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Mutation Research 677 (2009) 59–65

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Mutation Research/Genetic Toxicology and
Environmental Mutagenesis
journal homepage: www.elsevier.com/locate/gentox
Community address: www.elsevier.com/locate/mutres

Isothiocyanate from the Tunisian radish (Raphanus sativus) prevents
genotoxicity of Zearalenone in vivo and in vitro
Jalila Ben Salah-Abbès a,∗ , Samir Abbès a , Zouhour Ouanes b , Mosaad A. Abdel-Wahhab c ,
Hassen Bacha b , Ridha Oueslati a
a
b
c

Laboratory of Immunology, Environmental Microbiology and Cancerology, Faculty of Sciences Bizerte, 7021 Zarzouna, Tunisia
Laboratory of Research on Biologically Compatible Compounds, Faculty of Dentistry, Avicenne street, 5019 Monastir, Tunisia
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 several 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 report is the first to provide evidence of a statistically significant decrease
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 lymphocyte cultures. The MTBITC alone was safe 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
humans and animals. It is an estrogenic fungal metabolite that contaminates animal feed and human food. ZEN poses a health risk 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 edible 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 incidence
of 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: jalila.bensalah@yahoo.fr (J. Ben Salah-Abbès),
oridha2003@yahoo.fr (R. Oueslati).
1383-5718/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrgentox.2009.05.017

exposure [3]. Moreover, zearalenone and its estrogenic metabolites 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 pubertal changes have been observed in young children in whom ZEN
was detected in plasma [10]. Furthermore, recently Abbès et al. [11]
reported a significant cytogenetic effect on Balb/c mice treated with
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 endogenous 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

DB-5 column (25 m × 0. The test was carried out using the ZEN dose of 40 mg/kg bw.w).5 ml plastic tube. 5:1. lymphocytes were transferred to round-bottom tubes. injection. Folsom. arresting the cell cycle and causing apoptotic cell death in vivo [14.2 mm. a minimum of 18 × 106 cells were seeded in each 25 cm2 plastic flask containing 6 ml of complete culture medium: RPMI 1640 with 25 mM Hepes. and n-hexane. a minimum of 4 × 108 lymphocytes were recovered and cultured in a humidified atmosphere containing 5% CO2 at 37 ◦ C. which leads to detoxification and accelerated excretion of toxicants. Micronucleus test In order to analyse micronuclei in binucleated lymphocytes. 10% LAK supernatant. but may give rise to the appearance of signs of toxicity used in our previous work [11. The purity of the MTBITC was 96. The n-hexane extract was dried in a Buchi model RE 121 rotary evaporator at 50–60 ◦ C under vacuum and then dissolved in CH2 Cl2 .1. Japan) model GC-12A instrument with flame ionization detector (FID-GC). 50 ␮M 2 mercaptoethanol. gaps. respectively.3 ␮M) as a positive control for micronuclei and chromosome aberrations. It is known from the literature that the amount of MTBITC produced in grated radish reaches a maximum at 10 min after grating and then remains the same for another 50 min [19]. The material was incubated for 30 min at 25 ◦ C in order to increase the MTBITC level.7% as estimated by HPLC. Sodium pyruvate. v/v). positive controls and treatment groups. Germany).13]. (4) treated with ZEN (40 mg/kg bw) and (5) with the extract (5 mg/kg bw) + ZEN (40 mg/kg b.60 J. The material was freeze-dried and the desired dose (mg of lyophilized aqueous extract/kg body weight) was then prepared in distilled water.4.3.3. The temperature was 22 ± 2 ◦ C.3. culture medium was supplemented with cytochalasin B.3. the femurs were removed. France.3 ␮M) or Mitomycin C (0. On average. For the micronucleus test and the chromosome aberration assay. Dosing The test was performed in compliance with the European Commission Directive 2000/32/EC and the OECD Guideline 474 [21]. J&W Scientific. and the solution was extracted three times.17]. chloroform. 