Chemical reduction of the mycotoxin beauvericin using allyl isothiocyanate

Food and Chemical Toxicology 50 (2012) 1755–1762

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Chemical reduction of the mycotoxin beauvericin using allyl isothiocyanate G. Meca a,⇑, F.B. Luciano b, T. Zhou b, R. Tsao b, J. Mañes a a b

Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Av. Vicent Andrés Estellés s/n, 46100 Burjassot, Spain Guelph Food Research Centre, Agriculture and Agri-Food Canada, 93 Stone Rd. W, Guelph, ON, Canada N1G 5C9

a r t i c l e

i n f o

Article history: Received 14 December 2011 Accepted 22 February 2012 Available online 3 March 2012 Keywords: Beauvericin Allyl isothiocyanates Mycotoxins reduction LC-DAD LC-MS/MS

a b s t r a c t Beauvericin (BEA) is a bioactive compound produced by the secondary metabolism of several Fusarium strains and known to have various biological activities. This study investigated the reduction of BEA present in the concentration of 25 mg/kg on a solution model (phosphate buffer saline at pH 4 and 7) and in wheat flour using allyl isothiocyanate (AITC) as a reactant. The concentration of the mycotoxin studied was evaluated using liquid chromatography coupled to the diode array detector (LC-DAD), whereas adducts formed between the BEA and AITC were examined by liquid chromatography coupled to mass spectrometry-linear ion trap (LC-MS-LIT). In solution, BEA reduction ranged from 20% to 100% on a time-dependent fashion and no significant differences were evidenced between the two pH levels employed. In the food system composed by wheat flour treated with gaseous AITC (50, 100 and 500 lL/L), the BEA reduction varied from 10% to 65% and was dose-dependent. Two reaction products between the bioactive compounds employed in this study were identified, corresponding to BEA conjugates containing one or two AITC molecules. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Beauvericin (BEA) is a cyclic hexadepsipeptide consisting of an alternating sequence of three D-a-hydroxy-isovaleryl and three N-methyl-L-phenylalanyl groups. It was originally isolated from Beauveria bassiana (Hamill et al., 1969) and has been detected in various fungal species, including Fusarium spp. which is a common contaminant of cereals and cereal-containing products (Jestoi 2008). The cytotoxicity of BEA has been studied by several authors in rodent, monkey, porcine, insect and human cell lines (Tomoda et al., 1992; Macchia et al., 1995; Nash et al., 1998; Nilanonta et al., 2002; Calo et al., 2003; Fornelli et al., 2004; Ruiz et al., 2011) using different determination techniques such as the tetrazolium salt (MTT), the trypan blue dye exclusion, Alamar Blue, BrdU and sulforhodamine B (SRB) assays. BEA is considered a common contaminant of cereals and also of products composed by cereals (Zinedine et al., 2011). It is noteworthy that the presence of BEA in food commodities reported during the last decade in some European countries (i.e. Finland, Norway, Spain, Slovakia, Croatia, Switzerland and Italy), the USA and South Africa (Zinedine et al., 2011; Jestoi 2008; Meca et al., 2010a,b Ritieni et al., 1997; Munkvold et al., 1998; Shephard et al., 1999) were at higher concentrations (milligrams per kilograms) then those

⇑ Corresponding author. Tel.: +34 963544959; fax: +34 96354954. E-mail address: [email protected] (G. Meca). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2012.02.070

