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Mutation Research 677 (2009) 5965

Contents lists available atScienceDirect

Mutation Research/Genetic Toxicology andEnvironmental 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

Isothiocyanate from the Tunisian radish (Raphanus sativus) preventsgenotoxicity of Zearalenonein vivoandin vitro

Jalila Ben Salah-Abbsa,, Samir Abbs a, Zouhour Ouanes b, Mosaad A. Abdel-Wahhab c,Hassen Bacha b, Ridha Oueslati a

a Laboratory of Immunology, Environmental Microbiology and Cancerology, Faculty of Sciences Bizerte, 7021 Zarzouna, Tunisiab Laboratory of Research on Biologically Compatible Compounds, Faculty of Dentistry, Avicenne street, 5019 Monastir, Tunisiac Food Toxicology & Contaminants Department National Research Centre, Dokki, Cairo, Egypt

a r t i c l e i n f o

Article history:

Received 6 January 2009

Received in revised form 28 January 2009

Accepted 14 May 2009

Available online 6 June 2009

Keywords:

Isothiocyanates

Raphanus sativus

Zearalenone

Bone-marrow

Splenocytes

Micronuclei

Chromosome aberrations: Mitotic index

DNA fragmentation

a b s t r a c t

Zearalenone (ZEN) is a naturally occurring contaminant of animal feed that has been implicated in sev-

eral mycotoxicoses in farm livestock. Recently some information has become available indicating that

ZEN caused cancer or at least increased its prevalence, although the mechanism of action is unknown.

Many papers mentioned that exposure to ZEN results in genotoxicity and DNA damage. Therefore, we

investigated the chemo-preventive role of 4-(methylthio)-3-butenyl isothiocyanate (MTBITC) extracted

from Tunisian Raphanus sativus(radish) on the cytogenetic effect of ZEN in Balb/c mice and in in vitro

cultures of mouse lymphocytes isolated from mouse spleen. We determined chromosome aberrations

and micronuclei as well as the mitotic index and DNA fragmentation following ZEN treatment alone or in

combination with MTBITC. This reportis thefirst to provide evidence of a statistically significantdecrease

of structural chromosome aberrations and micronuclei associated with an augmentation of the mitotic

index and prevention of DNA fragmentation in all mice treated with ZEN-MTBITC and in mouse lympho-

cyte cultures. The MTBITCalone wassafe and succeeded in reducing the toxicity of ZEN by counteracting

its deleterious effect, thus protecting against the genotoxicity and clastogenicity from ZEN.

2009 Elsevier B.V. All rights reserved.

1. Introduction

Zearalenone (ZEN) represents a class of dietary contaminant

mycotoxins that can cause genotoxicity and carcinogenicity to

humansand animals. It is an estrogenic fungal metabolite that con-

taminates animal feed andhuman food. ZEN poses a healthrisk not

only to humans but also to livestock and, as a consequence, may

cause economic losses either by unfavourable effects on domestic

animals themselves or by an increased potential for health effects

in human beings from consuming mycotoxin-contaminated edi-

ble animal products. ZEN has been detected in meat and milk. It

has been implicated in several mycotoxicoses in humans and farm

livestock[1]. In an NTP cancer bioassay a dose-related incidenceof hepatocellular adenomas and endometrial adenocarcinomas

were seen in female mice [2] and a hyperplastic or neoplastic

endometrium has been reported in humans at normal levels of

Abbreviations: MTBITC, 4-(methylthio)-3-butenyl isothiocyanate; DCCM,

dextran-coated charcoal medium; LAK, lymphokine-activated killer; MNPCEs,

micronucleated polychromatic erythrocytes; ZEN, zearalenone. Corresponding author. Tel.: +216 72 59 19 06x202; fax: +216 72 59 05 66.

E-mail addresses:[email protected](J. Ben Salah-Abbs),

[email protected](R. Oueslati).

exposure [3]. Moreover, zearalenone and its estrogenic metabo-

lites showed a positive DNA-damaging effect in Bacillus subtilis,

SOS repair in bacteria, cell cytotoxicity[4]and teratological effects

in mouse embryos [5]. Pfohl-Leszkowicz et al. [6] demonstrated

that ZEN caused DNA adducts in female mice and rats treated by

intraperitoneal injection or orally. In addition, ZEN induced sister

chromatid exchange, chromosome aberrations and polyploidy in

Chinese hamster ovary cells in vitro in the absence of exogenous

metabolic activation and in bovine lymphocyte cultures[7]. It also

produced DNA fragmentation, cell-cycle arrest and apoptosis [8].

