Food and Chemical Toxicology 66 (2014) 113–121

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Absence of in vitro genotoxicity potential of the mycotoxin deoxynivalenol in bacteria and in human TK6 and HepaRG cell lines Natsuko Takakura a,1, Fabrice Nesslany b,2, Valérie Fessard a,1, Ludovic Le Hegarat a,1,⇑ a b

ANSES, French Agency for Food, Environmental and Occupational Health & Safety, Fougères Laboratory, Toxicology of Contaminants Unit, Fougères, France Institut Pasteur de Lille, Laboratory of Toxicology, EA 4483 Lille, France

a r t i c l e

i n f o

Article history: Received 17 December 2013 Accepted 18 January 2014 Available online 24 January 2014 Keywords: Deoxynivalenol Genotoxicity Micronucleus Comet TK6 HepaRG

a b s t r a c t Deoxynivalenol (DON) is the major mycotoxin detected in cereal foods and a risk for human health following DON ingestion could not be excluded due to high level exposure. In this light, the hazard of DON must be carefully evaluated. Therefore, the aim of this study is to perform in vitro genotoxicity tests with DON using the Salmonella typhimurium reverse mutation assay (Ames’ test), the comet assay and the micronucleus test in accordance with the OECD test guideline 487 in two human cell lines: the lymphoblastoid TK6 and the hepatoma HepaRG cells. DON gave negative results in the Ames’ test performed, both with and without rat liver S9 on three strains TA98, TA100 and TA102. DON elicited cytotoxicity in TK6 and HepaRG cells but did not induce primary DNA damage. DON failed also to induce MN formation in TK6 cells with or without human and rat liver S9. After 24 h of treatment, DON induced micronucleus formation in TK6 cells but only at concentrations producing more than 55 ± 5% cytotoxicity. In HepaRG cells, DON highly increased the caspase-3/7 activity but no micronucleus induction was observed. Taken together, our results suggest that DON could be considered as a non in vitro genotoxin. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Deoxynivalenol (DON) is a type B trichothecene produced by Fusarium species and the major mycotoxin detected in cereal foods such as barley, wheat and corn based products (AFSSA, 2009; EFSA, 2004). The French Agency for Food, Environmental and Occupational Health and Safety (ANSES) undertook two Total Diet Studies (TDSs), one between 2000 and 2004 (TDS1) and one between 2006

Abbreviations: ANOVA, analysis of variance; ANSES, French Agency for Food, Environmental and Occupational Health and Safety; BN, binucleated; CPA, cyclophosphamide; Cyt B, cytochalasin B; DMSO, dimethyl sulphoxide; DON, deoxynivalenol; EFSA, European Food Safety Authority; FCS, fetal calf serum; MEM, minimum essential medium; MMS, methylmethanesulfonate; MN, micronucleus; MTDI, maximum tolerable daily intake; OECD, organisation for economic cooperation and development; PBS, phosphate-buffered saline; TDS, Total Diet Study. ⇑ Corresponding author. Address: Anses Laboratoire de Fougères, Unité de Toxicologie des contaminants, 10 B rue Claude Bourgelat – Javené CS 40608, 35306 Fougères Cedex, France. Tel.: +33 (0)2 99 17 27 47; fax: +33 (0)2 99 94 78 80. E-mail addresses: [email protected] (N. Takakura), [email protected] (F. Nesslany), [email protected] (V. Fessard), [email protected] (L. Le Hegarat). 1 Anses Laboratoire de Fougères, 10 B rue Claude Bourgelat – Javené CS 40608, 35306 Fougères Cedex, France. 2 Institut Pasteur de Lille, Laboratory of Toxicology 1, rue du professeur Calmette, BP 245, 59019 Lille Cedex, France. http://dx.doi.org/10.1016/j.fct.2014.01.029 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.