permitted to the classically legislated Fusarium mycotoxins as the fumonisins or the trichothecenes. Isothiocyanates (ITC) are products of enzymatic hydrolysis of glucosinolates, which are sulfur-containing glucosides characteristic from plants of the order Brassicales. This order includes commonly consumed vegetables of family Brassicaceae, such as broccoli, cabbage, cauliflower, brussels sprouts, mustard, horseradish and rocket salad (Nugon-Baudon and Rabot, 1994). Isothiocyanates are considered to be amongst the most potent naturally occurring bioactive compounds intervening in the process of cancer development (Holst and Williamson, 2004). Indeed, numerous epidemiological studies have shown that consumption of vegetables from Brassicaceae family is inversely associated with incidence of cancer, particularly of lung, stomach, colon, and rectum (Van Poppel et al., 1999; Verhoeven et al., 1996). Furthermore, it has been reported that intake of these vegetables may reduce the risk of breast, prostate and bladder cancer development (Ambrosone et al., 2004; Fowke et al., 2003; Tang et al., 2008; Traka et al., 2008). The anticarcinogenic properties have been attributed to the ability of ITC to inhibit the activity of phase I xenobiotic metabolizing enzymes, and/or to induce the activity of phase II detoxification enzymes (Fahey and Talalay, 1999). Isothiocyanates contribute to the characteristic pungent taste of Brassicaceae vegetables (Engel et al., 2002), and have been reported as potent antimicrobials (Luciano and Holley, 2009). Allyl isothiocyanate (AITC), which is the most studied ITC, was found to inhibit the growth of yeast, mould and bacteria at very low levels (Isshiki et al., 1992).

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ITC are characterized by the presence of a –N@C@S group, the central carbon of which is strongly electrophilic (Zhang, 2004). This electrophilic nature enables ITC to readily bind to thiol and amino groups of amino acids, peptides and proteins, forming conjugates (Luciano and Holley, 2009), dithiocarbamate and thiourea structures (Cejpek et al., 2000). BEA is a peptide mycotoxin that may serve as an electron donor to the electrophile carbon present in isothiocyanates. The aim of this study was therefore to test the reactability of the mycotoxin BEA with AITC, and to assess the potential of using AITC to reduce or detoxify BEA.

water (<18 MX cm resistivity) was obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA). Chromatographic solvents and water were degassed for 20 min using a Branson 5200 (Branson Ultrasonic Corp., CT, USA) ultrasonic bath. 2.2. Solution model preparation Beauvericin (25 mg/L) was added to screw-capped tubes containing 10 mL of phosphate saline buffer at pH 4 or 7. AITC at 1 mM was added to all tubes, which were subsequently tightly closed and shaken at 200 rpm and 23 °C. Aliquots were drawn at 0, 4, 8, 24 and 48 h and directly injected into the LC system for BEA analysis. 2.3. Wheat flour experiments

2. Materials and methods 2.1. Materials BEA (98% purity), phosphate buffer saline (PBS) at pH 7, formic acid (HCOOH) and AITC were obtained from Sigma–Aldrich (St. Louis, USA). Acetonitrile, and methanol were purchased from Fisher Scientific (New Hempshire, USA). Deionized

Two petri-dish bottoms (50 mm diameter) containing 2 g of all-purpose white wheat flour were placed into 1 L Mason jars (Analisis Vinicos, Spain). Then, 200 lL of a BEA stock solution (dissolved in methanol) was added to the flour to achieve a final concentration of 25 mg/kg. A 2.5  2.5 cm paper-filter soaked with AITC was inserted into the jars, giving final concentrations of 50, 100 and 500 ll/L in the gaseous phase after total volatilization of the essential oil. The

Fig. 1. Schematic representation of chemical beauvericin (BEA) reduction employing allyl isothiocyanate (AITC) in a food system composed by white wheat flour. Two plastic petri dishes containing 2 g of wheat flour contaminated with 25 mg/kg of BEA were introduced inside a glass bottle. The glass bottle was also introduced with a paper filter containing different concentrations of the volatile compound AITC. The gaseous AITC reacts with BEA present in the flour, reducing the presence of this food contaminant.

Fig. 2. Beauvericin reduction in model solution during the reaction with the allyl isothiocyanate at pH 4 (white) and 7 (black).

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2.4. Mycotoxin extraction procedure and clean-up BEA was extracted according to the procedures described by Jestoi (2008). Briefly, 2 g of wheat flour samples were extracted with 20 mL methanol using an Ultra Ika T18 basic Ultraturrax (Staufen, Germany) for 3 min. The mixture was centrifuged at 4500g for 5 min and then the supernatant was evaporated to dryness with a Büchi Rotavapor R-200 (Postfach, Switzerland). The residue was re-dissolved in 2 mL of extraction solvent. The extract was cleaned up using a Strata C18-E cartridge (6 mL, 1 g). The cartridge was first activated with 2  2 mL of methanol and conditioned with 2  2 mL of water before the extract was loaded. The cartridge was then washed with 2  2 mL of water at a flow rate of 0.5 mL/min till all water was out. BEA was eluted using 1 mL of methanol. Both the methanolic eluate and the water washing were filtered through 0.22 lM nylon filters purchased from Análisis Vínicos (Tomelloso, Spain) before LC and LC-MS analyses (Fig. 1). 2.5. HPLC analysis of BEA