Hsia et al. [9]indicated that ZEN and other fusarotoxin induced

a high risk for oesophageal cancer. In addition, precocious puber-

tal changes have been observed in young children in whom ZENwas detected in plasma [10]. Furthermore, recently Abbs et al. [11]

reporteda significant cytogeneticeffect on Balb/c micetreatedwith

ZEN after 48 h.

Until now, the carcinogenicity of ZEN is still questionable since

it is classified by IARC in Group 3, but the protection and the risk

reduction by deliberate intervention to enhance mostly endoge-

nous mechanisms that reduce the risk arising from the potential

genotoxicity, mutagenicity and carcinogenicity of this estrogenic

mycotoxin, are axiomatic. The protection of humans or high-risk

animals from DNA damage through identification and application

of chemo-protective agents of low toxicity that are already present

1383-5718/$ see front matter 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.mrgentox.2009.05.017
http://www.sciencedirect.com/science/journal/13835718http://www.elsevier.com/locate/gentoxhttp://www.elsevier.com/locate/mutresmailto:[email protected]:[email protected]://localhost/var/www/apps/conversion/tmp/scratch_1/dx.doi.org/10.1016/j.mrgentox.2009.05.017http://localhost/var/www/apps/conversion/tmp/scratch_1/dx.doi.org/10.1016/j.mrgentox.2009.05.017mailto:[email protected]:[email protected]://www.elsevier.com/locate/mutreshttp://www.elsevier.com/locate/gentoxhttp://www.sciencedirect.com/science/journal/13835718


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60 J. Ben Salah-Abbs et al. / Mutation Research 677 (2009) 5965

in the human diet is a challenging task. Isothiocyanates are found

in cruciferous vegetables such as broccoli and radish (R. sativus). 4-

(Methylthio)-3-butenyl isothiocyanate (MTBITC) is one of the most

extensively studied compounds in this class, and recent epidemi-

ological and experimental studies show that MTBITC and other

isothiocyanates may possess promising cancer chemo-preventive

activities [12,13]. In regard to prostate cancers, MTBITC has been

found to reduce tumour cell growth by affecting signalling path-

ways, arresting the cell cycle and causing apoptotic cell death in

vivo [14,15]or in vitro [16,17].However, the exact mechanisms of

its chemo-preventive effects are not fully understood. The most

important principles of chemoprevention by cruciferous vegeta-

bles are inhibition of phase-I enzymes and induction of phase-II

enzymes, which leadsto detoxificationand accelerated excretion of

toxicants.

In the present study, we investigated the chemo-preventive role

of 4-(methylthio)-3-butenyl isothiocyanate (MTBITC) extracted

from Tunisian R. sativus on the genotoxic effect of ZEN in Balb/c

mice and inin vitrocultures of mouse lymphocytes.

2. Materials and methods

2.1. Chemicals

ZEN and methyl-isothiocyanate were purchased as pure crystals from

SigmaAldrich, France. The MTBITC was extracted and purified from the root ofR.

sativus according to the method described previously by Esaki and Onozaki [18]

with a slight modification. The purity of the MTBITC was 96.7% as estimated by

HPLC. Foetal calf serum, RPMI 1640, Hepes and l-glutamine were purchased from

Gibco, Life Technology. Sodium pyruvate, 2-mercaptoethanol and antibiotics (peni-

cillin, streptomycin sulphateand amphotericin) were obtained fromSigmaAldrich,

France. All other chemicals were of the highest purity commercially available.

2.2. Extraction, measurement and isolation of isothiocyanate from R. sativus

Whole roots ofR. sativusgrowing in Tunisia were washed with water, peeled,

and then cut into 2 cm3 cubes. The material was incubated for 30 min at 25 C in

order to increase the MTBITC level. It is known from the literature that the amount

ofMTBITC produced ingrated radishreachesa maximum at 10minafter grating and

then remains the same for another 50 min[19].A 1020 g aliquot of the gratedR.

sativus wasfiltered, andthe residuewas extractedtwicewith 30 ml ofdistilledwater.