and 2010 (TDS2), on a large panel of substances present in food including mycotoxins. In these studies, DON dietary exposure was estimated by measuring its level in food products representative of a normal diet. TDSs results showed that the total intake of DON for the French population has increased from TDS1 to TDS2 exceeding the health-based guidance value (maximum tolerable daily intake (MTDI) = 1 lg/kg body weight/day) (Leblanc et al., 2005; Sirot et al., 2013). Therefore, these studies indicate that consumers are routinely exposed to DON at doses where a risk for human health could not be excluded. Then, considering the chronic exposure to DON, carcinogenicity is one of the main human health hazards that need to be clarified. As mutagenicity is a key effect leading to carcinogenicity, the mutagenic potential of this mycotoxin must be carefully characterised. Whereas previous studies indicated the formation of primary DNA damage by the comet assay both in human intestinal Caco2 cells and hepatoma HepG2 cells with DON (Bony et al., 2006; Zhang et al., 2009), as well as chromosomal aberrations in primary cultures of rat hepatocytes (Knasmuller et al., 1997), clastogenicity has never been studied on human cells even with the OECD test guideline 487 detecting the in vitro micronucleus (MN) formation. The objective of the present study was to address the in vitro genotoxicity of DON according to the recommendations of the scientific opinion of European Food Safety Authority (EFSA) (EFSA,

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2011) using the Ames’ test on three Salmonella typhimurium strains as well as the comet and the MN assays on two human cell models: the TK6 lymphoblastoid cell line, a common cell line used in genetic toxicology and the metabolic competent HepaRG cell line since liver is a common target for xenobiotics and the main site of their bioactivation. 2. Materials and methods 2.1. Chemicals Deoxynivalenol (DON), benzo[a]pyrene (B[a]P), dimethyl sulphoxide (DMSO), cyclophosphamide (CPA), human insulin and glutamine from Sigma–Aldrich (Saint-Quentin Falavier, France). Methylmethanesulfonate (MMS) was purchased from Acros Organics (Geel, Belgium). Hydrocortisone hemisuccinate was purchased from Upjohn Pharmacia (Guyancourt, France). Fetal calf serum (FCS) for HepaRG cells was purchased from Perbio (Brebières, France). RPMI 1640 medium with Glutamax™, MEM with Glutamax™, Williams E medium, phosphate-buffered saline (PBS), penicillin, streptomycin, and FCS for

TK6, were supplied by Invitrogen Corporation (Illkirch, France). All other chemicals were of the highest quality available. DON was dissolved in DMSO at 5 mg/ml, liquoted and stored at 20 °C. The final concentration of DMSO in all assays did not exceed 0.6%.

2.2. Microscale bacterial reverse mutation assay (Ames’ test) Taking into account the limited amount of compound, the current assay was performed using the Ames’ test in micromethod (Courty et al., 2008; Nesslany et al., 2009) in S. typhimurium strains TA98, TA100, and TA102 from 0.03 to 500 lg of DON /plate. After enzymatic induction with Arochlor 1254, rat liver S9 was prepared according to (Ames et al., 1975) and (Maron and Ames, 1983). The S9-mix included 10% S9 fraction, 8 mM MgCl2, 32 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP and 100 mM sodium phosphate (pH7.4). Briefly, concentrated bacterial suspension and DON with and without rat liver S9 (5% S9 final concentration) were incubated at 37 °C under stirring for 90 min before plating. Then, the content of each well was transferred in 2 ml of top agar (to which 10% of 0.5 mM biotin histidine solution was added), maintained in a state of superfusion at 45 °C. The contents of each tube were shaken, prior to spreading out in a Petri plate containing 20 ml of minimum agar. After 48-h incubation at 37 °C, the number of

Fig. 1. Treatment schedule for the micronucleus (MN) test on TK6 and HepaRG cells.

Relative increase in cell count (RICC), RICC =

Increase in number of treated cells (final — initial) x100 Increase in number of control cells (final — initial)

Relative population doubling (RPD) PD (Population doubling) = Number of PD in treated cells

RPD =

log (final cell number/initial cell number) log 2 x100

Number of PD in control cells Relative cell counts (RCC) Final cell number in treated culture Final cell number in control culture

RCC =

x100

Replicative index (RI) RI =

Number of binucleated cells + 2 x polynucleated cells Total cell number

Relative RI =

RI of treated culture RI of control culture

X100

Fig. 2. The different methods to calculate cytotoxicity from cell counting as defined in the OECD guideline No. 487.