0. Metaphases with chromosome breaks. 1 mM sodium pyruvate and antibiotics (100 IU/ml penicillin. were obtained from the Animal house of the Pasteur Institute.4. 4(Methylthio)-3-butenyl isothiocyanate (MTBITC) is one of the most extensively studied compounds in this class. The obtained MTBITC was identified by GC–MS and NMR spectroscopy analyses of desulfoderivates (EEC. from each of three replicates (300 metaphases per dose level) for negative controls. 2. and humidity was 55 ± 15%. the exact mechanisms of its chemo-preventive effects are not fully understood. Animals were placed in groups of 10 in polypropylene cages in a ventilated cabinet. 24 h after initiation. The level of total MTBITC was measured using the method of Zhang et al. 50 ␮l of n-hexane extract from R. Chromosome aberrations were identified according to criteria described by Savage [23]. Cultures to be used for chromosome aberration analyses were incubated with 5-bromo-2 -deoxyuridine at a final concentration of 5 ␮M for a total of 24 h. described as the highest dose that causes no mortality. 10% heat-inactivated foetal bovine serum. carrier gas. In the present study. France. 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).1. Dosing was by the oral route during ten successive daily treatments followed by one 24 h sampling time. 2. Once pelleted.2. CA). 100 ␮g/ml streptomycin sulphate and 2.45 ml of 50 mM Na2 B4 O7 –HCl buffer (pH 8. five l of 6 M HCl was added to 0. 2. 250 ◦ C. sativus was diluted with a mixture of 0. aged 7–8 weeks at the time of dosing. The solution was dried on anhydrous Na2 SO4 and filtered.3 ml of the aqueous sample extract. centrifuged and treated with cold (4 ◦ C) hypotonic solution (KCl 0. After random distribution. The smears were stained with a derived May–Grünwald–Giemsa technique. sativus). The suspension was used at 40 mg/kg bw for the toxicity and genotoxicity tests. respectively in order to concentrate the MTBITC. sativus on the genotoxic effect of ZEN in Balb/c mice and in in vitro cultures of mouse lymphocytes. [20] with a slight modification. Materials and methods 2. each time with 150 l of ethyl acetate. To avoid degradation of MTBITC in alkaline pH prior to this analysis. The frequency of micronuclei was assessed by scoring 1000 binucleated lymphocytes from each treatment. 180 kPa He. RPMI 1640.5). Chromosome aberration test in mouse bone-marrow Concerning the chromosome aberration study. respectively. at a final concentration of 6 ␮g/ml. and then the MTBITC content was calculated by measuring the absorbance of the sample at 365 nm and comparing with a linear standard equation derived from the absorbance readings of a serial dilution of known methyl-isothiocyanate concentrations. For detection of micronuclei. The aqueous extracts were combined and diluted to 100 ml to prepare a crude sample solution in which the concentration of MTBITC was analysed. 2. 50% ethanol in water.075 M). The filtrate was concentrated under a stream of nitrogen until dryness. column temperature 70 ◦ C for 70 s then increasing to 170 ◦ C at 3 ◦ C/min.0 ␮l. we investigated the chemo-preventive role of 4-(methylthio)-3-butenyl isothiocyanate (MTBITC) extracted from Tunisian R. In regard to prostate cancers. Chemicals ZEN and methyl-isothiocyanate were purchased as pure crystals from Sigma–Aldrich.5 ␮g/ml amphotericin). The MTBITC extract in n-hexane was measured by gas chromatography on a Shimadzu (Kyoto. Micronucleus test in mouse bone-marrow After coding the slides. Hepes and l-glutamine were purchased from Gibco.2. The lighting time provided was 12 h per day from 8 am to 8 pm. The MTBITC was extracted and purified from the root of R. sativus growing in Tunisia were washed with water. 