Safrole-2 ',3 '-oxide induces cytotoxic and genotoxic effects in HepG2 cells and in mice

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Environmental

Mutagenesis

j

o

u r

n a l

h o m e p

a g

e :

w w w . e l s e v i e r . c o m / l o c a t e / g e n t o x

C o

m m

u n i t

y

a d d

r e

s s :

w w w . e l s e v i e r . c o m / l o c a t e / m u t r e s

Safrole-2



,3



-oxide

induces

cytotoxic

and

genotoxic

effects

in

HepG2

cells

and

in

mice

Su-yin

Chiang

a

,

∗

_,

_Pei-yi

_Lee

a

_,

_Ming-tsung

_Lai

b

,

c

_,

_Li-ching

_Shen

d

_,

_Wen-sheng

_Chung

d

_,

Hui-fen

Huang

a

_,

_Kuen-yuh

_Wu

e

_,

_Hsiu-ching

_Wu

f

a_{GraduateInstituteofChineseMedicine,ChinaMedicalUniversity,Taichung,Taiwan} b_{SchoolofMedicine,Chung-ShanMedicalUniversity,Taichung,Taiwan}

c_{DepartmentofPathology,Chung-ShanMedicalUniversityHospital,Taichung,Taiwan} d_{DepartmentofAppliedChemistry,NationalChiaoTungUniversity,Hsinchu30050,Taiwan} e_{InstituteofOccupationalMedicineandIndustrialHygiene,NationalTaiwanUniversity,Taipei,Taiwan}

f_{SchoolofPost-BaccalaureateChineseMedicine,ChinaMedicalUniversity,ChinaMedicalUniversity,Taichung,Taiwan}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received28May2011

Receivedinrevisedform9August2011 Accepted25September2011 Available online 1 October 2011

Keywords: Safrole-2_,3_-oxide Genotoxicity Cometassay Micronucleustest HepG2cells Peripheralblood

a

b

s

t

r

a

c

t

Safrole-2,3-oxide(SAFO)isareactiveelectrophilicmetaboliteofthehepatocarcinogensafrole,themain componentofsassafrasoil.Safroleoccursnaturallyinavarietyofspicesandherbs,includingthe

com-monlyusedChinesemedicineXixin(AsariRadixetRhizoma)andDongquai(Angelicasinensis).SAFOis

themostmutagenicmetaboliteofsafroletestedintheAmestest.However,littleornodataareavailable

onthegenotoxicityofSAFOinmammaliansystems.Inthisstudy,weinvestigatedthecytotoxicityand

genotoxicityofSAFOinhumanHepG2cellsandmaleFVBmice.UsingMTTassay,SAFOexhibiteda

dose-andtime-dependentcytotoxiceffectinHepG2cellswithTC50valuesof361.9␮Mand193.2␮Mafter24

and48hexposure,respectively.Inaddition,treatmentwithSAFOatdosesof125␮Mandhigherfor24h

inHepG2cellsresultedina5.1–79.6-foldincreaseinmeanComettailmomentbythealkalineComet

assayanda2.6–7.8-foldincreaseinthefrequencyofmicronucleatedbinucleatedcellsbythe cytokinesis-blockmicronucleusassay.Furthermore,repeatedintraperitonealadministrationofSAFO(15,30,45,and 60mg/kg)tomiceeveryotherdayforatotaloftwelvedosescausedasignificantdose-dependentincrease

inmeanComettailmomentinperipheralbloodleukocytes(13.3–43.4-fold)andinthefrequencyof

micronucleatedreticulocytes(1.5–5.8-fold).RepeatedadministrationofSAFO(60mg/kg)tomicecaused

liverlesionsmanifestedasarimofballooningdegenerationofhepatocytesimmediatelysurrounding

thecentralvein.OurdataclearlydemonstratethatSAFOsignificantlyinducedcytotoxicity,DNAstrand

breaks,micronucleiformationbothinhumancellsinvitroandinmice.Morestudiesareneededtoexplore theroleSAFOplaysinsafrole-inducedgenotoxicity.

© 2011 Elsevier B.V. All rights reserved.

1.

Introduction

Safrole

(4-allyl-1,2-methylenedioxybenzene,

CAS

Number:

00094-59-7),

the

main

component

of

the

essential

oil

in

the

root

bark

and

the

fruit

of

sassafras

plants,

occurs

naturally

in

various

amounts

in

numerous

edible

herbs

and

spices:

e.g.,

basil,

nutmeg,

star

anise,

mace,

cinnamon

leaves

[1]

.

Besides,

safrole

is

found

in

the

essential

oil

of

commonly

used

Chinese

medicine,

such

as

Xi

xin

(Asari

Radix

et

Rhizoma,

up

to

5300

ppm

of

dried

herb)

[2]

and

Dong

Abbreviations: SAFO,safrole-2_,3_{-oxide;CBPI,cytokinesis-blockproliferation}

index;MNRETs,micronucleatedreticulocytes;RETs,reticulocytes.

∗ Correspondingauthorat:GraduateInstituteofChineseMedicine,ChinaMedical University,No.91,Hsueh-ShihRoad,Taichung40402,Taiwan.

Tel.:+886422053366x3305;fax:+886422032295. E-mailaddress:sychiang@mail.cmu.edu.tw(S.-y.Chiang).

quai

(Angelica

sinensis,

up

to

40

ppm)

[3]

.

In

Taiwan,

about

2

million

people

(10%

of

the

population)

chew

betel

quid,

a

mild

stimulant

comprising

areca

nut,

slaked

lime,

and

piper

betel

inflorescence

[4]

.

High

levels

of

safrole

(15.4

mg/g

wet

weight)

found

in

Piper

betel

inflorescence

can

lead

to

extremely

high

levels

of

safrole

exposure

(up

to

420

␮M)

in

saliva

during

betel

quid

chewing

[4]

.

Safrole

was

banned

by

the

United

States

Food

and

Drug

Administration

for

use

as

flavorings

and

food

additives

in

1960

(Federal

Register

1960,

25

FR

12412)

because

it

caused

hepatocarcinoma

in

rats

when

fed

in

the

diet

at

doses

of

390

and

1170

ppm

for

2

years

[5,6]

.

In

1976,

the

International

Agency

for

Research

on

Cancer

classified

safrole

as

a

Group

2B

carcinogen

(possible

human

carcinogen)

[7]

.

