxicitydeterminedinvitrobymorphological alterationsandneutralredabsorption.Toxicol.Lett.24,119–124.
Borenfreund,E.,Puerner,J.A.,1987.Short-termquantitativeinvitrocytotoxicity assayinvolvinganS-9activatingsystem.CancerLett.34,243–248.
Chesis,P.L.,Levin,D.E.,Smith,M.T.,Emster,L.,Ames,B.N.,1984.Mutagenicityof quinones:pathwaysofmetabolicactivationanddetoxification.Proc.Natl.Acad. Sci.81,1696–1700.
Coelho, R.G., Vilegas, W., Devienne, K.F., Raddi, M.S.G., 2000. A new cyto-toxicnaphthopyronedimer fromPaepalanthusbromelioides. Fitoterapia71, 497–500.
Cole,S.D.,Madren-Whalley,J.S.,Li,A.P.,Dorsey,R.,Salem,H.,2014.Highcontent analysisofaninvitromodelformetabolictoxicity:resultswiththemodel toxi-cants4-aminophenolandcyclophosphamide.J.Biomol.Screen.19,1402–1408. Cragg, G.M., Newman,D.J., 2005. Plants asa source ofanti-cancer agents. J.
Ethnopharmacol.100,72–79.
Gonzalez,F.J.,2005.RoleofcytochromesP450inchemicaltoxicityandoxidative stress:studieswithCYP2E1.Mutat.Res.569,101–110.
Greene,N.,Aleo,M.D.,Louise-May,S.,Price,D.A.,Will,Y.,2010.Usinganinvitro cytotoxicityassaytoaidincompoundselectionforinvivosafetystudies.Bioorg. Med.Chem.Lett.20,5308–5312.
Gurib-Fakim,A.,2006.Medicinalplants:traditionsofyesterdayanddrugsof tomor-row.Mol.Asp.Med.27,1–93.
Guterres,Z.R.,daSilva,A.F.,Garcez,W.S.,Garcez,F.R.,Fernandes,C.A.,Garcez,F.R., 2013.MutagenicityandrecombinagenicityofOcoteaacutifolia(Lauraceae) apor-phinoidalkaloids.Mutat.Res.757,91–96.
Harvey,A.L.,2008.Naturalproductsindrugdiscovery.DrugDiscov.Today13, 894–901.
Hill,H.Z.,Hill,G.L.,1984.Invitroactivationofcyclophosphamideforaninvitro chemosensitivityassay.J.Surg.Oncol.26,225–229.
Kitagawa, R.R., Raddi, M.S.G., Santos, L.C., Vilegas, W., 2004. A new cyto-toxic naphthoquinone from Paepalanthus latipes. Chem. Pharm. Bull. 52, 1487–1488.
Kitagawa,R.R.,Fonseca,L.M.,Ximenes,V.F.,Khalil,N.M.,Vilegas,W.,Raddi,M.S.G., 2008.Ascorbicacidpotentiatesthecytotoxicityofthenaphthoquinone 5-methoxy-3,4-dehydroxanthomegnin.Phytochemistry69,2205–2208. Kitagawa, R.R., Vilegas, W., Carlos, I.Z., Raddi, M.S.G., 2011. Antitumor and
immunomodulatory effects of the naphthoquinone 5-methoxy-3,4-dehydroxanthomegnin.Braz.J.Pharmacogn.21,1084–1088.
Kitagawa,R.R.,Bonacorsi,C.,Fonseca,L.M.,Vilegas,W.,Raddi,M.S.G.,2012. Anti-Helicobacterpyloriactivityandoxidativeburstinhibitionbythenaphthoquinone 5-methoxy-3,4-dehydroxanthomegninfromPaepalanthuslatipes.Braz.J. Phar-macogn.22,53–59.