Fig. 3. Beauvericin reduction in a food system composed by wheat flour contaminated with 25 mg/kg of the Fusarium toxin and treated with 50, 100 or 500 ll/L of allyl isothiocyanate.

control group did not receive any treatment. Jars were hermetically closed and kept at room temperature (23 °C) for 48 h. Then, they were opened inside a fume hood and left for 30 min allowing AITC gas to escape. The flour was used for further analysis.

BEA was analyzed using a Shimadzu LC system equipped with LC-10AD pumps and a diode array detector (DAD) from Shimadzu (Japan). The DAD was set at 205 nm for BEA detection, and the separation was in a Gemini (150  4.6 mm, 5 lm) (Phenomenex, Torrance, CA, USA) column. The mobile phase was acetonitrile–water (70:30 v/v) and delivered isocratically at a flow rate of 1.0 mL/min. The injection volume was 20 lL. The identification of BEA was performed by comparing retention times and UV spectra of peaks in extracted samples with those of the standard. Quantification of BEA was done using a calibration curve generated from serial dilutions of the standard (0.5–50 mg/L). 2.6. LC-MS-LIT identification of the BEA adducts An Applied Biosystems/MDS SCIEX Q TRAP TM linear ion traps mass spectrometer (Concord, Ontario, Canada), coupled with a Turbo Ion Spray source, was used. This instrument is based on a triple-quadrupole path (QqQ) in which the third

Fig. 4. LC-DAD chromatograms of (a) Beauvericin treated with 500 ll/L of allyl isothiocyanate in a food system. The figure denotes the presence of two new peaks after the toxin peak, which were not found in the (b) control.

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quadrupole can also be operated as a linear ion trap (QqLIT) with improved performance. In the QqLIT configuration the Q TRAPTM can also operate in enhanced resolution scan (ER) and in enhanced product ion scan (EPI) modes. Applied Biosystem/ MDS SCIEX Analyst software version 1.3.2 was used for data acquisition and processing. A Gemini (150  2.0 mm, 5 lm) Phenomenex column was used. LC was set up using a constant flow of 0.2 ml/min and acetonitrile/water (70:30 v/v with 0.1% of HCOOH) as mobile phases in isocratic condition. The instrument was operated in the positive ion electrospray mode using the following parameters: cone voltage 40 V, capillary voltage 3.80 kV, source temperature 350 °C, desolvation temperature 270 °C and collision gas energy 5 eV. The analyses were carried out using the following procedure:

(4 and 7) and incubation times of 4, 8, 24 and 48 h. As shown on Fig. 2, pH showed no significant effect on the extent of the reaction. At both pHs, BEA disappeared rapidly, averaging 77% reduction after 4 h, 85% at 8 h and 97.5% after 24 h. BEA was not detected after 48 h incubation with AITC, indicating a total disappearance of this mycotoxin. This result suggests that BEA, a mycotoxin otherwise stable at pH 4 or 7, can be either degraded or form new adduct compound(s) in the presence of AITC. 3.2. BEA reduction in wheat flour

(a) Characterization of the neo formed compounds with the modality of Enhanced Resolution Scan (ER), utilizing the mass range from 400 to 1200 Da. (b) Mass spectra characterization of the peak obtained in the ER scan with the modality enhanced product ion scan (EPI) to obtain a MS2 scan of a fragment of the adducts.

The potential reduction or detoxification of BEA by AITC as observed in the above mentioned model system was confirmed and validated in real food, in this study, wheat flour. The flour was artificially contaminated with BEA to a level of 25 mg/kg, and then exposed to gaseous AITC at 50, 100 and 500 ll/L during 48 h. BEA in extracts of wheat flour samples were also analyzed by LC and the reduction rates calculated as stated in the Section 2. As shown in Fig. 3, exposure to AITC for 48 h significantly reduced the amount of BEA in wheat flour. The effect was dose-dependent; AITC at 50 ll/L and 100 ll/L led to 46.6% and 52.4% reduction of BEA, respectively. The highest reduction of the mycotoxin observed in this study was 64% by 500 ll/L of AITC at the end of 48-h incubation. BEA in the blank control was stable and not degraded or reduced in concentration.