2 mM l-glutamine. the number of polychromatic erythrocytes harboring one or more micronuclei polychromatic erythrocyte cells (MNPCEs) was determined for at least 2000 polychromatic erythrocytes. injector and detector temperature. Isothiocyanates are found in cruciferous vegetables such as broccoli and radish (R. Animals and experimental design Young male and female Balb/c mice. Extraction and treatment of mouse spleen lymphocytes for the micronucleus and chromosome aberration tests Lymphocytes from spleen were isolated by gently rubbing the spleen from non-treated animals aseptically through sterile (100 ␮m) nylon mesh. Cell cultures were distributed in five treatment groups as follows: (1) the negative control. cells were gently re-suspended in the fixative (methanol:acetic acid. However.2. All tests were performed in triplicate. (2) with 25 ␮M MTBITC. In vitro study 2. from each animal. then 50 ␮l of 8 mM 1. just before administration.2-benzenedithiol was added and mixed well in a 1. 2. 2. The 50% growth-inhibiting concentrations (IC 50) of ZEN and MTBITC in lymphocytes were found to be 15 ␮M and 50 ␮M.22].15] or in vitro [16. 2.3. rings and centric fusions (robertsonian translocations) were recorded and expressed as percentage of total metaphases per group. and the residue was extracted twice with 30 ml of distilled water. The volume of added solutions never exceeded 1% of the culture medium (30 ␮l in 3 ml) to avoid possible toxicity of the vehicle. and then cut into 2 cm3 cubes. and the cells were spread on slides. 1990). (3) treated with MTBITC (5 mg/kg bw). Ben Salah-Abbès et al. Life Technology. (6) treated with Mitomycin C (1 mg/kg bw) or Colchicin 3 mg/kg as positive controls for chromosome aberrations and micronuclei. supplemented with 30% serum-free medium DCCM-1. The tube was heated at 65 ◦ C for 1 h.3. Isolated lymphocytes were washed in RPMI1640 medium supplemented with 1% foetal calf serum. air-dried slides were prepared according to a standard protocol. 100 cells colchicin-arrested in metaphase were examined for chromosome abnormalities at a magnification of 1000× with an optical microscope (Carl Zeiss. and centrifuged immediately. and recent epidemiological and experimental studies show that MTBITC and other isothiocyanates may possess promising cancer chemo-preventive activities [12.33 ␮m film thickness. the animals were sacrificed by CO2 asphyxia. measurement and isolation of isothiocyanate from R. All other chemicals were of the highest purity commercially available. sativus according to the method described previously by Esaki and Onozaki [18] with a slight modification. in a 5% CO2 incubator at 37 ◦ C and relative humidity (100% nominal). In brief.5. . The operating conditions for gas chromatography were as follows: injection volume. Tunisia.4.45 ml of methanol and 0. MTBITC has been found to reduce tumour cell growth by affecting signalling pathways. the cell suspensions were centrifuged. 1. ZEN was suspended in olive oil. free from any sources of chemical contamination.4. (4) with 15 ␮M ZEN + 25 ␮M MTBITC and (5) treated with colchicin (0. respectively. 2-mercaptoethanol and antibiotics (penicillin. sodium bicarbonate 2 mg/ml. In vivo study 2. A 10–20 g aliquot of the grated R. The most important principles of chemoprevention by cruciferous vegetables are inhibition of phase-I enzymes and induction of phase-II enzymes.3. 2. (3) with 15 ␮M ZEN. Extraction. streptomycin sulphate and amphotericin) were obtained from Sigma–Aldrich. split-less (closed for 70 s). sativus was filtered. The aqueous extracts were prepared daily. Lymphocytes were cultured for a total time of 48 h. / Mutation Research 677 (2009) 59–65 in the human diet is a challenging task.1. Foetal calf serum. peeled. 2. the bone-marrow was extracted. sativus Whole roots of R. Slide preparation and counting At the sampling times. After three additional washes with freshly prepared fixative.