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N. Takakura et al. / Food and Chemical Toxicology 66 (2014) 113–121 Table 1 Ames’ test in micromethod on Salmonella typhimurium strains TA98, TA100 and TA102 with DON. Metabolic activation

S9-mix

Treatment

Dose (lg/plate)

TA98

TA100

TA102

DON

0.00 0.03 0.08 0.23 0.69 2.06 6.17 18.52 55.56 167.00 500.00

20.8 19.7 19.3 25.7 23.3 22.7 19.3 19.7 18.3 22.3 19.3 984

143 122.7 137.7 136.3 132.3 136.0 135.0 131.0 125.0 132.3 156.7 2992

154.5 145.0 184.0 177.7 186.7 180.0 171.0 177.0 179.3 183.0 211.7 2114.7

0.00 0.03 0.08 0.23 0.69 2.06 6.17 18.52 55.56 167.00 500.00

26.7 27.7 34.7 31.0 34.0 31.0 30.3 31.3 28.0 37.0 33.0 858.7

151.0 147.3 143.3 133.7 165.0 145.0 146.0 146.3 153.7 135.7 141.3 1461.3

185.8 201.0 251.3 272.3 287.3 275.7 202.0 219.3 203.0 206.3 209.7 1538.7

Positive controla +S9-mix

DON

Positive controlb a b

TA98: 2-nitrofluorene 0.5 lg/plate; TA100: MethylNitroNitrosoGuanidine 0.25 lg/plate; TA102: Mitomycine C 0.0625 lg/plate. TA98, TA100: 2-anthramine 0.5 lg/plate; TA102: Benzo[a]pyrene 4 lg/plate.

few hours, HepaRG cells took an elongated shape with active division until they reached confluence. After 2 weeks, they were shifted to the same culture medium including 2% DMSO for two more weeks to obtain differentiated cells. In the present study, HepaRG cells were used between passages 14 and 19. Differentiated HepaRG cells were exposed to DON in a FCS-free medium for 3 and 24 h.

Table 2 Comet assay with DON on TK6 and HepaRG cells. Cells

Agent

lM

% Tail DNAa

Cytotoxicityb (%)

TK6

DON

0.00 0.25 0.50 0.75 1.00 1.50 45.00

0.95 ± 0.27 0.50 ± 0.16 0.83 ± 0.18 0.99 ± 0.39 1.00 ± 0.11 0.41 ± 0.06 5.27 ± 0.76*

0.0 4.4 ± 1.8 20.7 ± 9.6 35.0 ± 5.5 43.4 ± 3.3 56.0 ± 6.4 11.9 ± 4.8

0 5 10 20 25 30 35 1000

2.30 ± 0.01 2.16 ± 0.14 1.64 ± 0.01 2.60 ± 0.14 1.83 ± 0.00 Toxic Toxic 5.48 ± 0.35*

0.0 17.4 ± 14.3 11.7 ± 0.5 15.4 ± 10.3 37.9 ± 1.4 59.7 ± 3.1 52.5 ± 3.6 12.9 ± 0.3

MMS HepaRG

DON

CPA a

Mean of the medians from two independent experiments. 100 – RCC (Relative cell count) after 24-h treatment. p < 0.05, ND: not determined.

b *

Revertants/plate

revertant colonies was scored for each plate. Each experimental point was tested in triplicate. Appropriate positive reference controls were also tested (without S9: 2nitrofluorene (0.5 lg/plate) for TA98, methylnitronitrosoguanidine (0.25 lg/plate) for TA100, and mitomycine C (0.0625 lg/plate) for TA102; with liver S9: 2-anthramine (0.5 lg/plate) for TA98 and TA100, and B[a]P (4 lg/plate) for TA102). 2.3. Cell culture The human TK6 lymphoblastoid cell line was maintained as suspension culture in RPMI 1640 medium with Glutamax™ and supplemented with 10% FCS, 100 IU/ml penicillin and 100 lg/ml streptomycin. The average doubling time of the TK6 cells was 10–12 h. TK6 cells (2  105 cells/ml) were treated with DON in 5% FCS culture medium for 3 and 24 h. The human hepatoma HepaRG cell line (Biopredic international, Rennes, France) was cultured as previously described (Bazin et al., 2010; Le Hegarat et al., 2010). Briefly, they were seeded at 2.1  104 cells/cm2 in Williams E medium supplemented with 10% FCS, 100 IU/ml penicillin, 100 lg/ml streptomycin, 5 lg/ml insulin, 2 mM L-glutamine and 25 lM hydrocortisone hemisuccinate. Within a