Theaqueousextractswerecombinedand dilutedto 100ml topreparea crudesamplesolution in which the concentration of MTBITC was analysed. To avoid degradation

of MTBITCin alkaline pHpriorto this analysis,fivel of6M HCl was added to0.3ml

of theaqueoussampleextract,and thesolutionwas extractedthreetimes, eachtime

with 150l of ethyl acetate, chloroform, and n-hexane, respectivelyin order to con-

centrate the MTBITC. The n-hexane extract wasdriedin a Buchi model RE121rotary

evaporator at 5060 C under vacuum and then dissolved in CH2Cl2. The solution

was dried on anhydrous Na2SO4 and filtered. The filtrate was concentrated under

a stream of nitrogen until dryness. The level of total MTBITC was measured using

themethod ofZhanget al. [20] witha slightmodification. Inbrief, 50l ofn-hexane

extract from R. sativus wasdilutedwitha mixture of0.45ml ofmethanol and0.45ml

of 50mM Na2B4 O7HCl buffer (pH 8.5), then 50l of 8 mM 1,2-benzenedithiol was

added and mixed well in a 1.5 ml plastic tube. The tube was heated at 65 C for

1 h, and then the MTBITC content was calculated by measuring the absorbance

of the sample at 365nm and comparing with a linear standard equation derived

from the absorbance readings of a serial dilution of known methyl-isothiocyanate

concentrations.

The MTBITC extract in n-hexane was measured by gas chromatography on aShimadzu (Kyoto, Japan) model GC-12A instrument with flame ionization detec-

tor (FID-GC). The operating conditions for gas chromatography were as follows:

injection volume, 1.0l; injector and detector temperature, 250 C; DB-5 column

(25m0.2mm, 0.33m film thickness; J&W Scientific, Folsom, CA); column tem-perature 70 C for 70s then increasing to 170 C at 3 C/min; carrier gas, 180kPa

He; injection, split-less (closed for 70s). The obtained MTBITC was identified by

GCMSand NMRspectroscopyanalysesof desulfoderivates(EEC,1990).The material

was freeze-dried and the desired dose (mg of lyophilized aqueous extract/kg body

weight) was then prepared in distilled water. The aqueous extracts were prepared

daily, just b efore administration.

2.3. In vivo study

2.3.1. Dosing

The testwas performedin compliancewith the EuropeanCommission Directive

2000/32/EC andthe OECDGuideline 474 [21]. Dosing wasby theoral route during ten

successive dailytreatmentsfollowed by one24 h samplingtime. ZENwas suspended

inolive oil. Thesuspension wasusedat 40mg/kgbw forthetoxicityand genotoxicity

tests.

2.3.2. Animals and experimental design

Young male and female Balb/c mice, aged 78 weeks at the time of dosing,

wereobtained fromthe Animal house of thePasteurInstitute,Tunisia.Animals were

placed in groups of 10 in polypropylene cages in a ventilated cabinet, free from any

sources of chemical contamination. The temperature was 22 2 C, and humidity

was 5515%. The lighting time provided was 12h per day from 8 am to 8 pm.

The test was carried out using the ZEN dose of 40 mg/kg bw, described as the

highest dose that causes no mortality, but may give rise to the appearance of signsof toxicity used in our previous work [11,22].

After random distribution, animals were distributed into six groups of 10 mice

and treated orally for 10 days as follows: groups (1) and (2) with the vehicle alone

(distilled water or olive oil); (3)treated with MTBITC(5 mg/kg bw); (4)treatedwith

ZEN (40mg/kg bw) and (5) with the extract (5 mg/kg bw)+ ZEN (40mg/kg b.w); (6)

treated with Mitomycin C (1mg/kgbw) or Colchicin3 mg/kg as positive controlsfor

chromosome aberrations and micronuclei, respectively.

2.3.3. Slide preparation and counting

At the sampling times, the animals were sacrificed by CO2asphyxia; the femurs

were removed, the bone-marrow was extracted, the cell suspensions were cen-

trifuged,and thecells were spreadon slides. Thesmearswerestainedwitha derived

MayGrnwaldGiemsa technique.

