Genotoxicity assessment of deoxynivalenol in the Caco-2 cell line model using the Comet assay

Toxicology Letters 166 (2006) 67–76

Genotoxicity assessment of deoxynivalenol in the Caco-2 cell line model using the Comet assay Sylvie Bony a,∗ , Monique Carcelen a , Laurence Olivier a , Alain Devaux b a

b

UMR INRA-DGER Mycotoxines et Toxicologie Compar´ee des X´enobiotiques, Ecole Nationale V´et´erinaire de Lyon, 1, av. Bourgelat, F-69280 Marcy l’Etoile, France INRA D´epartement EFPA, Laboratoire des Sciences de l’Environnement, Ecole Nationale des Travaux Publics de l’Etat, Rue Maurice Audin, F-69513 Vaulx en Velin, France Received 23 March 2006; received in revised form 28 April 2006; accepted 28 April 2006 Available online 3 June 2006

Abstract The genotoxic risk associated with deoxynivalenol (DON), a prevalent trichothecene mycotoxin which contaminates cereal-based products has not yet been deeply explored. In this work, the alkaline version of the Comet assay was used to evaluate DNA damage stemming from DON exposure in both dividing and differentiated Caco-2 cells, an epithelial intestinal cell line. To avoid false positive results, cytotoxic and apoptotic thresholds were firstly established using the MTS and neutral red assays and the Hoestch staining method, respectively. Dividing cells were found to be more sensitive to DON than differentiated cells and the lowest IC10 (0.5 ␮M) obtained for dividing cells exposed for 72 h was used as the highest working concentration in the genotoxicity study. Both differentiated and dividing cells responded with a dose-dependant relationship to DON in terms of DNA damage in the 0.01–0.5 ␮M range. These results demonstrated the existence of a genotoxic potential for DON at low concentrations compatible with actual exposure situations and calls for additional studies to determine the functional consequences which could be taken into account for the risk assessment of this food contaminant. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Mycotoxin; Deoxynivalenol; Comet assay; Genotoxicity; Cytotoxicity

1. Introduction Deoxynivalenol (DON) is identified as by far the most common trichothecene mycotoxin in cereal crops contaminated by field fungi of the Fusarium genus. Recently, data on DON contamination became available in surveys targeted towards cereals and grain products intended for human food as stated in the SCOOP Report

∗ Corresponding author. Tel.: +33 4 78 87 25 29; Fax: +33 4 78 87 00 39. E-mail address: [email protected] (S. Bony).

on Fusarium toxins (EC, 2003). In these studies, DON levels averaged in the order of hundreds of micrograms but tended to vary greatly. In addition to its presence in raw cereals, DON is particularly stable towards most processed food and thus can remain in significant amount in manufactured products (Hazel and Patel, 2004). Acute exposure, which is rather scarce, can lead to abdominal pains, vomiting and diarrhea (Rotter et al., 1996). However when exposure is chronic, which is more prone to occur, the result usually leads to reduced feed consumption, growth retardation and alteration of the immune response. Regarding its carcinogenicity, the role of DON in the high incidence of esophageal cancer in Africa