means with the same letter are not significantly different (P ≤ 0. The significance of differences was based on a probability of P ≤ 0.005. To determine the frequencies of micronuclei in binucleated mouse lymphocytes. A significant increase in the incidence of MNPCE was observed in the ZEN.2. Treatment with MTBITC of ZEN-treated mice resulted to a significant decrease in the total chromosomal aberration induced by ZEN.8%. lymphocytes were transferred to centrifuge tubes and treated with hypotonic solution (KCl 0.2.33 1. Chromosome aberration analysis Structural aberrations included centric fusions.33 1. in bone-marrow cells. DNA fragmentation analysis Mouse lymphocytes were cultured in medium containing ethanol as a vehicle. the results show that the mean number of MNPCE in bone marrow was not affected by treatment with MTBITC.2.00 0.00 0.00 5.4.33 0. Statistical analysis Data on micronuclei and cytotoxicity were analyzed using Student’s t-test.21 47.00 5. corresponding to 7. 2.075 M) for 5 min at 37 ◦ C and fixed with chilled methanol:acetic acid (4:1). / Mutation Research 677 (2009) 59–65 61 Table 1 Identification. The data show that treatment with ZEN alone resulted in a significant increase in chromosome aberrations.66 ± ± ± ± ± ± 0.33 ± ± ± ± ± ± 0. In vivo study 84.66 1. No significant differences were observed in the group treated with MTBITC alone compared with controls.2 ␮mol/100 mg of fresh weight of the R. After a 48 h incubation DNA was extracted and analyzed by electrophoresis on a 1% agarose gel with Tris–Borate–EDTA. 3.62b 1.05).17b 0.4.66 0.2 50 The amount of MTBITC reached 38.98 ± 4.33 0. 3.33 0. The frequency of micronuclei in binucleated cells was significantly increased after treatment with ZEN. Ben Salah-Abbès et al.2%.12b 0.8 Despite the evident signs of genotoxicity shown by the animals in ZEN group. means with the same letter are not significantly different (P ≤ 0. Chromosome aberration test For analysis of chromosome aberrations.05 and 0.98 ± 4.66 0.5. In this study 1/2 IC50 was used in the cell cultures.48 3.05). sativus extract.33 1.27a 6. the results show that the mean number of micronucleated PCE (MNPCE) in bone marrow was not affected by treatment with MTBITC.66 11. Name of isothiocyanate Isothiocyanate side chain 4-(Methylthio)-3-butenyl isothiocyanate Table 2 Mean value of micronuclei detected in polychromatic erythrocytes of bone-marrow cells in Balb/c mice treated with ZEN alone or in combination with MTBITC (means ± SE).33 0. 1.88 0. A significant increase in the incidence of MNPCE was observed in ZEN.1.19 0.1. An important quantity of total MTBITC was present in the R.6. air-dried preparations were made according to standard procedures and stained with an aqueous Giemsa solution (Gurr R-66). Despite the evident signs of genotoxicity shown by the animals in the ZEN group.81%.27 0.4.33 65 2.27 0. 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]. gaps. mainly centric fusions and chromosome breaks. sativus. sativus.01 3.27a 0. 2.65 55.and colchicin-treated groups. Groups Structural aberrations Centric fusion Control water Control olive oil Mitomycin C 1mg/kg bw MTBITC 5 mg/kg bw ZEN 40 mg/kg bw MTBITC + ZEN 0. Phytochemical study The phytochemical results summarized in Table 1 show the presence of an important quantity of total MTBITC in R.65 3.32 ± ± ± ± ± ± 0. Within each column. compared with control. in bone-marrow cells.and in colchicin-treated groups compared with the controls.26 1. The amount of MTBITC reached 38.33 0.33 1.66 3. 3.23a 5.3.3. 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. Micronucleus analysis The results of the micronucleus test in animals treated with ZEN and MTBITC are summarized in Table 2. After three additional washes with freshly prepared fixative.11 ± ± ± ± ± ± 0.03 0.57 0.23 3.99 40.33 0. structure and activity of MTBITC extracted from R. 3.33 19. 