2.3.4. Micronucleus test in mouse bone-marrow

Aftercodingthe slides, thenumberof polychromaticerythrocytes harboringone

or moremicronuclei polychromaticerythrocyte cells(MNPCEs) was determined for

at least 2000 polychromatic erythrocytes.

2.3.5. Chromosome aberration test in mouse bone-marrow

Concerning the chromosome aberration study, 100 cells colchicin-arrested in

metaphase were examined for chromosome abnormalities at a magnification of

1000with an optical microscope (Carl Zeiss, Germany), from each of three repli-

cates (300 metaphases per dose level) for negative controls, positive controls and

treatment groups. Chromosome aberrations were identified according to criteria

described by Savage [23]. Metaphases with chromosome breaks, gaps, rings and

centric fusions (robertsonian translocations) were recorded and expressed as per-

centage of total metaphases per group.

2.4. In vitro study

2.4.1. Extraction and treatment of mouse spleen lymphocytes for the micronucleus

and chromosome aberration testsLymphocytes from spleen were isolated by gently rubbing the spleen from

non-treated animals aseptically through sterile (100m) nylon mesh. Isolated lym-

phocytes were washed in RPMI1640 medium supplemented with 1% foetal calf

serum. On average, from each animal, a minimum of 4 108 lymphocytes were

recovered and cultured in a humidified atmosphere containing 5% CO 2 at 37C.

For the micronucleus test and the chromosome aberration assay, a minimum of

18106 cells were seeded in each 25cm2 plastic flask containing 6 ml of complete

culture medium: RPMI 1640with 25mM Hepes, sodiumbicarbonate2 mg/ml,2 mM

l-glutamine, supplemented with 30% serum-free medium DCCM-1, 10% LAK super-

natant, 10% heat-inactivated foetal bovine serum, 50M 2 mercaptoethanol, 1 mM

sodium pyruvate and antibiotics (100 IU/ml penicillin, 100g/ml streptomycin sul-phate and 2.5g/ml amphotericin).

The50% growth-inhibiting concentrations(IC 50)of ZENand MTBITC in lympho-

cyteswerefoundto be15Mand50M, respectively. Cellculturesweredistributed

infive treatment groups asfollows:(1) thenegative control,50% ethanolin water;(2)

with 25M MTBITC; (3) with 15M ZEN; (4) with 15M ZEN+ 25M MTBITC and

(5) treated with colchicin (0.3M) or Mitomycin C (0.3M) as a positive control formicronuclei and chromosome aberrations, respectively. All tests were performed in

triplicate. The volume of added solutions never exceeded 1% of the culture medium

(30l in 3 ml) to avoid possible toxicity of the vehicle. Cultures to be used for chro-

mosome aberration analyses were incubated with 5-bromo-2-deoxyuridine at a

final concentration of 5M for a total of 24h, in a 5% CO2 incubator at 37 C andrelative humidity (100% nominal).

2.4.2. Micronucleus test

In order to analyse micronuclei in binucleated lymphocytes, culture medium

was supplemented with cytochalasin B, 24h after initiation, at a final concentra-

tion of 6g/ml. Lymphocytes were cultured for a total time of 4 8 h. For detection of

micronuclei, lymphocytes weretransferred to round-bottomtubes, centrifugedand

treated with cold (4 C) hypotonic solution (KCl 0.075M), and centrifuged immedi-

ately. Oncepelleted,cells weregently re-suspended in the fixative (methanol:acetic

acid,5:1, v/v).After three additionalwashes with freshly preparedfixative,air-dried

slides wereprepared according to a standardprotocol. The frequencyof micronuclei

was assessed by scoring 1000 binucleated lymphocytes from each treatment.


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J. Ben Salah-Abbs et al. / Mutation Research 677 (2009) 5965 61

Table 1

Identification, structure and activity of MTBITC extracted from R. sativus.An important quantity of total MTBITC was present in the R. sativusextract. The amount of MTBITC

reached 38.984.2mol/100mg and the IC50 activity of MTBITC in mouse lymphocytes was 50M.