0378-4274/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2006.04.010

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and China was suspected but not clearly demonstrated (Marasas et al., 1979; Luo et al., 1990). Since IARC (1993) has maintained DON in group 3, “not classifiable as to its carcinogenicity to humans” because of the lack of evidence particularly concerning in vivo studies. In a 2-year study with B6C3F1 mice, no effect of dietary DON levels was obtained on tumor incidence (Iverson et al., 1995) but recently, a high incidence of lung adenocarcinoma and of dysplasia of glandular stomach was observed in NIH mice orally exposed to the toxin over 24 weeks (Huang et al., 2004). DON toxicity is mainly explained by its capacity to interfere with protein and nucleic acid synthesis (Ueno et al., 1973) and more recently DON and other trichothecenes were also shown to alter cell signaling and induce apoptosis. Thus, the cytotoxic and immunotoxic effects of DON are well established (for a review see Pestka and Smolinski, 2005). However, its genotoxicity remains fairly equivocal. DON is not mutagenic in bacteria but cytogenetic effects were obtained as chromosomal aberrations and SCE were observed both in vitro and in vivo suggesting the existence of a genotoxic potential for DON (Hsia et al., 1988; Bilgrami et al., 1993; Knasmuller et al., 1997). In this context, we have realized an in vitro evaluation of the genotoxic potential of DON on the human intestinal Caco-2 cell line chosen as a target cell type since oral exposure is the route of contamination by DON. The choice of the model was also driven by the fact that the intestinal epithelium characterized by rapid cell renewal is known to be one of the main target tissues for trichothecene toxicity. Derived from a colon carcinoma, the Caco-2 cell line is widely used as a model system for the study of enterocytic functions as it differentiates at post-confluency into a polarized monolayer with properties of ileal epithelial cells (Pinto et al., 1983). In the present work, this cell line was used both in its proliferative undifferentiated phase and after confluency in its differentiated physiological state exhibiting typical dome formation, to assess the potential of deoxynivalenol to induce DNA damage using the Comet assay. Alkaline version of the Comet assay or single-cell gel electrophoresis (SCGE) assay is a sensitive tool widely used for the measurement of DNA damage at the individual cell level both in vivo and in vitro (Tice et al., 2000). As many studies have highlighted the fact that strand break assays such as alkaline elution, alkaline unwinding or sucrose-gradients and Comet assay are prone to false positive responses due to cytotoxicity that can rapidly lead to DNA fragmentation stemming from cell necrosis and/or apoptosis (Hartmann et al., 2001; Collins, 2004), the present DON genotoxic dose–response study was

monitored at strictly controlled non-cytotoxic DON concentrations in order to assign unequivocally observed effects to genotoxicity. 2. Materials and methods 2.1. Chemicals Dulbecco’s Modified Eagle’s Medium (DMEM), nonessential amino acids, antibiotics, trypsin-EDTA, sodium pyruvate and phosphate buffered saline (PBS), were purchased from Invitrogen Life Technologies. Fetal calf serum (FCS) was purchased from Perbio sciences. Deoxynivalenol, neutral red, Trypan blue, phenazine methosulfate (PMS), methyl methanesulfonate (MMS), Triton X-100, EDTA, DMSO, agarose type I and type VII, TRIS, ethidium bromide, Hoechst staining solution and paraformaldehyde were all purchased from Sigma–Aldrich (Saint Quentin Fallavier, France). The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) was provided by Promega France-Charbonnieres-les-Bains (CellTiter 96® Aqueous MTS reagent powder ref. G1112). All other chemicals used were purchased locally and were of analytical grade. 2.2. Cell culture and DON exposure The human colonic carcinoma Caco-2 cells were obtained from the American Type Culture Collection (ATCC No. HTB-37). Cells were sub-cultured weekly in DMEM containing 4.5 g/l glucose, 10% heat-inactivated fetal bovine serum, 1% non-essential amino acids, penicillin (100 IU/ml) and streptomycin (100 ␮g/ml) and 1% sodium pyruvate under an atmosphere of 95% air and 5% CO2 at 37 ◦ C. For the experiments, Caco-2 cells (passage 24–38) were harvested at 80% confluency using trypsin (0.5%) EDTA (0.02%) and seeded at a density of 2.104 cells/cm2 in multi-well plates (Becton Dickinson, Meylan, France). Culture medium was changed three times a week and systematically before DON exposure. A 1 mM stock solution and subsequent working solutions of deoxynivalenol were prepared in absolute ethanol and distributed into wells so that the final ethanol concentration was 1%. Two exposure times (24 and 72 h) were tested with dividing undifferentiated cells (from day 3 or 4 post-seeding) and with differentiated cells treated at least 10 days post-confluency (from day 17 or 18 post-seeding). A DON concentration range (0, 0.25, 1, 2.5, 5 and 10 ␮M) was first applied in order to determine the IC10 and IC50 values by the mean of two cytotoxicity tests (see below) and the presence of apoptotic figures by the mean of Hoescht staining (see below). Then, cells were exposed to a low non-cytotoxic DON concentration range (0, 0.01, 0.05, 0.1 and 0.5 ␮M) in order to study the genotoxic potential of DON. Triplicate plates were realized and within a plate, each dose was applied in four and eight replicate wells for 24- and 96-well plates, respectively,