In

vitro

mouse

and

rat

hepatic

microsomal

studies

and

in

vivo

studies

have

shown

that

safrole

is

metabolized

by

the

cytochrome

P-450

pathway

to

an

electrophilic

epoxide

metabolite,

safrole-2

-3

-oxide

(SAFO)

[8–10]

;

a

presumed

prox-imate

carcinogenic

metabolite,

1

-hydroxysafrole

[10–12]

;

and

1383-5718/$–seefrontmatter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2011.09.014

a

reactive

oxygen

species

(ROS)-forming

metabolite,

hydroxy-chavicol

(1,2-dihydroxy-4-allylbenzene)

[13,14]

.

A

physiologically

based

biokinetic

(PBBK)

model

for

safrole

in

rats

developed

using

in

vitro

metabolic

parameters

has

shown

that,

at

a

dose

of

300

mg/kg

safrole,

the

percentage

of

safrole

metabolized

to

2

,3

-dihydroxysafrole

(derived

from

SAFO),

1

-hydroxysafrole,

3

-hydroxysafrole,

and

1,2-dihydroxy-4-allylbenzene

was

6.9%,

10.0%,

7.7%,

and

74.0%

of

the

dose,

respectively

[10]

.

The

carcinogenic

effects

of

SAFO

and

1

-hydroxysafrole

have

been

reported

in

mice

[15,16]

,

and

1

-hydroxysafrole

is

considered

to

be

the

main

prox-imate

carcinogenic

metabolite

of

safrole

[12]

.

SAFO

can

react

directly

with

calf

thymus

DNA

in

vitro

to

produce

at

least

eight

SAFO-DNA

adducts

as

measured

by

_{P-postlabeling}

_analysis

_[17]

_.

However,

none

of

these

DNA

adducts

were

detected

in

liver

tis-sues

of

male

Balb/C

mice

treated

with

a

single

intraperitoneal

dose

(600

␮mol/kg)

of

SAFO

or

safrole

[17]

,

probably

because

of

the

rapid

metabolic

inactivation

of

the

compound

by

cytosolic

and

micro-somal

epoxide

hydrolase

and

cytosolic

glutathione

S-transferase

[18,19]

.

These

data

led

to

the

assumption

that

the

metabolism

of

safrole

via

epoxidation

may

not

play

a

major

role

in

the

genotoxicity

of

safrole

[17]

.

The

contribution

of

SAFO

to

the

genotoxicity

of

safrole

still

arouses

interest

because

of

its

structural

similarity

to

other

known

epoxide

carcinogens

like

styrene

oxide;

its

sufficient

electrophilic

reactivity

to

form

DNA

adducts

in

vitro

[17,20]

;

its

apparent

for-mation

in

safrole-exposed

rats,

guinea

pigs,

and

man,

as

evidenced

by

the

detection

of

dihydrodiol

metabolites

in

urine

[8,21–23]

;

its

considerable

persistence

in

urine

of

SAFO-exposed

rats

and

guinea

pigs

[8]

;

its

pronounced

mutagenicity

in

the

Ames

test

[16,24]

,

and

its

tumorigenicity

in

mice

[15]

.

Miller

et

al.

reported

that

CD-1

adult

female

mice

given

24

topical

applications

(2

mg/application)

of

SAFO,

followed

by

twice-weekly

applications

of

croton

oil,

induced

skin

papillomas

and

keratoacanthomas

in

36%

of

SAFO-treated

mice,

compared

to

7%

in

control

mice

[15]

.

Moreover,

SAFO

was

the

most

active

metabolite

of

safrole

tested

in

the

Ames

test,

exert-ing

dose-dependent

mutagenic

activities

in

S.

typhimurium

strains

TA100

[16]

and

TA1535

[16,24]

in

the

absence

of

metabolic

acti-vation,

with

specific

mutagenic

activities

of

6000

and

6000–7100

revertants/

␮mol,

respectively.

Although

SAFO

has

been

shown

to

be

efficiently

detoxified,

SAFO

was

detected

not

only

in

urine

of

rats

and

guinea

pigs

treated

with

SAFO

[8]

,

but

also

in

urine

of

rats

treated

with

safrole

[9]

.

The

above

evidences

suggest

that

some

of

SAFO

can

escape

detoxification

that

in

turn

has

the

potential

to

cause

DNA

damage

in

vivo.

Currently,

there

is

little

or

no

published

data

on

the

muta-genicity

and

genotoxicity

of

SAFO

in

mammalian

systems

either

in

vitro

or

in

vivo.

This

study

investigated

the

cytotoxicity

and

geno-toxic

effects

of

SAFO

by

MTT,

alkaline

Comet,

and

micronucleus

assays

in

HepG2

cells

and

by

the

histopathological

microscopy

in

liver,

alkaline

Comet,

and

micronucleus

assays

in

peripheral

blood

cells

of

FVB

mice

to

better

understand

the

role

that

SAFO

plays

in

the

genotoxicity

of

safrole.

In

HepG2

cells,

SAFO

induced

cyto-toxicity

in

a

dose-

and

time-dependent

manner,

and

resulted

in

a

dose-dependent

increase

in

mean

Comet

tail

moment

and

the

frequency

of

micronucleated

binucleated

cells.

In

male

FVB

mice,

repeated

administration

of

SAFO

caused

liver

damage,

typically

manifested

as

a

rim

of

ballooning

degeneration

of

hepatocytes

immediately

adjacent

to

the

central

vein.

SAFO

caused

a

signif-icant

dose-dependent

increase

in

mean

Comet

tail

moment

of

peripheral

blood

leukocytes

and

in

the

frequency

of

micronucleated

reticulocytes.

Our

data

demonstrate

that

SAFO

exhibits

significant

Fig.1. Chemicalstructureofsafrole2_,3_{-oxide(SAFO).}

cytotoxic

and

genotoxic

effects

both

in

human

cells

in

vitro

and

in

mice.

2. Materialsandmethods

2.1. Chemicals

FetalbovineserumwasobtainedfromHyClone(Logan,Utah,USA).Dulbecco’s ModifiedEagle’sMedium(DMEM),penicillin,andstreptomycinwerepurchased from Life Technologies (Gaithersburg,Maryland, USA). Acridineorange (AO), dimethylsulfoxide(DMSO),propidiumiodide(PI),MTT,agarose(normalandlow meltingpoint),andcytochalasinBwerepurchasedfromSigma(St.Louis,MO,USA). Allotherchemicalsandsolventswereofanalyticalgradeandwerealsoobtained fromSigma.