2.4. Comet assay The comet assay was performed only on cells treated with DON for 24 h as described previously by Le Hegarat et al. (2010). DNA was stained with propidium iodide (2.5 lg/ml in PBS) just before scoring with a fluorescence microscope (Leica DMR) equipped with a CCD-200E video camera. Fifty to one hundred cells per slide and 2 slides per concentration were analysed using the Comet Assay IV software (Perceptive Instruments, Haverhill, UK). The extent of DNA damage in individual cells was evaluated by the percentage of tail DNA. Two independent experiments (each one performed with duplicate cultures) were conducted. 2.5. Caspase-3/7 activity The activity of caspases 3 and 7 was determined as an indicator of apoptosis by an Apo-ONE Homogeneous caspase-3/7 assay (Promega) according to the manufacturer’s instructions. At the end of DON treatment, the caspase-3/7 substrate Z-DEVD-R110 was added to cell cultures for 1.5 h. Fluorescence was measured with excitation and emission wavelengths of 485 and 520 nm respectively, using a spectrophotometer (Fluostar Optima, BMG Labtech). Data were expressed as fold change in caspase-3/7 activity relative to solvent control. Two independent experiments on duplicate cultures were conducted. 2.6. Micronucleus test The protocol was in accordance with the OECD guideline No. 487 (OECD, 2010) with some modifications as previously described (Bazin et al., 2010; Fessard and Le Hegarat, 2010; Le Hegarat et al., 2010), according to the treatment schedule described in Fig. 1. Two independent experiments (on duplicate cultures) were conducted. 2.7. MN test on TK6 cells Human (from a single donor) and rat (b-naphtoflavone and phenobarbital-induced) liver S9 fractions were purchased from Biopredic international (Rennes, France). The composition of S9 mix was the same as the one described for the Ames’ test. For short time exposure (3 h), cells were treated with DON in the absence or in the presence of S9 (2% S9 final concentration). Then the treatment medium was replaced by 10% FCS fresh medium and cells were collected 21 h later. For the long

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time exposure, TK6 cells were treated with DON for 24 h or for 18 h prior to 18 h recovery. B[a]P (8 lM) and MMS (45 lM) were chosen as positive controls with and without S9 mix, respectively. Cells were collected by centrifugation (136 g for 5 min) and counted for cytotoxicity evaluation: relative increase in cell count (RICC), relative population doubling (RPD) and relative cell counts (RCC) were calculated according to the OECD Guideline N° 487 (Fig. 2). For micronucleus scoring, the cell pellets were treated for 4 min with a hypotonic solution (RPMI medium: distilled water = 1:1 (v/v)) prior to fixation (methanol: acetic acid = 3:1 (v/v)) for 10 min at room temperature. Cells were centrifuged again, resuspended in the fixative solution, spread on glass slides and stained with acridine orange (67 lg/ml in PBS). Micronuclei were scored in 2000 mononucleated cells per experiment under a fluorescence microscope (Leica DMR, Germany). 2.8. Cytokinesis-block MN test on HepaRG cells CPA (1 mM) was used as positive control as previously published (Le Hegarat et al., 2010). At the end of treatment, cells were washed twice with PBS, trypsinized and seeded at 1.4  104 cells/cm2 in 2 chambers Lab-Tek slide system. After 24 h, the cells were incubated in a medium containing 10% FCS and 4.5 lg/ml of cytochalasin B (Cyt-B) for 24 h. After twice washing with PBS, the cells were allowed to recover for 1.5 h in a 10% FCS-fresh medium prior to fixation (methanol: acetic acid = 9: 1 (v/v)) for 10 min. Then, the slides were stained with acridine orange and scored under a fluorescence microscope (Leica, DMR). The criteria used for identifying micronuclei were those recommended by the HUMN project (KirschVolders et al., 2003). Micronuclei have been scored in 1000 binucleated cells from each chamber. Cytotoxicity was determined from scoring 500 cells (binucleated, mononucleated and polynucleated) per culture. Cytostasis was calculated from the replicative index (RI) (Fig. 2) and cytotoxicity (%) was expressed as 100 – relative RI. 2.9. Statistical analysis Statistical analyses were carried out using GraphPad PRISM 5 (GraphPad Software Inc., San Diego, CA, USA). For the Ames’ test results, statistical analysis was performed with the Dunnett’s test allowing the comparison of the mean value for each dose to the mean value for the corresponding solvent control (Mahon et al., 1989). For the comet assay, mean of the medians from the two independent experiments were analysed with one-way Anova followed by the Dunnett’s test. Fold changes in caspase 3/7 activity relative to solvent were analysed with a one-sample Student t test, comparing the group’s means with the fixed value of 1.0. Comparison of the proportion of micronucleated cells in treated and solvent control cultures was performed by a Chi-square test for HepaRG, and with Yates’ like correction for TK6 cells. In all tests, data were considered significantly different when p < 0.05.