Li,A.P.,Uzgare,A.,LaForge,Y.S.,2012.Definitionofmetabolism-dependent xenobio-tictoxicitywithco-culturesofhumanhepatocytesandmouse3T3fibroblasts inthenovelintegrateddiscretemultipleorganco-culture(IdMOC) experimen-talsystem:resultswithmodeltoxicantsaflatoxinB1,cyclophosphamideand tamoxifen.Chem.Biol.Interact.199,1–8.
Ma,X.,Wang,Z.,2009.Anticancerdrugdiscoveryinthefuture:anevolutionary perspective.DrugDiscov.Today14,1136–1142.
Maron,D.M.,Ames,B.N.,1983.RevisedmethodsfortheSalmonellamutagenicity test.Mutat.Res.113,173–225.
Mininel,F.J.,Junior,C.S.L.,Espanha,L.G.,Resende,F.A.,Varanda,E.A.,Leite,C.Q.F., Vilegas,W.,Santos,L.C.,2014.Characterizationandquantificationofcompounds inthehydroalcoholicextractoftheleavesfromTerminaliacatappaLinn (Com-bretaceae)andtheirmutagenicactivity.Evid.BasedCompl.Alt.,1–11. Nesslany,F.,Simar-Meintières, S.,Ficheux,H.,Marzin,D.,2009.
Aloe-emodin-inducedDNAfragmentationinthemouseinvivocometassay.Mutat.Res.678, 13–19.
Newman,D.J.,Cragg,G.M.,2012.Naturalproductsassourcesofnewdrugsoverthe 30yearsfrom1981to2010.J.Nat.Prod.75,311–335.
Ponsoni,K.,Raddi,M.S.G.,Almeida,D.V.,Almeida,A.E.,Alécio,A.C.,2013.Effectsof liverS9enzymesonsomalargineandsolasodinecytotoxicityandmass spectro-metricfragmentation.Eur.FoodRes.Technol.237,179–184.
Resende, F.A., Vilegas, W., Santos, L.C., Varanda, E.A., 2012. Mutagenicity of flavonoidsassayedbybacterialreversemutation(Ames)test.Molecules17, 5255–5268.
Rietjens,I.M.C.M.,Boersma,M.G.,Vanderwoude,H.,Jeurissen,S.M.F.,Schutte,E.M., Alink,G.M.,2005.Flavonoidsandakenylbenzenes:mechanismsofmutagenic actionandcarcinogenicrisk.Mutat.Res.574,124–138.
Sacoman,J.L.,Monteiro,K.M.,Possenti,A.,Figueira,G.M.,Foglio,M.A.,Carvalho,.J.E., 2008.Cytotoxicityandantitumoralactivityofdichloromethaneextractandits fractionsfromPothomorpheumbellata.Braz.J.Med.Biol.Res.41,411–415. Santos,L.C.,Piacente,S.,Pizza,C.,Albert,K.,Dachtler,M.,Vilegas,W.,2001.Planifolin,
anewnaphthopyranonedimerandXavonoidsfromPaepalanthusplanifolius.J. Nat.Prod.64,122–124.
Santos,F.V.,Colus,I.M.S.,Silva,M.A.,Vilegas,W.,Varanda,E.A.,2006.Assessment ofDNAdamageinducedbyextractsandfractionsofStrychnospseudoquina,a Brazilianmedicinalplantwithantiulcerogenicactivity.FoodChem.Toxicol.44, 1585–1589.
Soares,V.C.,Varanda,E.A.,Raddi, M.S.G.,2006.Invitrobasaland metabolism-mediatedcytotoxicityofflavonoids.FoodChem.Toxicol.44,835–838. Srivastava,V.,Negi,A.S.,Kumar,J.K.,Gupta,M.M.,Khanuja,S.P.S.,2005.Plant-based
anticancermolecules:achemicalandbiologicalprofileofsomeimportantleads. Bioorg.Med.Chem.13,5892–5908.
Sudan,S.,Rupasinghe,H.P.,2014.Quercetin-3-O-glucosideinduceshumanDNA topoisomeraseIIinhibition,cellcyclearrestandapoptosisinhepatocellular carcinomacells.AnticancerRes.34,1691–1699.