2.2. PreparationofSAFO

SAFOwassynthesizedfollowingpreviouslypublishedprocedures[25].Briefly, m-chloroperbenzoicacid(30g,0.17mol)in200mLchloroformwasslowlyaddedto asolutionofsafrole(22.7mL,0.15mol)in50mLchloroformat0◦_{Candwasstirredat}

roomtemperatureovernight.Thereactionwasterminatedwith10%sodiumsulfite. Thereactionproductwasextracted3timeswith250mLof5%NaHCO3and2times

with200mLofwater.Theorganiclayerswerecombinedandevaporatedtodryness. Theresiduewassubjectedtocolumnchromatographywithneutralsilicageland elutedwithhexane/EtOAc(10:1),resultinginthefinalproductasalightyellow liquid.Theyieldwas39.7%.Theproductwasfurthercharacterizedandconfirmed asSAFObyliquidchromatographytandemmassspectrometry(LC–MS/MS)and nuclearmagneticresonance(NMR)spectroscopy.ESI+/MS:m/z179([M+H]+_);1_H

NMR(300MHz,inCDCl3):ı2.51(dd,1H,H-␥,J=2.6Hz,J=4.9Hz),2.70–2.81(m,

3H,H-␥_,H␣_,H␣_{),3.06−3.11(m,1H,H-␤),5.91(s,2H,CH2),6.66–6.69(m,1H,}

Ar-CH),6.73–6.75(m,2H,Ar-CH).13_{CNMR(75.4MHz,inCDCl}

3):ı46.7(C-␣),52.5(C-␤),

38.3(C-␥),108.2(Ar-CH),130.7(Cq),122.8(Ar-CH),109.4(Ar-CH),146.2(Cq),147.6 (Cq),100.8(C-g).ThepurityofthesynthesizedSAFOwasestimatedbyHPLC–UV chromatographytobe96.5%(theratioofitspeakareatothetotalareaofallpeaks). ThechemicalstructureofSAFOisshowninFig.1.

2.3. Cellcultureandexposure

AhumanhepatomaHepG2celllinewasobtainedfromtheBioresource Collec-tionandResearchCenter(Hsinchu,Taiwan).CellsweremaintainedinDulbecco’s ModifiedEagle’sMedium(DMEM)supplementedwith10%heat-inactivatedfetal bovineserum,100U/mlpenicillin,and100␮g/mlstreptomycinin75cm2_tissue

cultureflasksinahumidifiedincubatorat37◦Cunderanatmospherecontaining5% CO2.Cellsinthelogarithmicgrowthphaseweretreatedwithdifferent

concentra-tionsofSAFO(0,125,250,312.5and375␮M)for24or48hbeforeMTTanalysisand for24hbeforethealkalineCometassayandmicronucleustestwereperformed. 2.4. MTT(3-[4,5-dimethylthi-azol-2-yl]-2,5-diphenyltetrazoliumbromide)assay

CellviabilitywasdeterminedbyaMTTcolorimetricassay.MTTwaspurchased fromSigmaanddissolvedinphosphate-bufferedsaline(137mMNaCl,1.4mM KH2PO4,4.3mMNa2HPO4,2.7mMKCl,pH7.2).Briefly,cellswereseededin

96-wellcultureplatesandincubatedovernightbeforebeingtreatedwithdifferent concentrationsofSAFO(0,125,250,and375␮M)dissolvedinDMSO.The maxi-mumDMSOconcentrationinculturewas0.1%(v/v).Aftera24-hincubationperiod, aone-tenthvolumeof5mg/mlMTTwasaddedtotheculturemedium,and incu-batedfor4hinthedark.Anequalvolumeofsolubilizationsolution(10%SDS/0.1N HCl)wasthenadded,andtheabsorbancewasmeasuredatawavelengthof570nm and650nmbyaSpectraMax®_{340PC384AbsorbanceMicroplateReader(Molecular}

Devices,Sunnyvale,CA).Therelativesurvivalratewascalculatedusingthefollowing equation:

Relativecellsurvivalrate(%)= (AbsorbanceofSAFO-treatedcells−Absorbanceofmediumonly) (AbsorbanceofDMSO-treatedcells−Absorbanceofmediumonly)×100%

TheTC50values(50%toxicconcentration)weredeterminedasthe

concentra-tionofSAFOrequiredtoreducecellviabilityby50%relativetountreatedcontrol cells.Valuesarereportedasmean±standarddeviationofthreeindependent exper-iments.

2.5. Cometassay(alkalinesingle-cellgelelectrophoresis)inHepG2cells[26]

After24hexposuretoSAFO,thealkalineversionoftheCometassaywas per-formed.A10␮laliquotofcellsuspension(1×104_{cells)wasgentlymixedwith60␮l}

of0.5%normal-meltingagaroseinPBSat37◦_{C.Then,65␮lofthiscellsuspensionin}

normal-meltingagarosewasplacedonamicroscopeslidethathadbeenpre-coated with75␮lof1%solidifiedlow-meltingagarose.Acoverslipwasplacedonthecell suspensioninagaroseandwasallowedtosetfor10minonice.Aftertheagarosehad solidified,thecoverslipwasremovedandtheslidewasimmersedinalysisbuffer (2.5MNaCl,100mMNa2EDTA,10mMTris,1%TritonX-100,and10%DMSO,pH=10)

at4◦_{Cfor1h.Afterlysis,theslideswereplacedinahorizontalgelelectrophoresis}

tankcontainingacoldfreshlypreparedalkalinebuffer(300mMNaOHand1mM Na2EDTA,pH>13)for20mintoallowDNAtounwind.Thereafter,

electrophore-siswasperformedat25V(1.15V/cm),whichwasadjustedbyraisingorlowering thebufferlevelinthetankfor20min.Afterelectrophoresis,theslideswere imme-diatelyneutralizedwith0.4MTrisbuffer(pH=7.5)at4◦_{Cfor15min,fixedwith}

methanolfor5min,andallowedtodryatroomtemperature.Finally,theslideswere stainedwithpropidiumiodide(5␮g/ml)andobservedunderaninverted fluores-cencemicroscope(OlympusCKX41andU-RFLT50)(OlympusCo.Ltd.,Tokyo,Japan) witha20objective.Cometimageswerecapturedbyadigitalcharge-coupleddevice (CCD)camera(OlympusDP70,OlympusCo.Ltd.,Tokyo,Japan)underfluorescence microscope.