The utilization of the mass spectrometry associated at the detection with the linear ion trap utilized in these two modalities has permitted to obtain a total characterization of the compounds isolated (Meca et al., 2011). 2.7. Calculation and statistical analysis All experiments were performed three times. Statistical analysis was carried out using analysis of variance (ANOVA), followed by Dunnet’s multiple comparison tests. Differences were considered significant if p < 0.05.

3. Results and discussion 3.3. Potential mechanism of BEA reduction 3.1. BEA reduction in model solution Reduction of BEA can be caused by accelerated degradation in the presence of AITC, or by forming new adductive product(s) with AITC. The former possibility is low considering that BEA itself was

The reaction between BEA (25 mg/L) and AITC (0.1 mM) was monitored by LC in PBS buffer solutions at two different pH levels

TIC of +EMS: from Sample 15 (50 2 m) of pepe.wiff (Turbo Spray), Smoothed, Smoothed, Smoothed, Smoothed, Smoothed

Max. 3.7e8. cps

16.56

3.7e8 3.6e8

Peak 2

3.4e8

Peak 1

3.2e8 3.0e8 2.8e8

Intensity, cps

2.6e8 13.15

2.4e8 2.2e8

14.77 15.02

2.0e8

6.28

18.91

1.8e8 1.6e8 1.4e8

18.64 20.47 0.36

0.75

2.95

1.2e8

22.96

12.57

1.49 7.12

4.95 5.80

9.77

17.59

22.17

10.90

25.42

26.17

27.73

28.78

9.04

4.43

1.0e8 3.53

8.0e7 6.0e7 4.0e7 2.0e7 0.0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

Time, min Fig. 5. LC-MS-LIT chromatogram of the wheat flour extract. The flour was contaminated with 25 mg/kg BEA and exposed to gaseous allyl isothiocyanate for 48 h at room temperature.

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G. Meca et al. / Food and Chemical Toxicology 50 (2012) 1755–1762 +EMS: 13.092 to 13.347 min from Sample 17 (100 2 m) of pepe.wiff (Turbo Spray)

Max. 4.7e6 cps. 1039.7

4.6e6 4.4e6

(806+2H2O+2AITC)++

4.2e6 4.0e6

(784.6-(HyLv+1-N-Me-Phe))+

3.8e6 3.6e6 3.4e6 3.2e6

(822-(Phe+HyLv+Phe))+

3.0e6 520.6

Intensity, cps

2.8e6 2.6e6 2.4e6 2.2e6 2.0e6 1.8e6 1.6e6 1.4e6

502.5

1.2e6

(BEA+Na)+

1.0e6

(BEA+H)+

8.0e5 6.0e5

(BEA+K)+

806.6

4.0e5 443.5

2.0e5 0.0 400

483.5 542.5 573.4

450

500

550

1058.5

623.6

600

784.6 650

700

750

800

1092.4

1043.6

822.6 850

900

950

1000

1050

1100

1150

1200

m/z, Da

Fig. 6. ER mass spectrum (m/z 400–1200) of the Peak 1 evidenced in Fig. 5.

+EMS: 16.412 to 16.867 min from Sample 17 (100 2 m) of pepe.wiff (Turbo Spray)

Max. 1.1e7 cps.

496.2

1.09e7 1.05e7

(784.6-(Phe-HyLv-Phe))+

1.00e7 9.50e6 9.00e6 8.50e6

991.4

(784.6+HPO4+1AITC)+

8.00e6 7.50e6

Intensity, cps

7.00e6 6.50e6 6.00e6 5.50e6 5.00e6 4.50e6

478.2

4.00e6 3.50e6 3.00e6

(BEA+Na)+

2.50e6 2.00e6

(BEA+H)+

1.50e6 1.00e6 5.00e5 0.00

459.1 806.2 518.1

419.2

400

573.1

510.2 523.1 589.3

450

500

550

(BEA+K)+

599.3 772.3 784.3

623.2

600

650

700

750

800

1013.4 1044.2

850

900

950

1000

m/z, Da Fig. 7. ER mass spectrum (m/z 400–1200) of the Peak 2 evidenced in Fig. 6.