3. Results 3.1. DON did not induce gene point mutations in microscale bacterial reverse mutation assay DON, at dose range from 0.03 lg to 500 lg/plate, induced no statistically significant increases in the number of revertants

3.2. DON did not induce primary DNA damage in the comet assay on TK6 and HepaRG cells No primary DNA damage was detected in TK6 and HepaRG cells treated for 24 h with DON using the comet assay (Table 2) whereas positive controls, MMS and CPA, clearly induced DNA migration in TK6 and HepaRG cells, respectively. DON was cytotoxic to both cell lines although proliferative TK6 cells were more sensitive than quiescent HepaRG cells; 55% cytotoxicity was induced with 1.5 lM DON in TK6 cells, when up to 30 lM DON was required to induce 60% cytotoxicity in HepaRG cells. No caspase-3/7 activity increase was detected in TK6 cells after 3 h and 24 h DON treatment (data not shown), whereas a large increase of caspase-3/7 activity was found in HepaRG cells after 3 and 24 h of treatment up to 12.5 lM DON (Fig. 3). 3.3. DON induced MN formation on TK6 cells only at cytotoxic concentrations After a 3 h treatment in the absence of S9, DON induced a statistically significant increase in the MN formation only in the experiment 1 at the highest concentration tested (25 lM) (Fig. 4 and Table 3). However, 25 lM DON induced also a cytotoxicity level (64.6%, 80.7% and 64.6% for RCC, RICC and RPD, respectively), exceeding the OECD guideline 487 recommended limit (55 ± 5%) (OECD, 2010). The positive control, MMS, induced a significant MN formation in the two experiments, 18.1 and 19.5‰ micronucleated cells, compared to the solvent control, 4.5 and 3.5‰ (Table 3). After a 3 h treatment in the presence of rat liver S9, DON did not induce MN formation in TK6 cells in the two independent experiments even for a concentration inducing more than 55% cytotoxicity (Fig. 4 and Table 3). Concurrently, B[a]P significantly increased MN formation in the two experiments (Table 3). After a 3 h treatment in the presence of human liver S9, DON induced a statistically significant increase in the MN formation in TK6 cells at the two highest concentrations of 12.5 and 25 lM (6.5 and 6.0‰ micronucleated cells, respectively), only in the second experiment (Fig. 4 and Table 3). These data were statistically significant probably due to the low level of MN frequency in the

30

A

Fold increase

Fold increase

30

irrespective of the S. typhimurium strains TA98, TA100 and TA102 either in the absence or in the presence of liver S9-mix (Table 1).

20

10

**

***

***

25.0

50.0

0

B

***

20

*** 10

*** 0

0

1.6

6.4

12.5

DON (µM)

0

1.6

3.2

6.4

12.5

25.0

50.0

DON (µM)

Fig. 3. Caspase 3/7-induced activity in HepaRG cells with DON. Caspase 3/7 activity in HepaRG cells after 3 h (A) and 24 h (B) DON treatment. Results are expressed as the mean ± SE of two independent experiments with duplicate cultures **p < 0.01. ***p < 0.001.