Name of isothiocyanate Isothiocyanate side chain Abbreviation Isothiocyanate (mol/100 mg) IC50 activity (M)

4-(Methylthio)-3-butenyl isothiocyanate MTBITC 38.98 4.2 50

Table 2

Mean valueof micronuclei detected in polychromatic erythrocytesof bone-marrowcells in Balb/c mice treated with ZEN alone or in combination with MTBITC

(meansSE).

Treatments Micronucleus Prevention (%)

Control water 1.33 0.27a

Control olive oil 1.33 0.27a

Colchicin 3 mg/kg bw 65 6.12b

MTBITC 5 mg/kg bw 2.21 0.23a

ZEN 40 mg/kg bw 47.23 5.17b

MTBITC + ZEN 3.11 0.27a 84.8

Despite the evident signs of genotoxicity shown by the animals in ZEN group, the

results show that the mean number of MNPCE in bone marrow was not affected

by treatment with MTBITC. A significant increase in the incidence of MNPCE was

observed in ZEN- and colchicin-treated groups, compared with control. Treatment

with MTBITC significantly decreased the frequencies of MNPCEs and this reduction

reached 84.8%.Withineach column, meanswith thesame letter arenot significantly

different (P0.05).

2.4.3. Chromosome aberration test

For analysis of chromosome aberrations, lymphocytes were transferred to cen-

trifuge tubes and treated with hypotonic solution (KCl 0.075 M) for 5 min at 37C

and fixedwith chilled methanol:acetic acid(4:1). Afterthreeadditional washes with

freshly prepared fixative, air-dried preparations were made according to standard

procedures and stained with an aqueous Giemsa solution (Gurr R-66).

2.4.4. Mitotic index analysis

The mitotic index was evaluated by counting at least 1000 cells per treatment:

the number of dividing cells (prophases and metaphases) was divided by the total

number of cells.

2.4.5. DNA fragmentation analysis

Mouse lymphocytes were cultured in medium containing ethanol as a vehicle,

MTBITC (25M) and ZEN alone (15M) or in combination with MTBITC (25M).After a 48 h incubation DNA was extracted and analyzed by electrophoresis on a 1%

agarose gel with TrisBorateEDTA.

2.4.6. Statistical analysis

Data on micronuclei and cytotoxicity were analyzed using Studentst-test. The

differences in mean percentages between treated and control groups and among

treated groups for numerical aberrations were evaluated with the Chi-square test

[24]. Thesignificanceof differenceswas based ona probabilityofP0.05 and0.005.

3. Results

3.1. Phytochemical study

Thephytochemicalresultssummarized in Table1 showthe pres-

ence of an important quantity of total MTBITC in R. sativusextract.

The amount of MTBITC reached 38.984.2mol/100 mg of fresh

weight of the R. sativus, corresponding to 7.17 mg (7%), and theIC50 activity of MTBITC in mouse lymphocytes was 50M. In thisstudy 1/2 IC50was used in the cell cultures.

3.2. In vivo study

3.2.1. Micronucleus analysis

The results of the micronucleus test in animals treated with

ZEN and MTBITC are summarized in Table 2. Despite the evi-

dent signs of genotoxicity shown by the animals in the ZEN

group, the results show that the mean number of micronucle-

ated PCE (MNPCE) in bone marrow was not affected by treatment

with MTBITC. A significant increase in the incidence of MNPCE

was observed in the ZEN- and in colchicin-treated groups com-

pared with the controls. Treatment with MTBITC significantlydecreased the frequency of MNPCEs and this reduction reached

84.81%.

3.2.2. Chromosome aberration analysis

Structural aberrations included centric fusions, gaps, rings and

chromosomal breaks (Table 3). The data show that treatment

with ZEN alone resulted in a significant increase in chromosome

aberrations, mainly centric fusion and chromosome breaks, in

bone-marrow cells. No significant differences were observed in the

group treated with MTBITC alone compared with controls. Treat-

ment with MTBITC to ZEN-treated mice resulted to a significant

decrease in the total chromosomal aberration induced by ZEN.

The reduction in chromosomal aberrations resulting from MTBITC

treatment of mice treated with ZEN reached 82.2%.