1050

1100

1150

1200

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extract was injected into the LC-MS system to identify the two newly formed peaks. The LC-MS-LIT total ion chromatogram confirmed the two peaks (Peak 1, Rt: 13.15 min; Peak 2, Rt: 16.56 min) shown in the HPLC profile (Fig. 5). Fig. 6 shows the mass spectrum of Peak 1 obtained in the positive ER mode. Information about this adduct can be obtained from several characteristic fragment ions, especially those indicative of the presence of BEA: m/z 784.3, 806.2, and 822.0 that represent [BEA + H]+, [BEA + Na]+ and [BEA + K]+, respectively (Fig. 6). The sodium adduct [BEA + Na]+ was the predominant ion among the three. Fragment ions at m/z 483.5 and 520.6 further

stable under in either buffered solutions at pH 4 and 7, or when spiked into wheat flour. Formation of new reaction products between BEA and AITC was considered the most likely mechanism as two new peaks were observed in both experiments. Figs. 4 and 5 are typical chromatographic profiles of the wheat flour extracts treated with 500 ll/L of AITC. Fig. 4a shows the BEA peak with two newly formed compounds eluted at later times (UV detection at 205 nm). These two peaks were not present in the extract of the control (wheat flour spiked with 25 mg/kg of BEA without exposure to AITC) (Fig. 4b). These two peaks are considered to be the BEA adducts with AITC. To test this hypothesis, the above

TIC of +EMS: from Sample 72 (500-1m) of pepe.wiff (Turbo Spray), Smoothed, Smoothed, Smoothed

Max. 4.6e6 cps.

4.89

4.6e6 4.4e6

(a)

AITC fragment in 991.4

4.2e6 4.0e6 3.8e6 3.6e6 3.4e6 3.2e6

Intensity, cps

3.0e6 2.8e6 2.6e6 2.4e6 2.2e6 2.0e6 1.8e6 1.6e6 1.4e6 1.2e6 1.0e6

3.73

8.0e5

6.03 6.28

6.0e5 4.0e5

7.94 9.78 11.00 11.53 12.05

1.57 2.37

2.0e5

14.73 15.06 15.41

17.39

19.56

22.41 23.48

20.35

23.85

25.28

26.75

29.68

0.0 2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

Time, min +EMS: 4.549 to 4.977 min from Sample 72 (500-1m) of pepe.wiff (Turbo Spray) 2.3e5

Max. 2.3e5 cps.

(b)

58.0

2.2e5

N=C=S

2.1e5 2.0e5 1.9e5 1.8e5 1.7e5 1.6e5

Intensity, cps

1.5e5 1.4e5 1.3e5 1.2e5 1.1e5 1.0e5 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4

AITC

4.0e4 3.0e4

59.0

2.0e4 1.0e4

91.0 77.0

103.0

0.0 50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

m/z, Da Fig. 8. (a) EPI chromatogram of the ion corresponding to m/z 991.4 and (b) EPI spectrum of the fragment 991.4 using the scan range m/z 50–1200.