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3 + 21 hrs w/o S9 Exp.1 20

MN/1000

RCC

RICC

3 + 21 hrs w/o S9 Exp.2 RPD

20

100

MN/1000

RCC

RICC

RPD

90

90

80

80

15

15 70

50 40

MN/1000

*

10

60 10

50 40 30

30 5

5

20

20 10 0

10 0

0 0

1.6

3.1

6.3

12.5

25.0

0 0

1.6

DON (µM)

MN/1000

RCC

3.1

6.3

12.5

25.0

DON (µM)

3 + 21 hrs with rat S9 Exp.1 20

RICC

3 + 21 hrs with rat S9 Exp.2 RPD

100

20

MN/1000

RCC

RICC

RPD

90

80

15

15

70

40

MN/1000

50

60 10

50 40

30

30

5

5

20

20

10 0

10

0 0

1.6

3.1

6.3

12.5

0

25.0

0 0

1.6

DON (µM)

MN/1000

RCC

3.1

6.3

12.5

25.0

DON (µM) 3 + 21 hrs with human S9 Exp.2

3 + 21 hrs with human S9 Exp.1 20

RICC

RPD

20

100

MN/1000

RCC

RICC

RPD

80

80

15

15

70

40 30 5

0 3.1

6.3

12.5

10

50 40

*

5

*

30

20

20

10

10

0 1.6

60

25.0

DON (µM)

0

Cytotoxicity

50

Cytotoxicity

10

MN/1000

70 60

100 90

90

0

Cytotoxicity

10

70

Cytotoxicity

60

100 90

80

MN/1000

Cytotoxicity

60

Cytotoxicity

MN/1000

70

MN/1000

100

0 0

1.6

3.1

6.3

12.5

25.0

DON (µM)

Fig. 4. Micronucleus test with DON on TK6 cells after a short treatment of 3 h without and with rat or human liver S9. Graphs represent the frequency of TK6 micronucleated cells. The cytotoxicity is indicated by RCC, RICC and RPD. The maximum toxicity recommended is the OECD guideline N°487 (55 ± 5%) is shown ( ). *p < 0.05. **p < 0.01. *** p < 0.001.

solvent control (2.0‰) compared to the historical negative control dataset (CI95% around 5‰) (data not shown), and were, therefore, considered as non-biologically relevant. Interestingly, B[a]P failed to induce MN formation in TK6 cells in the presence of human liver S9 (Table 3). After a 24 h continuous exposure, DON increased significantly MN formation at 1.6 and 3.2 lM compared to the negative control, in the two independent experiments (Fig. 5 and Table 4). In the

first experiment, DON increased the frequency of micronucleated cells up to 11.0 and 17.4‰ at 1.6 and 3.2 lM, respectively, compared to 5.0‰ for the control, and in the second experiment up to 9.0 and 11.5‰, respectively, versus 3.0‰ for the control (Table 4). However, MN induction was observed for cytotoxic DON concentrations (more than 60% for RICC). The clastogenic positive control MMS (45 lM) highly increased the frequency of micronucleated cells in the two experiments (22.4 and 21.5‰).

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Table 3 Micronucleus assay with DON on TK6 cells after a short treatment of 3 h without and with rat or liver S9.

24 hrs Exp.1 20

MN/1000

RCC

RICC

RPD

100

***

90 80

15 60

* 10

50 40

Cytotoxicity

MN/1000

70

30 5 20 10 0

0 0

0.4

0.8

1.6

3.2

DON (µM) 24 hrs Exp.2 20

MN/1000

RCC

RICC

RPD

100 90 80

15

*** 10

60 50

*

40

Cytotoxicity

MN/1000

70

30 5 20 10 0

0 0

0.4

0.8

1.6

3.2

DON (µM) 18 + 18 hrs 20

MN/1000

RCC

RICC

RPD

100 90 80

15

MMS (45 lM) was used as positive control in the absence of S9. b B[a]P (8 lM) was used as positive control with S9. ⁄ p < 0.05. ⁄⁄ p < 0.01. ⁄⁄⁄ p < 0.001.