1). Treatment with MTBITC significantly decreased the frequency of MNPCEs and this reduction reached 84. Treatment with MTBITC to ZEN-treated mice resulted to a significant decrease in the total chromosomal aberration induced by ZEN.33 29.33 1.66 13. The reduction in chromosomal aberrations resulting from MTBITC treatment of mice that had received ZEN reached 82.00 2. No significant differences were observed in the group treated with MTBITC alone compared with controls.33 9.98 ± 4.00 2.33 1.47a 0.00 Rings ± ± ± ± ± ± 0. MTBITC (25 ␮M) and ZEN alone (15 ␮M) or in combination with MTBITC (25 ␮M).99 7. Treatments Control water Control olive oil Colchicin 3 mg/kg bw MTBITC 5 mg/kg bw ZEN 40 mg/kg bw MTBITC + ZEN Micronucleus 1.11b 0.11a .00 0. rings and chromosomal breaks (Table 3). 3.1.4. Results 3.2%.4. and the IC50 activity of MTBITC in mouse lymphocytes was 50 ␮M.23 Total aberrations Chromosomal breaks 1.33 ± ± ± ± ± ± 0.2.17 mg (≈7%). mainly centric fusion and chromosome breaks. Micronucleus test The data for micronuclei in mouse lymphocyte cells are presented in Fig.3. in triplicate. In vitro study 2.27 0. The results indicate that treatment with ZEN alone results in a significant increase in chromosome aberrations.46a 4.2 ␮mol/100 mg and the IC50 activity of MTBITC in mouse lymphocytes was 50 ␮M.66 0. 1000 cells were analysed for each test concentration.33 2. sativus extract.27a Prevention (%) Abbreviation Isothiocyanate (␮mol/100 mg) IC50 activity (␮M) MTBITC 38.43a 3.00 1. The micronucleus frequency dropped by 93% in cells treated with ZEN+ MTBITC (Fig.J. 3.33 Gaps 1. Treatment with MTBITC significantly decreased the frequencies of MNPCEs and this reduction reached 84.66 1. Table 3 Effect of MTBITC and its protective role on chromosomal aberrations in bone-marrow cells of Balb/c mice treated with ZEN by the oral route.52 0.66 1. No significant difference was found for induction of micronuclei between the control and MTBITCexposed cells. Within each column. 2. The reduction in chromosomal aberrations resulting from MTBITC treatment of mice treated with ZEN reached 82.

13 0.4 Table 5 Evaluation of the mitotic index in mouse lymphocyte cultures treated for 48 h with ZEN alone or in combination with MTBITC. the co-treatment of ZEN + MTBITC decreased significantly the chromosome aberrations frequency compared with that observed in cells treated with ZEN alone. whereas a slight augmentation of the mitotic index was observed in the co-treatment.31 1. means with the same letter are not significantly different (P ≤ 0. The mean value of the mitotic index indicated a significant decreasing trend in the proliferating ability of lymphocytes treated with ZEN alone.07 10. the result of MTBITC-alone treatment indicated a normal value compared with the control.2. The fragmentation of mouse lymphocyte-DNA after treatment with ZEN.54 Chromosomal breaks Gaps 0. the co-treatment of ZEN + MTBITC decreased significantly the chromosome aberration frequency compared with that observed in cells treated with ZEN alone. Fig. Moreover. centric fusions and fragments) as well as in the number of cells with aberrations.8a 3. Within each column. 17.05).25 45. ZEN induced a statistically significant increase in the aberration frequency (chromosome breaks.55 0. Ben Salah-Abbès et al. The reduction reached to 87. MTBITC.2.65 32. No cytogenetic effect was observed in mouse lymphocyte cells treated with MTBITC alone. The DNA fragmentation by ZEN in the mouse lymphocytes was prevented by combined treatment with ZEN and MTBITC. Frequencies of MN in binucleated lymphocytes incubated for 48 h with ZEN alone or in combination with MTBITC.3.1 ␮M MTBITC 25 ␮M ZEN 15 ␮M MTBITC 25 ␮M + ZEN 15 ␮M Total aberrations (mean ± SD) Structural aberrations Centric fusion 0.17 1. compared with control.12 ± ± ± ± ± 0.52c 0. ± ± ± ± ± 8. The DNA ladder was added for compari- Fig. 63. The reduction reached 87.79 5. MTBITC. in triplicate.3.4%.55 11.22 0. 