G. Meca et al. / Food and Chemical Toxicology 50 (2012) 1755–1762

confirmed the existence of BEA moiety in the molecule, as they are apparent breakdown products of BEA evidenced by loss of hydroxyisovaleryl (HyLv) and N-methyl phenylalanine (N-MePhe), two principal components forming the molecule of BEA. The fragment of m/z 483.5 was due to the loss of one HyLv and one N-Me-Phe from the fragment m/z 784.6, while the fragment of m/z 520.6 was from the loss of one HyLv and two phenylalanine (Phe) moieties-Me-Phe from the fragment m/z 822 (Fig. 6). The ion at m/z 1039.7 was considered to be the molecular ion adduct with one sodium and two H2O molecules [M + 2H2O + Na]++ (Fig. 6). The molecular ion of Peak 1 [M]+ is therefore m/z 982, an adduct of one molecule of BEA (MW = 784) with two molecules of AITC (992 = 198). The MS2 (EPI mode) of Peak 1 indicated the presence of AITC in the molecule (Fig. 8b). Based on the above described fragmentation pattern, and the characteristic features of the BEA and AITC molecules, Peak 1 is therefore tentatively identified as an adduct of BEA with two AITC molecules. Its absolute molecular structure still needs to be confirmed with other analytical methods including NMR and IR. Similarly, the mass spectrum of Peak 2 showed an molecular adduct ion of m/z 991.4 which was indicative of a new compound consisting of one BEA and one AITC group (Fig. 7). The fragment ion with m/z 496.2 is considered to be from BEA with the loss of two phenylalanine (Phe) units and one hydroxyvaleric acid (HyLv). The MS2 spectrum of m/z 991.4 obtained in the EPI mode clearly showed a peak with a diagnostic fragment ion at m/z 58, which is considered to be from the N@C@S moiety of AITC (Fig. 8). Peak 2 was therefore tentatively identified as an adduct of BEA with one AITC. BEA is a mycotoxin of relatively minor importance in terms of its occurrence and toxicity; therefore methods for detoxifying or reducing BEA have not been developed. The present study presents a novel approach in reducing BEA contamination in foods using a naturally occurring food component AITC. In fact, to be best of our knowledge, it may be the first report for any mycotoxins. Our results suggest strongly that AITC reduces BEA by forming adduct products in a form BEA:nAITC (n = 1 for Peak 1; n = 2 for Peak 2). Although the exact structural features and the toxicity of these newly formed adducts are not know, this finding signifies as the first step toward a detoxification process for BEA. On the other hand, although the mechanism of the formation of the adducts or conjugates (Peaks 1 and 2) is not clear, the highly electrophilic central carbon atom (N@C@S) in the isothiocyanates such as AITC makes these compounds highly reactive even under mild conditions (Verma, 2003). Isothiocyanates can easily go through nucleophilic addition reactions with hydroxyls, amines, and thiols, generating products such as carbamates, thiourea, and thiocarbamates, respectively (Zhang and Talalay, 1994). AITC has been found to react with glutathione, amino acids, proteins, water, alcohol, and sulfites (Kawakishi and Namiki, 1969; Cejpek et al., 2000; Luciano et al., 2008) and was able to disintegrate the cystine disulfide bond through an oxidative process (Kawakishi and Namiki 1982). BEA contains some nucleophile groups in its structure that probably reacted with N@C@S in AITC forming the two newly discovered adducts in ambient conditions. This discovery may lead to the utilization of AITC or AITC-containing products such as mustard meal as an agent to reduce or detoxify BEA, and perhaps other mycotoxins in contaminated foods and/or animal feeds.

4. Conclusion Degradation of Fusarium mycotoxins has been extensively researched, and heat treatment and biotransfomation have shown promising results. BEA is a minor Fusarium toxin and, although it is commonly present in foods, it has yet to be legislated. Some