3.3. In vitro study

3.3.1. Micronucleus test

The data for micronuclei in mouse lymphocyte cells are pre-

sented in Fig. 1. To determine the frequencies of micronuclei in

binucleated mouse lymphocytes, 1000 cells were analysed for each

testconcentration, in triplicate. No significant difference was found

for induction of micronuclei between the control and MTBITC-

exposed cells. The frequency of micronuclei in binucleated cells

was significantlyincreased aftertreatment with ZEN. The micronu-

cleus frequency dropped by 93% in cells treated with ZEN+ MTBITC

(Fig. 1).

Table 3

Effect of MTBITC andits protective roleon chromosomal aberrations in bone-marrow cellsof Balb/c micetreatedwith ZENby theoral route. Theresultsindicatethat treatment

with ZEN alone results in a significant increase in chromosome aberrations, mainly centric fusions and chromosome breaks, in bone-marrow cells. No significant differences

were observed in the group treated with MTBITC alone compared with controls. Treatment with MTBITC of ZEN-treated mice resulted to a significant decrease in the total

chromosomal aberration induced by ZEN. The reduction in chromosomal aberrations resulting from MTBITC treatment of mice that had received ZEN reached 82.2%. Within

each column, means with the same letter are not significantly different ( P0.05).

Groups Structural aberrations Total aberrations

Centric fusion Chromosomal breaks Gaps Rings

Control water 0.33 0.27 1.66 0.27 1.33 0.00 0.66 0.27 3.65 0.47a

Control olive oil 0.33 0.66 0.66 0.00 1.66 0.00 1.33 0.00 3.65 0.43a

Mitomycin C 1mg/kg bw 29.33 0.88 13.66 1.33 3.66 0.33 9.33 0.33 55.01 3.11b

MTBITC 5 mg/kg bw 1.33 0.33 0.66 0.33 1.00 0.03 1.00 0.26 3.99 0.46a

ZEN 40 mg/kg bw 19.00 1.52 11.33 2.33 5.00 0.57 5.66 1.33 40.99 4.62b

MTBITC + ZEN 2.33 0.23 1.66 0.33 2.00 0.19 1.33 0.48 7.32 1.11a


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64 J. Ben Salah-Abbs et al. / Mutation Research 677 (2009) 5965

[4042]. In a human cross-over study, the impact of a crucifer-

ous vegetable diet on cytochrome P-450 1A2,N-acetyltransferase-

and xanthine oxidase activities was investigated [43]. The par-

ticipants received first a vegetable-free diet followed by a diet

enriched in cruciferous vegetables (raddish, cauliflower, broccoli

and cabbage). After a consumption period of 6 consecutive days, a

pronounced increase in CYP-450 1A2 activity was seen whereasN-acetyltransferase- and xanthine oxidase activities were not

affected.

The possiblemechanism of protectionofferedby MTBITC against

ZEN-induced genotoxicity is its ability to inhibit the oxidative

process by neutralizing reactive oxygen species as well as its inter-

action withoestrogen receptors thatare occupied by the mycotoxin

ZEN. In addition, it could not be excluded that MTBITC acts as

anti-genotoxicant, which enhances the DNA-repair system or DNA

synthesis, which is demonstrated by the disappearance of the new

DNA band caused by ZEN treatment. Further investigations are

needed to better understand the ways in which MTBITC exerts its

highly efficient prevention against epigenetic and genotoxic effects

of ZEN.

In summary, we have shown that MTBITC extracted from

TunisianR. sativus exerts its chemo-protective abilities by modu-

lating the activities of ZEN-sensitive enzymes and by protecting

DNA from ZEN-induced damage. These results may prove usefulin developing MTBITC-based chemoprotection regimes. However,

further work needs to be done to optimize the doses needed for

application in medicine, food additives and to determine the main

mechanism by which one could counteract the oxidative stress and

protect against the ZEN-induced genotoxicity. Especially, it is noted

that MTBITC itself appears to have no harmful effect and is able to

prevent ZEN-induced toxicityin vitroandin vivo.

Conflict of interest

The authors of this manuscript have no financial or personal

relationship with any organization which could influence the work

on the compound in this manuscript.

Acknowledgements

This research was supported by the Ministre de

lEnseignement Suprieur, la Recherche Scientifique et de la

Technologie , Tunisia (Laboratoire dImmunologie et Microbiolo-

gie Environnementale et cancrologie: IMEC). We would like to

thank Mr Kais Ben Othmen for his help to improve the English of

this manuscript.

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