1000 cells were analysed for each test concentration. Treatment Control Colchicin 0.55 1.78 ± ± ± ± ± 0.3a 3. 1.44 0. To determine the frequencies of micronuclei in binucleated mouse lymphocytes.12 0. Within each histogram.00 2. The reduction reached 93% in cells treated with ZEN+ MTBITC. 3.4%. Mitomycin C also induced a statistically significant enhancement of both structural chromosome aberrations and the number of aberrant cells. DNA fragmentation The data presented in Fig.3.43 3. The frequency of micronuclei in binucleated cells was significantly increased in ZEN-treated cells.66 4.12 0. MTBITC or ZEN + MTBITC in combination.8b 7.32a 3.66 ± 0.33 ± 0. 2 show fragmentation of mouse lymphocyte-DNA after treatment with ZEN. centric fusions and fragments) as well as in the number of cells with aberrations compared with the control. The DNA fragments in the ladder and the ZEN-treated cells were clearly visible after agarose-gel electrophoresis.05).00 7. 2. the result of MTBITC-alone treatment indicated a normal value compared with the control. or ZEN + MTBITC treatment cultures.23a 3. . means with the same letter are not significantly different (P ≤ 0.33 ± ± ± ± ± 0.13 1.66 ± 1.3. whereas a slight augmentation of the mitotic index was observed after the co-treatment.4.2.62 J.61a 87. As shown in Table 5 the reported mean value of the mitotic index indicated a significant decreasing trend in the proliferating ability of lymphocytes treated with ZEN alone. No cytogenetic effect was observed in mouse lymphocyte cells treated with MTBITC alone.7 64.33 1. However.12 1.7a 3. Chromosome aberration analysis Table 4 shows the results of the chromosome aberration assay in mouse lymphocytes cultured in vitro following treatments with ZEN.33 0. Moreover.02 0. means with the same letter are not significantly different (P ≤ 0.33 0.02 2.13 1.66 11.33 19.2b 7. visualized by agarose-gel electrophoresis. No significant difference was found for induction of micronuclei between the control and MTBITC-exposed cells.66 10. / Mutation Research 677 (2009) 59–65 Table 4 Frequencies of chromosome aberrations in splenocytes of mice after a 48 h treatment with ZEN alone or in combination with MTBITC.33 ± 2.00 3. ZEN induced a statistically significant increase in the aberration frequency (chromosome breaks. no specific DNA fragments were detected in the control.11b 0.33 1.05). or ZEN and MTBITC in combination.3 ␮M MTBITC 25 ␮M ZEN 15 ␮M MTBITC 25 ␮M + ZEN 15 ␮M Mitotic index (mean ± SD) 67. suggesting that MTBITC does not have any negative effect on lymphocyte cell growth.33 ± 0. ZEN alone or in combination with ZEN.5 13. MTBITC alone and ZEN + MTBITC. Treatment Control Mitomycin C 0. Mitotic index The mitotic index was determined for each mouse lymphocyte culture treated with MTBITC alone.25 0. Mitomycin C also induced a statistically significant enhancement of both structural chromosome aberrations and the number of aberrant cells. DNA fragmentation induced by ZEN (15 ␮M) alone and in combination with MTBITC (25 ␮M) in mouse lymphocytes. which suggests that MTBITC does not have any negative effect on lymphocyte cell growth.53 0.48 2. Moreover.66 4.12 1. Moreover. The DNA ladder was added for comparison and includes fragment sizes that vary between 750 and 2000 base pairs. Prevention (%) Rings ± ± ± ± ± 0. Within each column.