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for the cytotoxicity study and two wells per dose were realized in 24-well plates for the genotoxicity study. 2.3. Cell viability testing by the MTS assay MTS assay was used to assess cell viability in both dividing and differentiated cells. The MTS test is based on the capacity of viable cells to metabolize (via the mitochondrial succinate dehydrogenase) a yellow tetrazolium salt (MTS) in the presence of phenazine methosulfonate (PMS) acting as an electron coupling agent, to a purple formazan directly soluble in tissue culture medium and measured by the amount of 490 nm absorbance proportional to the number of living cells. The MTS test was performed in 96-well plate containing 110 ␮l of culture medium. At various time postseeding and post-exposure to DON as indicated above, 20 ␮l of a MTS/PMS extemporaly prepared solution (2 mg/ml and 45 ␮g/ml, respectively, in phosphate buffer) were added to each well and plates were returned to incubate for two additional hours at 37 ◦ C. The 490 nm absorbance was read using a Microplate reader (SpectraCount, Perkin-Elmer). Relative cell viability (in percentage) was expressed as (Abs490 treated cells/Abs490 control cells) × 100. The IC10 (and IC50 when possible) values were estimated by means of linear regression from a graph depicting cellular sensitivity versus DON concentration. 2.4. Cell viability testing by the neutral red assay Neutral red (NR) test was also performed to assess cytotoxicity of DON on dividing and differentiated Caco-2 cells (Babich and Borenfreund, 1991). Viable cells actively accumulate this supravital dye across the cell membrane into lysosomes. Thus after a subsequent lysis, absorbance at 540 nm is used to measure cell viability since toxin-injured plasma and lysosomal membranes cannot retain the dye after washing and fixation procedures. The stock solution of NR (3.3 g/l) was diluted in DMEM without phenol red and FCS to contain 50 ␮g NR/ml. The NR-containing media was pre-incubated overnight at 37 ◦ C, centrifuged (1500 × g for 10 min) and filtered through a Minisart GF filter (ref. 17824, Sartorius) in order to remove all precipitated dye crystals. This step was absolutely necessary to avoid unspecific binding of dye crystals to the cells. After exposure to DON in 24-well plates, the medium was replaced with 1 ml of the NR-containing medium and plates were returned to the incubator for 3 h. Thereafter, the cells were rapidly washed with 1 ml of 0.5% formaldehyde–1% CaCl2 followed by 2 ml of a 1% acetic acid–50% ethanol solution to extract the dye from cells. After 15 min of shaking, the absorbance was read at 540 nm using a Unicam 8625 UV–vis spectrometer. Relative cell viability (in percentage) was expressed as (Abs540 treated cells/Abs540 control cells) × 100. The IC10 (and IC50 when possible) values were estimated by means of linear regression from a graph depicting cellular sensitivity versus DON concentration.

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2.5. Hoechst staining of Caco-2 cells In order to visualize condensed chromatin and apoptotic bodies in treated cells, DON (0–10 ␮M) exposed Caco-2 cells were fixed directly in culture wells (12-well plates) with 4% paraformaldehyde for 15 min, washed three times with PBS and then permeabilized for 5 min with 0.2% Triton X-100 in PBS. After three washes in PBS, the cells were stained with Hoechst 33258 (5 ␮g/ml) for 1 h at 37 ◦ C (Cha¨ıbi et al., 2005). Following three last washes in PBS, cells were examined under an inverted microscope equipped with a fluorescent light source (Olympus iX-71) coupled to a high definition acquisition camera (DP70 Olympus). 2.6. Genotoxicity testing using the Comet assay Cells were seeded at 2.104 cells/cm2 in 24-well plates and were exposed to a low DON concentration range (0–0.5 ␮M) determined as non-cytotoxic in the previous cytotoxicity assessment step. Positive controls were performed by treating two wells per plate with 1 mM methyl methanesulfonate (MMS) for 1 h at 37 ◦ C. The cell content of each treated well was collected using trypsin (0.05%) EDTA (0.2%). Cell viability was checked by the Trypan blue method according to Boyse et al. (1964). Cell suspensions exhibiting a viability >90% were only used. The cell density was adjusted to 6.105 cells/ml. Comet assay was then carried out according to the Singh et al. procedure (1988) with slight modifications. 75 ␮l of the above cell suspension mixed with an equal volume of 1% low-melting agarose was spread on a microscope slide previously covered with a 0.8% normal-melting agarose layer. After agarose solidification on a chilled plate, 90 ␮l of 0.5% low-melting agarose was spread on the previous layer and allowed again for solidification. The cells were treated under dim light with a lysing solution (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, pH 10, 1% (v/v) Triton X-100 and 10% (v/v) DMSO) for 1 h at 4 ◦ C, then the DNA was allowed to unwind for 40 min in the electrophoresis buffer (0.3 M NaOH, 1 mM EDTA, pH > 13). The electrophoresis was run for 30 min at 25 V and 300 mA (0.75 V/cm). Slides were then neutralized using a Tris buffer solution (0.4 M Tris, pH 7.5) before staining with ethidium bromide and scored using an epifluorescence microscope (Axioskop, Zeiss) equipped with a CCD camera (Cohu) and using the image-analysis software Komet 4.0 (Kinetic Imaging Ltd.). From each concentration, 100 randomly selected cells (50 cells from each of the two replicate slides) were analysed and the Comet parameter retained was the Olive tail moment (OTM) found to be the most relevant parameter in our experiments. 2.7. Statistical analysis of data For the cytotoxicity study, IC10 and IC50 values were compared for the time effect and the physiological state effect using the Student t-test. For the genotoxicity study, since the distribution of the Comet figures did not follow a Gaussian distribution as described earlier (Bauer et al., 1998), both Kruskall–Wallis