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studies have shown BEA cytotoxicity, but no detoxification method has been reported. The present study showed the capacity of the naturally occurring allyl isothiocyanate to react with BEA and form two conjugates. This is the first report evidencing the reaction of an isothiocyanate and a mycotoxin. Further investigation may focus on the toxicity of the products generated by the reaction of BEA and AITC. The characterization of the toxicological parameters of these newly discovered substances is of paramount importance to evaluate the possible utilization of AITC fumigation to control the levels of BEA in cereals. Moreover, the reaction of AITC with other mycotoxins should also be examined. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements This research was supported by the Ministry of Science and Innovation (AGL2010-17024), the Developing Innovative Agricultural Products Program, Agriculture & Agri-Food Canada (Project ID: AGR-0479) and by the pre-PhD program of University of Valencia ‘‘Cinc Segles’’. References Ambrosone, C.B., McCann, S.E., Freudenheim, J.L., Marshall, J.R., Zhang, Y., Shields, P.G., 2004. Breast cancer risk in premenopausal women is inversely associated with consumption of broccoli, a source of isothiocyanates, but is not modified by GST genotype. J. Nutr. 134, 1134–1138. Calo, L., Fornelli, F., Nenna, S., Tursi, A., Caiaffa, M.F., Macchia, L., 2003. Beauvericin cytotoxicity to the invertebrate cell line SF-9. J. Appl. Genet. 44, 515–520. Cejpek, K., Valusek, J., Velisek, J., 2000. Reactions of allyl isothiocyanate with alanine, glycine, and several peptides in model systems. J. Agric. Food Chem. 48, 3560–3565. Engel, E., Batty, C., Le Corre, D., Souchon, I., Martin, N., 2002. Flavor-active compounds potentially implicated in cooked cauliflower acceptance. J. Agric. Food Chem. 50, 6459–6467. Fahey, J.W., Talalay, P., 1999. Antioxidant functions of sulforaphane: a potent inducer of phase II detoxication enzymes. Food Chem. Toxicol. 37, 973–979. Fornelli, F., Minervini, F., Logrieco, A., 2004. Cytotoxicity of fungal metabolites to lepidopteran (Spodoptera frugiperda) cell line (SF 9). J. inv pathol. 85, 74–79. Fowke, J.H., Chung, F.L., Jin, F., Qi, D., Cai, Q., Conaway, C., 2003. Urinary isothiocyanate levels, brassica, and human breast cancer. Cancer Res. 63, 3980–3986. Hamill, R.L., Higgens, C.E., Boaz, H.E., Gorman, M., 1969. The structure of beauvericin, a new depsipeptide antibiotic toxic to Artemia salina. Tetrahedron Let. 49, 4255–4258. Holst, B., Williamson, G., 2004. A critical review of the bioavailability of glucosinolates and related compounds. Nat. Prod. Rep. 21, 425–447. Isshiki, K., Tokuoka, K., Mori, R., Chiba, S., 1992. Preliminary examination of allyl isothiocyanate vapor for food preservation. Biosci. Biotechnol. Biochem. 56, 1476–1477. Jestoi, M., 2008. Emerging Fusarium-mycotoxins fusaproliferin, beauvericin, Enniatins, and moniliformin-a review. Crit. Rev. Food Sci. Nutr. 48, 21–49. Kawakishi, S., Namiki, M., 1969. Decomposition of allyl isothiocyanate in aqueous solution. Agric. Biol. Chem. 33, 452–459. Kawakishi, S., Namiki, M., 1982. Oxidative cleavage of the disulfide bond of cystine by allyl isothiocyanate. J. Agric. Food Chem. 30, 618–620. Luciano, F.B., Hosseinian, F.S., Beta, T., Holley, R.A., 2008. Effect of free-SH containing compounds on allyl isothiocyanate antimicrobial activity against Escherichia coli O157:H7. J. Food Sci. 73, 214–220. Luciano, F.B., Holley, R.A., 2009. Enzymatic inhibition by allyl isothiocyanate and factors affecting its antimicrobial action against Escherichia coli O157:H7. Int. J. Food Microbiol. 131, 240–245. Macchia, L., DiPaola, R., Fornelli, F., Nenna, S., Moretti, A., Napoletano, R., Logrieco, A., Caiaffa, M. F., Tursi, A., and Bottalico, A. 1995. Cytotoxicity of beauvericin to mammalian cells. Book of Abstracts for International Seminar on Fusarium:mycotoxins, taxonomy and pathogenicity, May 9–13, 1995, Martina Franca, Italy. Meca, G., Zinedine, A., Blesa, J., Font, G., Mañes, J., 2010a. Further data on the presence of Fusarium emerging mycotoxins enniatins, fusaproliferin and beauvericin in cereals available on the Spanish markets. Food Chem. Toxicol. 48, 1412–1416. Meca, G., Fernandez-Franzon, M., Ritieni, A., Font, G., Ruiz, M.J., Mañes, J., 2010b. Formation of fumonisin B1-glucose reaction product, in vitro cytotoxicity, and lipid peroxidation on kidney cells. J. Agric. Food Chem. 58, 1359–1365.