This new band resulted from genetic alteration in DNA after treatment with ZEN. [6] and Grosse et al. at a low dose (0. In vitro and in vivo studies had shown that MTBITC killed cancer cells by an ROS-dependent apoptotic mechanism. Radish is used by people with different gastrointestinal. biliary. The ZEN oxidation can be considered the main source by which DNA adducts are formed through a quinone/hydroquinone redox couple. we have shown the novel antioxidant property of R. and inhibits protein and DNA synthesis [30]. A striking and characteristic chemical property of cruciferous plants is their high content of glucosinolates.30]. Indeed. Ben Salah-Abbès et al. We cannot exclude that at higher concentrations it would be possible to obtain a more evident mitotic index reduction. together with an increased concentration of free iron within the cell stimulate the production of OH radical via the Fenton-like reaction due to mobilization of Fe2+ by Ca2+ . no specific DNA fragments were detected in the control. [8] indicated that ZEN caused DNA fragmentation. our data show a statistically significant decrease of cell growth in lymphocyte cultures (15 ␮M). In vitro and in vivo studies have reported that MTBITC affects many steps of cancer development including modulation of phase-I and -II detoxification enzymes.25].38]. Concerning this parameter. A lack of adequate supply of NAD(P)H and GSH to permit H2 O2 consumption by GSH-dependent glutathione peroxidase and NAD(P)H-dependent glutathione reductase. consumption of food products containing this compound could be beneficial. We stated in our previous work that ZEN does evoke oxidative stress. control of the cell cycle and reduction of Helicobacter infections [37. Mice and lymphocyte cells treated with ZEN showed a significant increase in chromosome aberrations accompanied with high micronucleus frequencies. induces lipid peroxidation and cell death. with micronucleus formation as a possible consequence. Indeed. The genomic DNA did not show any new band in cells treated with ZEN + MTBITC. we reported that administration of ZEN to mice enhanced lipid peroxidation. this genotoxicity can be explained on the one hand by the fact that ZEN causes damage as previously demonstrated by covalent binding to DNA [6]: the formation of these adducts can be the preceding step to DNA cleavage during repair processes. whereas the selective dose of ZEN was based on literature data [11.33]. or ZEN + MTBITC cultures. One new DNA band was caused by ZEN. induction of apoptosis.25]. Moreover. which are genotoxic by-products of many metabolic processes. Other studies estimated that oxidative damage seems to be a key determinant of ZEN-induced genotoxicity [26]. functioning as a direct or as an indirect antioxidant by phase-II enzyme induction. Previous evidence suggests that ZEN is a mutagenic and DNA-reactive carcinogen because the existing data demonstrated the formation of ZEN–DNA adducts in mice similar to results found by Pfohl-Leszkowicz et al. which directly results from free radical-mediated toxicity. Previously. modulating cell signalling. a marker of mitotic activity in MCF-7 cells. The results of this study confirm and extend previous data demonstrating that ZEN induces oxidative stress and genotoxicity. The latter is the most frequent mycotoxin causing a significant problem of great agro-economic importance. thus indicating a strong reduction of cell-proliferating ability compared with the control. The selective dose of MTBITC was based on our previous work [22]. Regarding the DNA fragmentation. Ascorbic acid and vitamin E were also reported to inhibit the binding of AFB1 or ZEN to DNA [27].35]. Considering that MTBITC has potential chemo-preventive value to human health. an epigenetic and thresholded mechanism [29]. sativus extract in Balb/c mice. In humans. which can be explained by the conjugation of GPX with ZEN or its metabolites. apoptosis. it decreased GPX and SOD. In the same way. Like other fusarotoxins ZEN may reduce DNA synthesis by binding to the ribosomal peptidyl-transferase. This results in further cell damage by oxidative stress and may be one of the mechanisms by which ZEN