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Fig. 1. Morphology of dividing (A: day 4 post-seeding) and differentiated (B: day 18 post-seeding) Caco-2 cells.

and Mann–Whitney non-parametric tests were used for data analysis.

3. Results In our culture conditions, cells reached confluency at about day 7, and cells referred as “differentiated” were exposed to deoxynivalenol 1 week later, when domes were microscopically visible, demonstrating a functionally polarized differentiation. Fig. 1 shows the morphology of non-confluent dividing cells (A) and of the post-confluent ones (B). 3.1. DON cytotoxicity assessment in Caco-2 cells The cytotoxicity of DON was evaluated using the MTS and neutral red assays both for dividing undifferentiated (Fig. 2A) and non-dividing differentiated cells (Fig. 2B), with a focus on a rather low concentration range (0–10 ␮M). As shown in Table 1, DON concentrations inhibiting 10% and 50% of cell viability were found to be in a very similar range when calculated with MTS or NR assays. DON was less cytotoxic for differentiated cells compared to dividing cells in the concentration range tested. No cytotoxicity was observed after a 24 h exposure in differentiated cells when IC10 values in the 0.9–1.2 ␮M range were measured for dividing cells. Exposure time (24 and 72 h) had a low impact on the dose response curve in dividing cells but a marked difference in IC10 values were obtained when compar-

Fig. 2. The effect of deoxynivalenol on Caco-2 cell viability after 24 and 72 h of exposure measured using the MTS and the neutral red assays, (A) dividing cells, (B) differentiated cells. Values are expressed as percent of control response and each value is a result of three experiments. Bars represent standard deviation.

ing 1 and 3 days of exposure in differentiated cells (Table 1). IC50 was not reached in the studied concentration range for differentiated cells exposed for 72 h but a cytotoxic effect was obtained and cell death caused by 10 ␮M DON could be evaluated in the range of 20–30% (Fig. 2B). According to the results of this cytotoxicity study assessed by two complementary methods, it was decided to undertake the subsequent genotoxicity study by using for both cellular stages and exposure time, the same DON concentration range peaking at 0.5 ␮M, corresponding to the lowest calculated IC10 (72 h exposure for dividing cells). 3.2. Apoptosis evaluation by Hoechst staining in Caco-2 cells exposed to DON In order to check the interference of DNA fragmentation related to apoptosis that could impair the validity of genotoxicity results obtained by the Comet assay, the apoptotic status of Caco-2 cells exposed to a wide

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Table 1 IC10 and IC50 values for deoxynivalenol in dividing and differentiated Caco-2 cells exposed for 24 and 72 h to the toxin measured by the MTS and the neutral red assays Cell status

IC10 (DON, ␮M)

IC50 (DON, ␮M)

MTS bioassay

NR bioassay

MTS bioassay

NR bioassay

Dividing cells 24 h exposure 72 h exposure

1.2 ± 0.4 0.8 ± 0.1

0.9 ± 0.2 0.5 ± 0.3

10.0 ± 2.8 4.3 ± 0.9a

3.7 ± 1.5 3.7 ± 1.1

Differentiated cells 24 h exposure 72 h exposure

>10 2.8 ± 0.8b

>10 2.2 ± 0.9b

>10 >10

>10 >10

Mean ± S.D. of three separate determinations. a Significantly different from the corresponding 24 h exposure value (p < 0.05). b Significantly different from the corresponding dividing cell value (p < 0.05).