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Meca, G., Soriano, J.M., Font, G., Mañes, J., Ruiz, M.J., 2011. Production, purification, and mass spectrometry characterization of the cyclohexadepsipeptide enniatin J3 and study of the cytoxicity on differentiated and undifferentiated Caco-2 cells. Toxicol. Environ. Chem. 93, 383–395. Munkvold, G., Stahr, H.M., Logrieco, A., Moretti, A., Ritieni, A., 1998. Occurrence of fusaproliferin and beauvericin in Fusarium-contaminated livestock feed in Iowa. Appl. Environ. Microbiol. 64, 3923–3926. Nash, P.B., Purner, M.B., Leon, R.P., Clarke, P., Duke, R.C., Curiel, T.J., 1998. Toxoplasma gondii-infected cells are resistant to multiple inducers of apoptosis. J. Immunol. 160, 1824–1830. Nilanonta, C., Isaka, M., Kittakoop, P., Trakulnaleamsai, S., 2002. Precursor-directed biosynthesis of nbeauvericin analogs by the insect pathogenic fungus Paecilomyces tenuipes BCC 1614. Tetrahedron 58, 3355–3360. Nugon-Baudon, L., Rabot, S., 1994. Glucosinolates and glucosinolate derivatives: implications for protection against chemical carcinogenesis. Nutr. Res. Rev. 7, 205–231. Ritieni, A., Moretti, A., Logrieco, A., Bottalico, A., Randazzo, G., Monti, S.M., Ferracane, R., Fogliano, V., 1997. Occurrence of fusaproliferin, fumonisin B1, and beauvericin in maize from Italy. J. Agric. Food Chem. 45, 4011–4016. Ruiz, M.J., Franzova, P., Juan-Garcia, A., Font, G., 2011. Toxicological interactions between the mycotoxin beauvericin, deoxinivalenol and T-2 toxin in CHO-K1 cells in vitro. Toxicon 58, 315–326. Shephard, G.S., Sewram, V., Nieuwoudt, T.W., Marasas, W.F.O., Ritieni, A., 1999. Production of the mycotoxins fusaproliferin and beauvericin by South African isolates in the Fusarium section Liseola. J. Agric. Food Chem. 47, 5111–5115.

Tang, L., Zirpoli, G.R., Guru, K., Moysich, K.B., Zhang, Y., Ambrosone, C.B., 2008. Consumption of raw cruciferous vegetables is inversely associated with bladder cancer risk. Cancer Epidemiol. Biomark. Prev. 17, 938–944. Tomoda, H., Huang, X.H., Cao, J., Nishida, H., Nagao, R., Okuda, S., Tanaka, H., Omura, S., Arai, H., Inoue, K., 1992. Inhibition of acyl-CoA: cholesterol acyltransferase activity by cyclodepsipeptide antibiotics. J. Antibiot. 45, 1626–1632. Traka, M., Gasper, A.V., Melchini, A., Bacon, J.R., Needs, P.W., Frost, V., 2008. Broccoli consumption interacts with GSTM1 to perturb oncogenic signaling pathways in the prostate. PLoS ONE 3, 2568–2582. Van Poppel, G., Verhoeven, D.T., Verhagen, H., Goldbohm, R.A., 1999. Brassica vegetables and cancer prevention. Epidemiology and mechanisms. Adv. Exp. Med. Biol. 472, 159–168. Verhoeven, D.T., Goldbohm, R.A., van Poppel, G., Verhagen, H., van den Brandt, P.A., 1996. Epidemiological studies on brassica vegetables and cancer risk. Cancer Epidemiol. Biomark. Prev. 5, 733–748. Verma, R.P., 2003. Synthesis and reactions of 3-oxobutyl isothiocyanate (OB ITC). Eur. J. Org. Chem. 3, 415–420. Zhang, Y., Talalay, P., 1994. Anticarcinogenic activities of organic isothiocyanates: chemistry and mechanisms. Cancer Res. 54, 1976–1981. Zhang, Y., 2004. Cancer-preventive isothiocyanates: measurement of human exposure and mechanism of action. Mutat. Res. 555, 173–190. Zinedine, A., Meca, G., Mañes, G., Font, G., 2011. Further data on the occurrence of Fusarium emerging mycotoxins enniatins (A, A1, B, B1), fusaproliferin and beauvericin in raw cereals commercialized in Morocco. Food Contr. 22, 1–5.