60

*

10

50 40

Cytotoxicity

a

MN/1000

70 Statistically significant MN induction ( ), cytotoxicity exceeding 60% ( ).

30 5 20 10 0

After a 18 h treatment, a statistically significant increase in the number of micronucleated cells was detected only at the highest DON concentration (3.2 lM) inducing also a high rate of cytotoxicity (93% RICC) (Fig. 5 and Table 4).

0 0

0.4

0.8

1.6

2.5

3.2

DON (µM) Fig. 5. Micronucleus test with DON on TK6 cells after a long-term treatment of TK6 with DON without S9. TK6 cells were exposed for 24 h without recovery or for 18 h prior to 18 h recovery. The maximum toxicity recommended is the OECD guideline N°487 (55 ± 5%) is shown ( ). *p < 0.05. **p < 0.01. ***p < 0.001.

3.4. DON did not induce MN formation on metabolic competent HepaRG cells

4. Discussion

Whereas the promutagen CPA clearly induced MN formation, DON did not increase the percentage of micronucleated cells irrespective of the treatment duration (3 h and 24 h) and the concentration of exposure (from 1.6 to 50 lM and from 5 to 35 lM) in two independent experiments (Table 5).

Food is a main source of contaminants including natural compounds for which mutagenicity must be characterised to assess the risk for human consumers. The Fusarium mycotoxin deoxynivalenol is commonly found in foodstuff and some consumers, children in particular; are exposed to DON levels close to the

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N. Takakura et al. / Food and Chemical Toxicology 66 (2014) 113–121 Table 4 Micronucleus assay with DON on TK6 cells after a long-term treatment of TK6 with DON without S9.

Statistically significant MN induction ( ), cytotoxicity exceeding 60% ( ). a

MMS (45 lM) was used as positive control. p < 0.05. p < 0.01. ⁄⁄⁄ p < 0.001. ⁄

⁄⁄

Table 5 Cytokinesis-block micronucleus assay with DON on HepaRG cells. Exposure time

Treatment

(lM)

Experiment 1 MNBNsa (%)

3h

DON

CPA 24 h

DON

CPA a ***

Experiment 2 RI (%)

MNBNsa (%)

RI (%)

0.0 1.6 6.3 12.5 25 50 1000

3.2 3.2 2.6 2.9 3.0 3.0 8.2***

0.66 2.23 3.68 3.40 2.89 7.21 14.82

2.6 3.0 3.9 3.0 2.5 3.1 5.3***

5.85 11.98 0.97 12.39 18.52 13.37 3.34

0 5 10 15 20 25 30 35 1000

2.8 3.5 2.9 2.9 2.6 2.3 1.8 2.3 13.5***

0.00 0.39 11.79 0.13 1.30 1.68 5.18 6.86 0.28

2.8 4.0 2.4 2.5 3.5 2.6 2.3 3.1 11.2***

0.00 3.77 3.22 1.21 0.38 11.46 5.99 6.27 1.45

MNBN = Micronucleated binucleated HepaRG cells. p < 0.001.

provisional MTDI (TDS2) (Sirot et al., 2013). However, the genotoxic potential of DON has not been fully elucidated. The scientific opinion of EFSA (2011) entitled ‘‘genotoxicity testing strategies applicable to food and feed safety assessment’’ recommended to use two in vitro tests as the first step: a bacterial reverse mutation assay (OECD, 1997) and an in vitro MN test (OECD, 2010). This combination covers largely the genetic endpoints of gene mutations, and both structural and numerical chromosome alterations (Kirkland et al., 2011). In the present study, following those recommendations, the genotoxicity of DON was investigated by the gene mutation Ames’ test and by the comet and MN assays on two human cell lines. We found that DON failed to exert any mutagenic activity in three S. typhimurium strains TA98, TA100 and TA102 at a concentration range of 0.03500 lg/plate in the presence and absence of rat liver S9. Our results are in accordance with those previously