TheextentofDNAdamagewasanalyzedin50randomlyselectedcells(25cells fromeachoftworeplicateslides)fromeachsampleusingTriTekCometScoreTM

ver-sion1.5software(TriTekCorp.,Sumerduck,VA,USA).Thebasicassumptionisthat theamountofDNAatalocationisproportionaltothepixelintensityatthat posi-tion.Tailmomentisdefinedinarbitraryunitsasthe%DNAintailmultipliedbythe taillength,dividedby100.Allslideswerecodedandexaminedinadouble-blind mannertoavoidobserverbias.SomeCometdatawerenotnormallydistributed, andthemediandifferedsubstantiallyfromthemean.Therefore,themedian val-uesofthetailmomentforeachexperimentalsamplewerecalculated.Valuesare reportedasmean±standarddeviationoffourreplicatesfromtwoindependent experiments.

2.6. Cytokinesis-blockmicronucleustestinHepG2cells

HepG2cellswereseededoncoverslipsovernightandthentreatedwith dif-ferentconcentrations ofSAFO(0, 125,250,and312.5␮M)for 24h. SAFOat 375␮Mwastootoxictobeevaluated.Aftera24hincubationperiod, cytocha-lasinBwasaddedtoreachafinalconcentrationof3␮g/mlandincubatedfor another16htoobtainbinucleatedcells.Then,thecellsoncoverslipswerefixed withmethanolat4◦_{Cfor30min,air-driedfor10min,and stainedwith}

acri-dineorange(0.2mg/ml)inthedarkfor10min.Finally,theslideswereexamined under afluorescencemicroscopeat an excitationwavelengthof488nm.For each treatment, the frequencyof micronucleiformation was scoredin 1000 binucleatedcells.Cytokinesis-blockproliferationindex(CBPI)wasusedasa param-eterforcytotoxicity andwascalculatedbyscreening500cells pertreatment groupforthefrequencyofcells withoneormorenucleiusingthefollowing formula.

CBPI=[M1+2(M2)+3(M3+M4)] N

whereM1–M4isthenumberofcellswith1–4nucleiandNisthetotalnumber ofcellsscored[27].Allslideswerecodedandexaminedinadouble-blindmanner toavoidobserverbias.Valuesarereportedasmean±standarddeviationoffour replicatesfromtwoindependentexperiments.

2.7. Animalsanddosing

Mouseexperimentswereconductedinaccordancewithethicsapprovalfrom ChinaMedicalUniversityAnimalEthicsCommittee.MaleFVBmice,6–7weeksold weighing20–25g,werepurchasedfromtheNationalLaboratoryAnimalCenter (Taipei,Taiwan).AnimalswereacclimatizedforaboutsevendayspriortoSAFO exposure.Themiceweredividedinto5groupsof4animalseach.SAFOwas dis-solvedinoliveoil,andthedosingvolumeofSAFOinoliveoilwas3.3ml/kgbody weight.SAFOwasadministeredbyintraperitonealinjectionatdosesof15,3045, and60mg/kgeveryotherdayfor24consecutivedays.Controlsreceivedequal vol-ume(3.3ml/kg)injectionsofoliveoil.Allanimalswereallowedfreeaccesstofood andwaterduringtheexperiment.Inaddition,allmicewerekeptunderobservation andweighedduringtheexperiment.

2.8. Histopathologicalexamination

MiceweresacrificedbyCO2asphyxiationsixdaysafterthelast

administra-tion.Bloodsampleswerecollectedbyintracardiacpunctureintoheparin-containing bloodcollectiontubes,andthencentrifugedat3000rpmfor10minat4◦_C.Plasma

levelsofglutamateoxaloacetatetransaminase(GOT),glutamatepyruvate transami-nase(GPT),plasmacreatinine,andplasmabloodureanitrogenweremeasuredwith anautoanalyzer.Atnecropsy,agrosspathologicalexaminationwasperformed.The

liverandlungweredissectedandtheirabsoluteandrelativeweightswere eval-uated.Theliverspecimenswerethenfixedinbufferedformalinandstainedwith hematoxylinandeosinforhistologicalexaminationAllliverbiopsysectionswere processedforlightmicroscopyexamination(HEstain).Histopathological evalua-tionwasperformedblindedbyanexperiencedpathologistfromtheDepartmentof Pathology,ChungShanMedicalUniversityHospital,Taichung,Taiwan.

2.9. Cometassayinmouseperipheralbloodleukocytes

At16hafterthelastadministration,theperipheralblood(10␮l)wascollected fromatailveinusingaheparin-coatedmicropipettetip.ThealkalineCometassay wasthenperformedasdescribedaboveforHepG2cells.

2.10. Micronucleustestwithmouseperipheralbloodreticulocytes

A10␮lvolumeofaqueousacridineorange(AO)solution(0.2mg/ml)wasspread homogeneouslyonaglassslidethathadbeenpre-heatedtoabout70◦_Candallowed

todryatroomtemperature.Thepreparedslideswerestoredinadarkanddry locationatroomtemperatureforatleast4hbeforeuse.At24hafterthelast admin-istration,about10␮lofperipheralbloodwascollectedfromatailveinthrougha smallcutmadewithasharp-pointedscissor,immediatelydroppedontothe cen-terofAO-coatedslides,andthencoveredwithacleancoverslip.Theslideswere subsequentlykeptat4◦_{Cinthedarkforatleast4handexaminedundera}

fluores-cencemicroscopeatanexcitationwavelengthof488nm.Atleasttwoslideswere examinedformicronucleiformationperanimal.Thefrequencyofmicronucleated reticulocytes(MNRETs)wasrecordedbasedontheobservationof1000reticulocytes (RETs)perslide.Allslideswerecodedfora“double-blind”analysis.