concentration range (0.01–10 ␮M) was visualized by Hoechst staining. No biological hallmarks characterizing apoptosis (i.e. condensed chromatin and apoptotic bodies) were seen in the genotoxicity range studied (0.01–0.5 ␮M) neither in dividing (Fig. 3B) nor in dif-

ferentiated cells (not shown) for which staining revealed only round and homogenously stained nuclei both for 24 and 72 h of DON exposure. Apoptosis figures were only seen in cells treated at much higher DON concentrations (≥5 ␮M for dividing cells and ≥10 ␮M for differentiated

Fig. 3. Assessment of nuclear fragmentation by Hoechst staining of dividing Caco-2 cells exposed for 24 h to various DON concentrations and to staurosporine as a positive control. Typical chromatin condensation and apoptotic bodies were not observed in control (A) and up to 0.5 ␮M DON exposed cells (B) but they were observed at cytotoxic 5 ␮M DON concentration (C). (D) The positive control obtained by treating cells for 24 h with 0.1 ␮M staurosporine. Magnification 400×.

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Fig. 4. Frequency of DNA damage distribution of Caco-2 cells exposed to various concentrations of deoxynivalenol. (A) Dividing cells 24 h exposure, (B) dividing cells 72 h exposure, (C) differentiated cells 24 h exposure and (D) differentiated cells 72 h exposure.

cells) for both exposure times following a dose–response effect (data not shown) as in the positive control cells treated with 0.1 ␮M staurosporine for 24 h for which asymmetric staining as a result of chromatin condensation and nuclear fragmentation is shown by arrows (Fig. 3C and D). 3.3. DNA damage in Caco-2 cells exposed to DON Genotoxicity of DON was assessed in the range 0.01–0.5 ␮M in undifferentiated and differentiated Caco-2 cells through the alkaline Comet assay. Under the described experimental conditions (three independent experiments) the major part of control cells (85%) exhibited a tail moment value < 1.0. MMS-treated cells used as a positive control showed a tail moment value always higher than 10. Thus, cells with a tail moment value from 1.0 were arbitrarily divided into four groups: 1–1.99, 2–3.99, 4–9.99 and >10. Fig. 4A and B show the distribution of observed DNA damage in dividing cells after 24 and 72 h exposure, respectively. Mean tail

moment values increased significantly in exposed Caco2 cells according to DON concentration starting from 0.01 to 0.05 ␮M DON depending on the exposure time (Table 2). Such an increase in DNA damage was clearly higher after 72 h compared to 24 h exposure, reaching, respectively, a 5- and 2.5-fold control value when cells were exposed to 0.5 ␮M DON. This general trend was confirmed in post-confluent Caco-2 cells for both exposure times (Fig. 4C and D). Both dividing and differentiated MMS-treated cells exhibit a marked level of DNA damage reaching about 23-fold negative control values (Table 2). 4. Discussion In this study, the results obtained with the Comet assay underline the existence of a genotoxic potential for the deoxynivalenol at low non-cytotoxic concentrations on the Caco-2 enterocyte-like cell model both during its undifferentiated proliferating phase and late after confluency in its highly differentiated state.

S. Bony et al. / Toxicology Letters 166 (2006) 67–76 Table 2 DNA damage in dividing and differentiated Caco-2 cells estimated as the Olive tail moment by the alkaline Comet assay after 24 and 72 h exposure to various DON concentrations Average tail moment ± S.D. Dividing cells 24 h exposure DON (␮M) 0 0.01 0.05 0.1 0.5 MMS (1 mM, 1 h) 72 h exposure DON (␮M) 0 0.01 0.05 0.1 0.5 MMS (1 mM, 1 h) *

1.12 2.29 2.37 3.35 3.88

± ± ± ± ±

0.27 0.13* 0.71 1.08* 1.62*

23.13 ± 3.13*

1.08 2.59 3.41 5.07 6.82

± ± ± ± ±

0.44 0.86 1.34* 1.66* 1.77*

24.35 ± 3.89*

Differentiated cells

1.23 2.06 2.70 4.11 4.93

± ± ± ± ±

0.73 0.94 1.21 1.53* 0.99*

26.56 ± 3.26*

1.09 3.48 4.31 4.60 7.05

± ± ± ± ±

0.31 1.11* 0.75* 0.81* 1.89*

28.58 ± 4.23*

Significantly different from the corresponding control (p < 0.05).