published by Wehner et al. (1978) and by Knasmuller et al. (1997), suggesting the absence of gene mutagenic potential of DON. Afterwards, DON in vitro genotoxic potential was investigated by the comet assay and the MN test in human lymphoblastoid TK6 cells and in human hepatoma HepaRG cells. TK6 cell line is p53-competent and karyotypically stable (Pfuhler et al., 2011; Schwartz et al., 2004), and has been included as optional cell model in the in vitro micronucleus test OECD guideline 487 (Bryce et al., 2013; Fowler et al., 2012a; Lorge, 2010; Nesslany and Marzin, 2010; Pfuhler et al., 2011). In the short-term treatment in the absence of S9, DON at 25 lM induced an inconsistent increase in the MN frequency in TK6 cells which could be due to a high cytotoxicity level exceeding 60% for RCC, RICC and RPD. In the presence of human or rat liver S9, DON did not induce any significant MN formation in TK6 cells. For a long-term treatment

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without metabolic activation including or not a recovery period, DON increased MN formation at concentrations inducing also more than 50% cytotoxicity as measured by RICC and RCC, and partially by RPD. Currently, RICC and RPD are preferred as cytotoxic parameters because RCC is known to underestimate cytotoxicity in the non-cytokinesis blocked in vitro MN assay (Fowler et al., 2012a; Lorge et al., 2008; Nesslany and Marzin, 2010). Very recently, it was shown that, if cells are harvested after more than 2.0 cell cycles, RPD values progressively decrease compared to RICC values due to log transformation, underestimating the cytotoxicity level (Honma, 2011; O’Donovan, 2012). In our study, as PD values for negative control cells varies between 1.9 and 2.7, RICC would be the most appropriate estimation of cytotoxicity. According to the OECD guideline No. 487, mutagenicity detected for concentrations inducing more than 55 ± 5% of cytotoxicity could be due to a secondary event. Our results suggest that MN induction observed for few DON concentrations in TK6 cells was due to cytotoxicity. Furthermore, DON decreased also the cell number dose-dependently after 24 h treatment without inducing primary DNA damage detected by the comet assay in TK6 cells. Taken together, we conclude that DON did not elicit chromosome damage in TK6 cells regardless the absence or presence of human or rat S9. In the present study, DON failed to induce primary DNA damage and MN in the metabolically competent HepaRG cells. These negative results suggest that DON was not bioactivated by metabolism enzymes expressed in HepaRG cells. On the contrary, a previous study in HepG2 cells (Zhang et al., 2009) showed that DON induced comet formation and that this DNA damage was due to oxidative stress. Unlike HepG2 cells, HepaRG cells express the majority of phase I and phase II human liver enzymes with levels of expression close to those found in primary human hepatocytes (Andersson et al., 2012; Aninat et al., 2006; Anthérieu et al., 2010, 2012; Gerets et al., 2012). Indeed, recently, the HepaRG cell line was reported as suitable to detect promutagenic compounds (Bazin et al., 2010; Jossé et al., 2012; Le Hegarat et al., 2010). As these two hepatic cell lines differ for their metabolism capacities, the primary DNA damage observed on HepG2 with DON could be explained by the higher sensitivity of this cell line to oxidative stress compared to HepaRG cells (Zhang et al., 2009). Because apoptosis could contribute to positive MN responses, it was proposed to measure caspase-3/7 activation to monitor apoptosis-related cytotoxicity (Fowler et al., 2012b; Shi et al., 2010). It has been reported that DON induced caspase activation and apoptosis via p53 expression in other cell lines (Bensassi et al., 2009; He et al., 2012; Ma et al., 2012). In our study, DON activated caspase3/7 dose-dependently without inducing MN formation in p53competent HepaRG cells whereas, in p53-competent TK6 cells, caspase-3/7 activity did not increase (data not shown), although cell numbers and PD decreased dramatically after DON treatment. Therefore, the mechanisms involving apoptosis and the p53 pathway in DON toxicity should be further investigated.

5. Conclusion In conclusion, the current study indicates that DON did not induce gene point mutations in bacteria, or chromosome damage in TK6 and HepaRG cells, suggesting that DON is devoided of in vitro mutagenic/genotoxic potential. Currently, in vivo genotoxic studies combining the MN and comet assays are conducted to confirm the results obtained in vitro.

Conflict of Interest The authors declare that there are no conflicts of interest.

Transparency Document The Transparency document associated with this article can be found, in the online version.

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