2.11. Statisticalanalysis

Thedatawereexpressedasamean±standarddeviation,andweretestedfor normalityusingtheShapiro–WilktestandtheKolmogorov–Smirnovtestusingthe StatisticalAnalysisSoftware(SAS)package(Version9.1,SASInstitute,Cary,NC). WithsomedatafromMTT,Comet,orMNassaysshowingnon-normal distribu-tion(P<0.05),alltwo-groupcomparisonswereperformedwiththenonparametric Wilcoxonrank-sumtestasprovidedinPROCNPAR1WAYofSASsoftwarepackage Version9.1.ThelevelforstatisticalsignificancewassetatP<0.05[28].

3.

Results

3.1.

Cytotoxicity

in

HepG2

cells

The

viability

of

HepG2

cells

exposed

to

SAFO

at

the

doses

of

125,

250,

312.5,

and

375

␮M

for

24

or

48

h

was

examined

using

a

MTT

assay.

SAFO

produced

toxicity

in

HepG2

cells

in

a

dose-

and

time-dependent

manner

(

Fig.

2

).

Based

on

the

survival

curves,

the

SAFO

concentrations

required

to

achieve

50%

cell

survival

after

24

or

48

h

exposure

were

estimated

to

be

approximately

361.9

␮M

and

193.2

␮M,

respectively.

Fig.2. SAFOinducedcytotoxicityinHepG2cellsasmeasuredbyMTTassay.HepG2 cellsweretreatedwithdifferentconcentrationsofSAFOfor24or48h.Themedium wasthenreplacedwithfreshmediumcontaining0.5mg/mlMTT.Theabsorbance at570and650nmoftestandcontrolwellswasreadtocalculatetherelative sur-vivalrate.Valuesarereportedasmean±standarddeviationofthreeindependent experiments.*p<0.05ascomparedtocontrolcells.

Fig.3.SAFOinducesDNAstrandbreakinHepG2cellsasmeasuredbyCometassay. HepG2cellsweretreatedwithdifferentconcentrationsofSAFOfor24h.Cellswere embeddedinagarose,subjectedtothealkalineCometassay,andstainedwith pro-pidiumiodide.ThemeanComettailmomentfrom50cellsineachtreatmentwas calculatedusingTriTekCometScoreTM_{version1.5software.Valuesarereportedas}

mean±standarddeviationoffourreplicatesfromtwoindependentexperiments. *p<0.05ascomparedtocontrolcells.

3.2.

Comet

assay

in

HepG2

cells

The

degree

of

SAFO-induced

DNA

damage

in

HepG2

cells

after

24

h

exposure

was

analyzed

by

the

alkaline

Comet

assay,

which

can

detect

DNA

single

strand

breaks,

alkali-labile

apyrimidinic/apurinic

sites,

and

transient

repair

sites.

The

Comet

tail

moment

in

SAFO-treated

cells

was

quantified

using

CometScore

software

and

then

summarized

as

the

median

of

50

cells.

Compared

with

background

DNA

damage

obtained

in

control

cells,

SAFO

resulted

in

a

significant

dose-dependent

increase

in

the

degree

of

DNA

damage

at

concen-trations

of

125

␮M

and

above

(

Fig.

3

).

Exposure

to

SAFO

at

doses

of

125

␮M

and

higher

resulted

in

a

5.1–79.6-fold

increase

in

mean

Comet

tail

moment.

3.3.

Cytokinesis-block

micronucleus

test

in

HepG2

cells

The

cytotoxic

and

genotoxic

effects

of

SAFO

were

further

evalu-ated

using

the

cytokinesis-block

micronucleus

test.

In

control

cells,

the

percentage

of

binucleated

cells

was

>75%

and

the

CBPI

val-ues

were

around

1.9.

The

background

frequency

of

binucleated

micronucleated

cells

ranged

from

1.0%

to

1.3%,

which

is

within

the

normal

range

as

reported

in

the

literature

or

within

our

lab-oratory’s

historical

data.

SAFO

treatment

decreased

the

percentage

of

binucleated

cells

in

a

dose–response

manner.

In

SAFO-treated

cells,

mean

CBPI

values

observed

at

doses

above

125

_␮M

were

sig-nificantly

lower

than

those

in

the

negative

control

cells

(P

<

0.05),

with

CBPI

values

of

1.3

at

312.5

␮M

(

Fig.

4

).

There

was

a

significant

dose-dependent

increase

in

the

frequency

of

binucleated

micronu-cleated

cells.

The

frequencies

of

micronucleated

binucleated

cells

were

significantly

increased

2.6-,

5.2-,

and

7.8-fold

at

125,

250,

and

312.5

␮M,

respectively.

3.4.

General

toxicity

evaluation

in

mice

Furthermore,

we

investigated

whether

SAFO

has

a

significant

cytotoxic

or

genotoxic

effect

in

vivo.

FVB

mice

were

subjected

to

intraperitoneal

injections

of

SAFO

(15,

30,

45,

and

60

mg/kg)

every

other

day

for

24

consecutive

days,

and

then

euthanized

by

CO

six

days

after

the

last

treatment.

During

the

experimental

period,

all

mice

survived

with

the

exception

of

one

mouse

in

the

group

that

received

60

mg/kg

SAFO

after

the

first

treatment.

This

mouse

was

probably

died

from

accidental

death

because

all

mice

in

a

Fig. 4. SAFO induced chromosome damage in HepG2 cells as measured by cytokinesis-blockmicronucleustest.HepG2cellsweretreatedwithdifferent con-centrationsofSAFOfor24h.Followingtreatment,cellswereexposedtocytochalasin B(3␮g/ml)for16htoobtainbinucleatedcells,andthencellswerefixedandstained withacridineorange(0.2mg/ml).Thenumberofmicronucleatedcellswasscored underfluorescentmicroscopy.Atotalof1000intactinterphasecellswerescoredfor eachtreatment.Valuesarereportedasmean±standarddeviationoffourreplicates fromtwoindependentexperiments.BNC:binucleatedcells;CBPI:cytokinesis-block proliferationindex.*p<0.05ascomparedtocontrolcells.

follow-up

study

were

survived

after

i.p.

administration

of

12

doses

of

60,

90,

or

120

mg/kg

SAFO.

The

behavior,

coat

color,

and

food

consumption

remained

normal

throughout

the

experiment.

In

addition,

there

were

no

sig-nificant

differences

in

changes

in

body

weight

(Data

not

shown).