Aware of the high cytotoxic and apoptotic capacities of DON demonstrated in many cell lines (Gutleb et al., 2002) we particularly focused our attention, in the design of this study, to avoid their possible influence on Comet assay results. Thus our first goal was to determine the DON concentrations, that would not reduce cell viability by more than the IC10 . As shown, dividing cells exhibited a higher sensitivity towards DON compared to differentiated cells. There is very limited information on the IC10 values for DON in literature, but concerning IC50, values obtained here with dividing cells (3.7–10 ␮M) are consistent with other results obtained with proliferating Caco-2 cells (Instanes and Hetland, 2004; Calvert et al., 2005; Cetin and Bullermann, 2005; Kouadio et al., 2005; Sergent et al., 2006). With the differentiated Caco-2 cells, no sign of toxicity was measurable in the concentration range tested, except that a mild effect was obtained after 72 h of exposure, with IC10 values being three to four times higher than those calculated with the undifferentiated cells. Similar differences were already observed with the HT-29-D4 cell line, another intestinal epithelium model (Maresca et al., 2002). This could be attributed to discrepancies in the underlying mechanisms driving the cytostatic and the actual cytotoxic effects of DON since the methods used do not enable differentiating static to lethal drug effects. However, it is probable that the greater sensitivity of proliferating cells remains due to DON capacity to inhibit protein synthesis and sub-

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sequently nucleic acid synthesis. Another explanation is the well known cyclical fluctuation of many enzymatic activities, particularly those related to the metabolizing capacities that are usually increased in Caco-2 cells at differentiation (Delie and Rubas, 1997). Among those, the UDP-glucuronosyltransferases (UGTs) for which DON can be a substrate as demonstrated in humans (Meky et al., 2003) and the glutathione S-transferases (GSTs) for which the 12–13 epoxide group characterizing DON and all epoxy-trichothecenes are putative targets. Additionally, it is important to note that the increased expression of transporters/efflux systems of the ABC family i.e. P-glycoprotein (P-gp) and non-Pgp carriers, occurring at differentiation in the Caco-2 model is prone to modulate greatly the intracellular concentration and subsequently the deleterious effects of toxic agents. Now, DON has recently been suggested as a noneffluxed compound in the Caco-2 cell line (Sergent et al., 2006) but this point needs additional investigations since controversial results were recently obtained (Lecoeur, personnal communication). Cell viability assays such as MTS and neutral red uptake might not respond very well to early apoptotic events. Moreover, early apoptosis hallmarks like nuclear chromatin condensation and its related DNA fragmentation (Allen et al., 1997) can be revealed by the alkaline version of the Comet assay. Thus, the threshold of apoptosis in Caco-2 cells exposed to DON was checked prior to the DNA damage evaluation. In the present work, condensed chromatin was not observed at DON concentration under 5 ␮M for dividing cells and 10 ␮M for differentiated cells and this result corroborates those recently obtained by Sergent et al. (2006). Furthermore, apoptotic nucleosomal fragmentation of DNA leads to characteristic highly damaged figures with the Comet assay, with no or very small heads and nearly all the DNA in the tail referred to as “clouds” or “hedgehogs”. Such highly damaged figures were very scarce in our study. Additionally, the wide distribution of the frequency of the damage levels obtained here is characteristic of a genotoxic effect which usually generates varying degrees of damage in a cell population, contrasting with the bimodal distribution of damage resulting from cytotoxicity and apoptosis. Thus, in this work we can reasonably assess that the dose–response DNA damage revealed by the Comet assay results from a genotoxic effect of DON. This is the first report for the use of the Comet assay to assess the genotoxicity of deoxynivalenol. The Comet assay has already been applied to the evaluation of the genotoxic potential of many mycotoxins, among which, nivalenol, another type B trichothecene (Tsuda et al., 1998). In this last work, both CHO cells and multiple organs of exposed