exerts its genotoxic effects. which often approaches 1% or more of their dry weight [34]. The targets of oxidative damage are usually critical biomolecules such as nucleic acids. ZEN appears to produce many genotoxic effects such as the increase of the permeability of the cell to Ca2+ [8. ZEN is known to be genotoxic. but this was not the aim of this study. The enhanced cellular concentration of Ca2+ and the presence of the pro-oxidant ZEN can uncouple oxidative phosphorylation. Discussion There is a worldwide contamination of foods and feeds with toxigenic fungi secreting mycotoxins such as Ochratoxin A and ZEN. the microflora of the gastrointestinal tract can efficiently convert glucosinolates to MTBITC [33. The chemoprotective effect of MTBITC seems to be more important than that of its parent compounds. protein and lipids [25]. hepatic.22]. administration of MTBITC extracted from Tunisian R. sativus on the genotoxicity of ZEN was investigated in vitro and in vivo. The beneficial influence of MTBITC noted in the present study can be attributed to the fact that this compound is a very efficient antioxidant and a scavenger of oxygen free radicals. which results in an increased leakage of electrons from the respiratory chain. / Mutation Research 677 (2009) 59–65 son and includes fragment sizes that vary between 750 and 2000 base pairs. and a reduced livestock production. The DNA fragments in the ladder and the ZEN-treated cells were clearly visible after agarose-gel electrophoresis.1 ␮M) [31]. On the other hand. Radish is not only a vegetable crop but also an important source of medicinal compounds. Similarly. From the in vitro study. In this study. and was capable of modulating biotransforming enzyme activity and preventing certain cancers [36]. urinary and respiratory disorders. In the current study. It was further shown that MTBITC increased neither ROS nor toxicity in normal cells. [28]. In the present study the increase of chromosome aberrations and micronucleus frequencies could be a consequence of severe cytotoxicity. ZEN was reported to modify lymphocyte proliferation and to induce phosphorylation of histone H3. and generate O2− and hence H2 O2 . In the previous reports from our laboratory.11].J. The antioxidant activity of MTBITC is higher than that of ascorbic acid and vitamin E [39]. sativus (radish. cell-cycle arrest and inhibition of macromolecule synthesis. which could be considered as a “diagnostic” marker attributed to the ZEN-treatment. The fragmentation of mouse lymphocyte DNA by ZEN was inhibited by the combined treatment with ZEN + MTBITC. ZEN may cause dysfunction of the mitotic spindle [27]. The DNA damage as well as the perturbation of the antioxidant-enzyme status are attributable to oxidative stress. the glucosinolates [32. the economic impact of mycotoxins includes toxic effects on humans and animals with increased costs for human health care and veterinary care. and in cardiovascular diseases such as hypertension. cruciferous) improved the parameters studied and alleviated the negative effects of ZEN. An important mechanism of protection against DNA damage and tumour formation by MTBITC is the inhibition of ␣-hydroxylation of nitrosamines . MTBITC. the present study indicated that ZEN induces alteration in DNA as demonstrated by appearance of a new band in treated mouse lymphocytes. Glucosinolates and their MTBITC hydrolysis products are well-known protectors against carcinogenesis. Balb/c mice and lymphocyte cell cultures used in this study are most sensible models to ZEN-induced genotoxicity and oxidative stress [7. 4. the effect of MTBITC extracted from Tunisian R. the main mechanistic point of view estimated that fusarotoxins bind to the ribosomal peptidyl-transferase and inhibit protein synthesis specifically and DNA synthesis consequently. and possibly underlying mechanisms were evaluated [22. Abid Essafi et al. which may contribute at least in part to ZEN-induced immunotoxicity and genotoxicity in Balb/c mice during long-term exposure [22. as was also evidenced by the 63 low mitotic index. it binds to 17-␤-estradiol receptors. However.

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