At

necropsy,

no

gross

abnormalities

were

found.

3.5.

Blood

chemistry

and

histopathology

There

were

no

significant

differences

in

the

relative

weights

(percentage

of

body

weight)

of

liver

between

the

experimental

and

control

groups.

We

also

observed

no

significant

differences

in

GOT,

GPT,

BUN,

or

creatinine

levels

in

serum

between

the

exper-imental

and

control

groups

(data

not

shown).

Histopathological

examination

showed

normal

architecture

in

the

liver

tissues

of

the

control

group.

There

were

no

significant

morphological

alter-ations

in

liver

sections

from

mice

treated

with

SAFO

at

the

doses

15,

30

or

45

mg/kg

every

other

day

for

24

days.

Furthermore,

in

the

highest

dose

group

(60

mg/kg),

SAFO

caused

prominent

balloon-ing

degeneration

and

cytoplasmic

vacuolation

in

the

single

layer

of

hepatocytes

immediately

surrounding

the

central

vein,

whereas,

the

hepatic

cells

around

the

portal

vein

area

remained

intact

(

Fig.

5

).

3.6.

Comet

assay

in

mouse

peripheral

blood

leukocytes

At

16

h

after

the

last

SAFO

administration,

10

␮l

of

peripheral

blood

was

collected

from

each

animal

and

subjected

to

the

alkaline

Comet

assay.

As

shown

in

Fig.

6

,

SAFO

at

all

doses

resulted

in

a

significant

increase

in

the

mean

Comet

tail

moment

relative

to

the

control

(P

<

0.05).

Exposure

to

SAFO

at

doses

of

15

mg/kg

and

higher

resulted

in

a

13.3–43.4-fold

increase

in

mean

Comet

tail

moment.

3.7.

Micronucleus

test

with

mouse

peripheral

blood

reticulocytes

We

also

evaluated

the

frequency

of

micronuclei

in

FVB

mice.

The

frequency

of

the

micronucleated

reticulocytes

provides

an

index

of

cytogenetic

damage

in

mice.

SAFO

treatment

resulted

in

a

signifi-cant

dose-dependent

increase

in

the

number

of

MNRETs

per

1000

Fig.5. PathologicalchangesinmouseliveraftertwelvedosesofSAFO(60mg/kg).RepresentativephotomicrographsofHEstainingofliversectionsfromcontrolmiceand SAFO-treatedmiceareshownattwomagnifications(40×and400×).InSAFO-treatedmouseliver,vacuolationandballooningdegenerationofhepatocytesimmediately adjacenttothecentralveinaremanifested.CV:centralvein,PV:portalvein.Vacuolizationofhepatocytes(arrows).

RETs

(

Fig.

7

).

The

frequency

of

MNRETs

increased

1.5-,

2.6-,

4-

and

5.8-fold

after

administration

of

15,

30,

45

and

60

mg/kg

of

SAFO,

respectively.

4.

Discussion

SAFO,

a

reactive

epoxide

metabolite

of

safrole,

has

been

shown

to

induce

DNA

adduct

formation

in

vitro

[17,20]

,

to

be

mutagenic

in

the

Ames

test

in

the

absence

of

metabolic

activation

[16,24]

,

and

to

cause

tumors

in

mice

[15]

.

Many

epoxides

are

very

reac-tive

electrophiles

that

react

readily

with

cellular

DNA

and

form

covalently

bound

DNA

adducts

[29]

.

Since

the

above

experimental

observations

consistently

point

to

direct

DNA-reactivity

of

SAFO,

we

hypothesized

that

SAFO

would

be

genotoxic

in

mammalian

sys-tems.

In

this

study,

we

showed

clear

evidence

that

SAFO

induced

pronounced

cytotoxicity

and

genotoxicity

in

human

cultured

cells

and

in

mice.

Safrole

has

been

shown

to

cause

liver

cancer

and

DNA

adduct

formation

in

rodents

[5,6,30]

;

however,

negative

mutagenic

and

genotoxic

results

have

been

reported

even

in

the

presence

of

metabolizing

rat

liver

homogenate

fraction

(S9

mix)

in

several

bio-logical

test

systems

in

vitro,

such

as

in

the

Ames

test

[16,24,31]

,

the

Chinese

hamster

V79/hprt

gene

mutation

assay

[32]

,

the

Chi-nese

hamster

V79/Na

_/K

_ATPase

_gene

_mutation

_assay

_[33]

_,

_and

_the

mouse

lymphoma

L5178Y/tk

assay

[34]

.

The

negative

findings

are

presumed

to

be

a

result

of

inadequate

metabolic

activation

in

the

test

systems.

Thus,

the

above

data

imply

that

adequate

metabolic

systems

are

required

when

the

genotoxicity

and

mutagenicity

of

safrole

or

its

metabolites

are

investigated.

The

metabolically

com-petent

human

hepatoma

HepG2

cell

line

retains

activities

of

various

Phase

I

and

Phase

II

enzymes

that

are

involved

in

the

activation

or

detoxification

of

environmental

genotoxicants.

That

cell

line,

there-fore,

better

represents

the

biotransformation

of

these

compounds

in

vivo

than

metabolically

incompetent

cell

lines

in

the

presence

of

exogenous

metabolic

activation

[35]

.

Hence,

in

this

study,

we

chose

HepG2

cells

as

an

in

vitro

model

system

to

examine

the

genotoxic

effects

of

SAFO.

Previous

work

with

HepG2

cells

showed

that

safrole

induced

the

formation

of

DNA

adducts

[36]

,

Comet

tails

[37]

,

micronuclei,

and

sister

chromatid

exchanges

[38]

.

Uhl

et

al.

found

that

expo-sure

of

HepG2

cells

to

safrole

at

a

concentration

of

4500

␮M

for

24

h

resulted

in

a

3.0-fold

increase

in

Comet

tail

length

relative

to

the

control

[37]

.

We

found

that

treatment

with

125

␮M

SAFO

for

24

h

led

to

a

16.7-fold

increase

in

Comet

tail

length

in

HepG2

cells

(

Fig.

3

).

HepG2

cells

have

relatively

normal

expression

of

most

Phase

II

enzymes,

but

have

lower

expression

of

cytochrome

P450

enzymes

relative

to

those

in

normal

human

liver

[39–41]

.