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mice were analysed and remarkably, among the various tissues examined, the DNA of the gastrointestinal tract and particularly colon, was preferentially damaged by nivalenol. In our study, the absolute damage level induced by DON remained moderate as compared with that caused by MMS (a strong alkylating agent). Despite this, the quality of cell preparation, as demonstrated by the very low DNA damage level in negative control cells, contributed to the capacity to discriminate with statistical significance between control and treated cells and confirmed the high sensitivity of the Comet assay to detect and quantify the genotoxic potential of toxicants (Tice et al., 2000). According to our knowledge, the data on genotoxic effects of trichothecenes and particularly of DON are scarce. Knasmuller et al. (1997) showed the clastogenic potential of DON. In that work, the toxin caused a moderate induction of micronuclei and a clear-cut dosedependent increase of chromosomal aberrations in primary culture of rat hepatocytes at non-cytotoxic concentrations (in the range 0.003–3.3 ␮M when cytotoxicity was in the range 33–330 ␮M). Earlier, Hsia et al. (1988) had found that a HPLC purified extract of DON induced chromosomal aberrations in V79 cells. In the same cell type, two other eight ketotrichothecenes: nivalenol and fusarenone X were found to have a mild effect revealed by the sister-chromatid exchange assay but at rather high exposure levels (10–50 ␮M) for which the antiproliferative effect of DON, related to the inhibition of protein and DNA synthesis, was marked with a strong cell cycle arrest effect (Thust et al., 1983). As far as we know, a direct chemical interaction of DON or other trichothecenes with DNA have never been explored. A direct interaction would first require DON to enter the Caco-2 cells in order to reach the DNA target. Recently, Sergent et al. (2006) working with a DON concentration range 0.5–10 ␮M were unable to detect the toxin at the intracellular level. Additionally, exploring the DON transport across the Caco-2 cells, these authors found that DON was not an effluxed compound and suggest a transcellular and/or a passive paracellular transport mechanism. However, it must be highlighted that this work was carried out during a very short exposure time (3 h). It would be interesting to explore longer exposure times. DNA damage could also result from an indirect genotoxic response. Like other trichothecenes such as the T-2 toxin, DON was shown to increase lipid peroxidation in rat liver (Rizzo et al., 1994) and this was recently confirmed using Caco-2 cells, for which an increase in the production of malondialdehyde, a biomarker of lipid peroxidation, was obtained after a 24 h DON exposure (Kouadio et al., 2005). Since lipid

peroxidation products are known to cause DNA damage (Vaca et al., 1988; Box and Maccubbin, 1997) it could be proposed that the DON genotoxicity is partly related to the production of free radicals and of reactive oxygen species (ROS) as already suggested to explain DONrelated DNA breaks in rat hepatocytes (Knasmuller et al., 1997). This point will need further investigation, for example by coupling the Comet assay with repair enzymes specific to DNA oxidative lesions (formamidopyrimidine glycosylase, endonuclease III) or by the use of free radical scavengers. Surprisingly, the genotoxic effect of deoxynivalenol was observed with the same order of magnitude in both dividing and differentiated Caco-2 cells. One could have expected differentiated cells to be less sensitive to DON genotoxicity than dividing cells. Indeed, post-confluent Caco-2 were found resistant to hydrogen peroxide in term of DNA strand breaks measured by the Comet assay when undifferentiated Caco-2 showed dose-related damage (Duthie and Collins, 1997) and a similar trend was observed in vivo, in rat colonocytes (OberreutherMoschner et al., 2005). In conclusion, although we cannot simply extrapolate these in vitro results to the in vivo situation, it is particularly interesting to underline that in the present experiment, the assayed DON concentration range is in accordance with the levels plausibly encountered in the gastrointestinal tract after consumption of moderately contaminated food (EC, 2003) and even still realistic in situations close to the (temporary) tolerable daily intake (1 ␮g/d/kg BW) sets by the Scientific Committee for Food (SCF, 2002; Pieters et al., 2002). The DONrelated DNA damage relationship demonstrated in this work, stresses the need for further investigations in order to fill the gap of trichothecenes genotoxicity studies as already suggested (Dirheimer, 1998; Larsen et al., 2004; Schlatter, 2004) and to reduce the uncertainties impairing the risk assessment for this ubiquitous food contaminant. Acknowledgments The authors thank Michelle Mazallon for her skilled assistance in cell culture, Dr. Samuel Buff for his kind help in the microscopic examination of Hoestch staining and Christine Farmer for her critical reading of the English manuscript. References Allen, R.T., Hunter III, W.J., Agrawal, D.K., 1997. Morphological and biochemical characterization and analysis of apoptosis. J. Pharmacol. Toxicol. 37, 215–228.

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