That

may

partly

explain

the

observation

that

treatment

of

HepG2

cells

with

safrole

led

to

a

significantly

positive

result

in

the

Comet

assay

only

at

concentrations

of

safrole

equal

to

or

higher

than

4500

␮M

[37]

.

Natarajan

and

Darroudi

observed

that

the

frequency

of

micronu-cleated

binucleated

cells

increased

by

about

2.2-fold

in

HepG2

cells

that

had

been

treated

for

28

h

with

300

␮M

safrole

[38]

.

By

compar-ison,

in

this

study,

treatment

with

312.5

␮M

SAFO

for

24

h

resulted

in

a

7.8-fold

increase

in

the

frequency

of

micronucleated

binucle-ated

HepG2

cells

(

Fig.

4

).

These

results

indicate

that

SAFO

is

more

genotoxic

than

its

parent

compound,

and

thus

may

partly

play

a

role

in

the

genotoxicity

of

safrole.

Many

epoxides

are

detoxified

by

epoxide

hydrolases

and

glu-tathione

S-transferases

[18,19,29]

.

The

lack

of

SAFO-DNA

adducts

in

mice

given

safrole

and

SAFO

are

generally

thought

to

occur

as

a

result

of

the

rapid

and

efficient

metabolic

inactivation

of

SAFO

[18,19]

.

Safrole

is

an

alkoxy

derivative

of

allylbenzene.

Guenth-ner

et

al.

compared

the

abilities

of

liver

homogenates

from

several

species

of

mammals,

including

guinea

pig,

rat,

mouse,

rabbit,

and

human,

to

detoxify

allylbenzene

2

,3

-oxide

and

found

that

human

livers

had

the

highest

allylic

epoxide

hydrolase

activity

among

the

species

tested

[19]

.

The

epoxide

hydrolase

activity

of

frozen

human

liver

was

about

seven

to

ten

times

higher

than

that

observed

in

mouse

and

rat

liver

[19]

.

In

addition,

kinetic

studies

showed

that

the

activity

of

microsomal

and

cytosolic

epoxide

hydrolases

in

mouse

liver

with

SAFO

as

the

substrate

was

similar

to

that

of

allylben-zene

2

,3

-oxide,

in

terms

of

Km

and

Vmax

[18,19]

.

These

data

imply

that

human

liver

may

provide

greater

protection

against

SAFO-induced

cytotoxicity

and

genotoxicity

than

mouse

or

rat

liver.

In

contrast,

human

glutathione

S-transferase

activities

toward

allylbenzene

oxide

are

only

two

to

four

times

lower

than

those

mea-sured

in

mouse

and

rat

liver

[19]

.

Although

Phase

II

enzyme

activity

Fig.6.SAFOinducesDNAstrandbreakinmouseperipheralbloodleukocytes.SAFOwasadministeredtomicebyintraperitonealinjectionatdosesof15,30,45and60mg/kg everyotherdayfor24days.Peripheralbloodcollectedfromatailveinat16hafterthelastadministrationwasembeddedinagarose,subjectedtothealkalineCometassay, andstainedwithpropidiumiodide.MeanComettailmomentwasscoredin100cellsineachanimal.Valuesareindicatedasmean±standarddeviation,*p<0.05ascompared tocontrolmice(n=4).

in

HepG2

cells

is

similar

to

that

in

primary

human

hepatocytes

[40,41]

,

we

still

observed

that

SAFO

significantly

induced

micronu-cleus

formation

in

binucleated

cells

and

extended

the

tail

length

even

at

the

lowest

dose

tested

(125

␮M)

(

Fig.

4

).

Therefore,

even

though

SAFO

was

reported

to

be

rapidly

and

efficiently

detoxified

by

microsomal

epoxide

hydrolase

and

glutathione

S-transferase

[18,19]

,

the

marked

genotoxic

effects

of

SAFO

observed

in

this

study

(

Figs.

4

and

5

)

suggest

that

SAFO

may

be

a

highly

reactive

molecule

capable

of

causing

DNA

damage

and

cytogenetic

changes

before

the

detoxification

reactions

occur.

Further

studies

are

needed

to

delineate

the

role

SAFO

plays

in

safrole-induced

genotoxicity

and

carcinogenicity.

Daimon

et

al.

found

that

safrole

was

genotoxic

in

vivo

[30]

.

In

their

study,

treatment

of

F344

rats

with

five

doses

of

either

62.5

and

125,

or

125

and

250

mg/kg

safrole

resulted

in

signif-icantly

increased

levels

of

safrole-DNA

adducts

and

increased

frequencies

of

sister

chromatid

exchanges

and

chromosome

aber-rations

in

the

hepatocytes

of

rats

[30]

.

Mughal

et

al.

demonstrated

a

positive

correlation

between

the

induction

of

micronuclei

frequency

in

the

peripheral

blood

erythrocytes

and

different

parameters

from

Comet

assay

in

the

peripheral

blood

lymphocytes

of

juvenile

rats

[42]

.

Using

these

techniques,

we

observed

a

sig-nificant

dose-dependent

increase

in

mean

Comet

tail

moment

in

peripheral

blood

leukocytes

(13.3–43.4-fold)

and

in

the

frequency

of

micronucleated

reticulocytes

(1.5–5.8-fold)

after

repeated

intraperitoneal

administration

of

SAFO

(15,

30,

45,

and

60

mg/kg)

to

mice.

In

contrast

to

our

positive

findings

on

the

genotoxicity

of

SAFO,

the

only

previous

study

that

attempted

to

detect

SAFO-DNA

adducts

in

vivo

by

_{P-postlabeling}

_analysis

_failed

_to

_detect

these

adducts

in

liver

tissue

isolated

24

h

after

intraperitoneal

injec-tion

of

a

single

dose

of

SAFO

(106.9

mg/kg)

or

safrole

(97.3

mg/kg)

to

male

Balb/C

mice

[17]

.

Further

studies

that

use

liquid

Fig.7.SAFOinducedMNRETsinmouseperipheralblood.SAFOwasadministered tomicebyintraperitonealinjectionatdosesof15,30,45and60mg/kgeveryother dayfor24days.Peripheralbloodwascollectedfromatailveinat24hafterthelast administration,andthenanalyzedusingamousemicronucleustest.Thenumberof micronucleatedreticulocyteswasrecordedbasedontheobservationof1000RETs permice.Valuesareindicatedasmean±standarddeviation,*p<0.05ascompared tocontrolmice(n=4).

chromatography/tandem

mass

spectrometry

analysis

to

quantitate

the

formation

of

SAFO-DNA

adducts

in

vivo

after

SAFO

or

safrole

exposure

may

be

needed

to

elucidate

the

discrepancy

between

the

previous

results

and

our

present

findings

[29]

.

We

observed

apparent

cytoplasmic

vacuolation

of

hepatocytes

around

the

central

vein

in

the

liver

from

mice

receiving

12

doses

of

SAFO

at

highest

dose

tested

(60

mg/kg)

(

Fig.

5

).

In

mice

that

received

45

mg/kg

SAFO,

the

cytoplasm

of

some

hepatocytes

adjacent

to

the

central

vein

area

was

finely

granular

and

lighter

than

that

of

con-trols.

In

mice

that

received

the

highest

dose

(60

mg/kg),

there

was

evidence

of

cytoplasmic

vacuolation

or

ballooning

degeneration

immediately

surrounding

the

central

vein,

whereas,

the

hepatic

cells

around

the

portal

vein

area

remained

intact

and

necrotic

cells

were

not

seen.

The

present

study

reported

that

SFAO-treated

mice

developed

liver

injury

as

manifested

by

a

rim

of

ballooning

degen-eration

of

hepatocytes

immediately

adjacent

to

the

central

vein.

This

characteristic

histopathological

change

induced

by

SAFO

is

very

different

to

that

seen

in

safrole-treated

mice

[43]

.

Hagan

et

al.

showed

that

safrole

administered

to

male

and

female

Osborne-Mendel

rats

at

doses

of

250,

500,

and

750

mg/kg/day

for

up

to

105

days

via

oral

intubation

and

that

safrole

administered

to

Swiss

mice

at

doses

of

250

and

500

mg/kg/day

for

60

days

induced

liver

changes

[43]

.

Microscopic

examination

revealed

evidence

of

hep-atic

cell

enlargement,

which

was

usually

focal

and

resulted

in

the

formation

of

nodules;

adenomatoid

hyperplasia;

cystic

necrosis;

fatty

metamorphosis;

and

bile

duct

proliferation

[43]

.

Although

the

mutagenicity

and

carcinogenicity

of

safrole

and

SAFO

have

been

known

for

more

than

30

years,

SAFO

has

recently

been

proposed

to

be

a

potential

candidate

for

cancer

therapy

because

of

its

anti-angiogenesis

and

apoptosis-inducing

activity

in

vitro.

SAFO

has

been

shown

to

have

anti-angiogenic

activity

by

trigging

apoptosis

via

a

mechanism

involving

the

overexpression

of

Fas,

integrin

beta4

and

P53,

attenuation

of

Ca

_-independent

phosphatidylcholine-specific

phospholipase

C

activity,

and

the

inhibition

of

intracellular

reactive

oxygen

species

generation

in

vascular

endothelial

cells

[44]

.

In

A549

human

cancer

cells,

SAFO

induced

apoptosis

by

up-regulating

Fas

and

FasL

[45]

and

activat-ing

caspase-3,

-8,

and

-9

[46]

.

Furthermore,

Yu

et

al.

reported

that

safrole

induced

apoptosis

in

human

oral

squamous

cell

carcinoma

HSC-3

cells

and

reduced

the

size

and

volume

of

HSC-3

solid

tumors

in

a

xenograft

athymic

nu/nu

mouse

model

[47]

.

These

data

sug-gest

that

SAFO

exhibits

anti-tumor

effects

in

vitro

and

in

mice.

Since

our

data

provide

the

clear

evidence

for

the

genotoxicity

of

SAFO

in

human

cells

in

vitro

and

in

mice,

the

potential

clinical

application

of

SAFO

in

cancer

treatment

needs

to

be

critically

re-evaluated.

In

conclusion,

we

show

clear

evidence

that

SAFO

induces

signif-icant

genotoxic

activity

in

vitro

and

in

vivo.

We

first

showed

that

SAFO

caused

significant

dose-dependent

increases

in

cytotoxicity,

mean

Comet

tail

moment,

and

micronucleated

binucleated

cells

in

human

HepG2

cells.

Moreover,

we

clearly

demonstrated

that

SAFO

exhibited

significant

genotoxic

effects

in

mice,

as

evidenced

by

sig-nificant

dose-dependent

increases

in

mean

Comet

tail

moment

in

peripheral

blood

leukocytes

and

in

the

frequency

of

micronucleated

reticulocytes

in

mouse

peripheral

blood.

SAFO

also

induced

liver

damage,

typically

manifested

as

a

rim

of

ballooning

degeneration

of

hepatocytes

immediately

adjacent

to

the

central

vein.

SAFO

should

not

be

used

directly

under

normal

circumstances;

therefore,

our

findings

strongly

suggest

the

need

to

carefully

evaluate

the

poten-tial

use

of

SAFO

in

cancer

therapy.

Our

data

will

provide

another

potential

mechanism

for

the

mutagenicity

and

carcinogenicity

of

safrole.

Further

mechanistic

studies

on

the

genotoxicity

and

muta-genicity

of

SAFO

may

be

needed

to

adequately

assess

its

role

in

safrole

carcinogenicity

and

to

help

assess

the

potential

health

risk

to

humans

due

to

daily

consumption

of

safrole

from

edible

herbs,

spices,

and

Chinese

medicinal

plants.

Conflict

of

interest

The

authors

declare

that

there

are

no

conflicts

of

interest.

Acknowledgements

We

express

our

deepest

gratitude

to

Professor

Tin-Yun

Ho

for

his

continuous

assistance

during

different

phases

of

this

study.

We

thank

Tsai-Chung

Li

and

Shin-Yuh

Yang

for

their

help

in

statisti-cal

analysis.

The

technical

assistance

from

Miss

Ju-Yu

Cheng,

Miss

Ya-Ying

Lin,

Miss

Hui-Ni

Cheng,

and

Dr.

Shun-Ting

Chou

is

grate-fully

acknowledged.

Part

of

the

experimental

work

was

supported

by

Grants

from

the

National

Science

Council

(NSC

97-2815-C-039-058-B)

and

China

Medical

University

(CMU

96-181)

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