Natural occurrence of zearalenone in Tunisian wheat grains

Food Control 25 (2012) 773e777

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Natural occurrence of zearalenone in Tunisian wheat grains Chiraz Zaied, Nidhal Zouaoui, Hassan Bacha*, Salwa Abid Laboratory for Research on Biologically Compatible Compounds (LRSBC), Faculty of Dentistry, Rue Avicenne, 5019 Monastir, Tunisia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 September 2011 Received in revised form 3 December 2011 Accepted 10 December 2011

Fusarium mycotoxins are worldwide occurring in cereals and they are frequently reported in fresh or stored grains. Cereals represent a staple food for the Tunisian population; it therefore has a high social, economic and nutritional relevance. Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by a variety of Fusarium fungi in temperate and warm countries. Fungi-producing ZEN contaminates corn, barley, wheat, sorghum and rice. A total of 205 samples of wheat were collected during the harvest year of 2010 from the major cropping areas in Tunisia and they were analyzed for zearalenone contamination. The aim of this study was to investigate for the first time the presence of ZEN in widelyconsumed cereals in Tunisia, especially durum and tender wheat, to compare the levels of contamination by ZEN with the European norms and to suggest some factors that can promote the production of ZEN in Tunisia. To perform this study, we developed and validated in our laboratory conditions an HPLC method for quantitative analysis of ZEN in solid cereal samples. Our results showed that the incidence of ZEN contamination was 75%. The levels of contamination determined in the positive samples ranged between 3 and 560 mg/kg with a mean value of 60 mg/kg. These important amounts of ZEN in wheat can be attributed to the Tunisian climate, warm temperature and prolonged wetness witch are favor to Fusarium growth and mycotoxin production during the cultivation and the final ripening period of wheat grains. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Zearalenone Prevalence Wheat Tunisia

1. Introduction Fusarium mycotoxins are worldwide occurring in small-grain cereals and they were frequently reported in fresh or stored grains (Bottalico, 1998). The main groups of Fusarium toxins commonly-found in grains are trichothecenes (including deoxynivalenol, nivalenol, T-2 and HT-2 toxins), zearalenone and fumonisins (B1, B2) (Logrieco, Mule, Moretti, & Bottalico, 2002). Fusarium toxins pose safety concerns for grains intended for direct consumption due to their harmful impact on the human health. Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced as a secondary metabolite of various Fusarium fungi including Fusarium graminearum and Fusarium culmorum (Bennett & Klich, 2003). ZEN is known for its numerous adverse effects on both males and females of different animal species (Ryu, Jackson, & Bullerman, 2002). In fact, ZEN is known to cause estrogenic effects, including infertility, reduced serum testosterone levels and sperm counts, reduced incidence of pregnancy, and a change in the progesterone levels (Shier, Sier, Xie, & Mirocha, 2001). ZEN was placed in group 3 according to the International Agency for Research on Cancer (IARC, 1993). Moreover, ZEN was found * Corresponding author. Tel./fax: þ216 73 42 55 50. E-mail address: [email protected] (H. Bacha). 0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2011.12.012

hepatotoxic; it induces adverse liver lesions (Conkova, Laciakova, Pastorova, Seidel, & Kovac, 2001; Maaroufi, Chekir, Creppy, Ellouz, & Bacha, 1996; Obremski et al., 1999). It is equally haematotoxic and it causes several alterations of immunological parameters (Abbes et al., 2006, 2007; Maaroufi et al., 1996; Murata, Sultana, Shimada, & Yashioka, 2003). Several studies have shown that ZEN is cytotoxic through the inhibition of cell viability and macromolecules synthesis and it induces the stress response and apoptosis in different cultured cell lines (Abid-Essefi et al., 2003, 2004; Boussema-Ayed et al., 2008; Hassen et al., 2005). A further set of experiments have demonstrated that ZEN exhibits several genotoxic effects such as the induction of micronuclei and chromosome aberrations, DNA strand breaks and DNA adducts formation (Abbes et al., 2006, 2007; Abid-Essefi et al., 2004; Boussema-Ayed, Ouanes, Bacha, & Abid, 2007; Chekir-Ghedira et al., 1998; Hassen, AyedBoussema, Azqueta, De Cerain Lopez, & Bacha, 2007; Ouanes et al., 2003; Ouanes, Ayed-Boussema, Baati, Creppy, & Bacha, 2005). Cereal crops such as barley, wheat, rice, sorghum and maize are susceptible to be contaminated by ZEN under prolonged cool and wet weather conditions in temperate and warm regions (Manova & Mladenova, 2009). Indeed, the occurrence of this toxin is influenced by environmental factors such as temperature, humidity and degree of rainfall during the pre-harvesting, harvesting and postharvesting periods (Torrado et al., 2010).

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In Tunisia, the agricultural system is much diversified. It includes mainly cereals which are frequently infected by mycotoxin-producing fungi (Kammoun, Gargouri, Barreau, Richard-Forget, & Hajlaoui, 2010). Weather conditions are favorable to Fusarium infection and ZEN production in grain. Wheat is the most important cereal cultivated in Tunisia covering about 700.000 ha per year, mainly located in the Northern part of the country (Bensassi, Zaied, Abid, Hajlaoui, & Bacha, 2010). Although consumption of wheat is usually increasing in Tunisia, up to now, few data are available concerning the occurrence of ZEN in wheat (Ghali, Hmaissia-khlifa, Ghorbel, Maaroufi, & Hedili, 2008). The Tunisian population consumes great amounts of cereals and cereal-based food. Indeed, large amounts of cereals commercialized in Tunisia are imported and little is known about eventual mycotoxin contamination (Zaied et al., 2009). Consequently, in Tunisia, there are no applicable norms concerning cereal contamination, particularly by ZEN, neither for local nor for imported cereals. Devegowda, Raju, and Swang (1998) reported that approximately 25% of cereals consumed in the world are contaminated by mycotoxins. In general, the extent of contamination is expected to be higher especially when climatic conditions are favorable to mycotoxin contamination. The European Commission specified the maximum levels of ZEN in different commodities. A maximum level of 100 mg/kg was fixed in unprocessed cereals other than corn and 350 mg/kg in unprocessed corn (European Commission, 2007). The aim of this study was to determine ZEN levels in the fresh harvested wheat in the principal cropping areas in Tunisia, to compare the levels of contamination by ZEN to the European norms, and to check off the factors affecting ZEN production in Tunisia. To perform this study, we validated an HPLC method for quantitative analysis of ZEN in solid cereal samples, involving ZEN extraction from the sample followed by clean-up step using an immunoaffinity column. 2. Materials and methods 2.1. Reagents ZEN standard was provided by Sigma Chemicals (St. Louis, MO, USA). The ZEN crystalline solution was reconstituted in methanol at a concentration of 5 mg/ml. The stock solution was stored at 20  C until use. The working standard solutions, ranging from 0.5 to 400 ng/ml, were prepared from suitable dilutions of the stock solution in the mobile phase acetonitrile: water: methanol (48:50:3, v/v/v) and stored at þ4  C. The used solvents such as water, methanol and acetonitrile were obtained from Fisher Scientific (Fisher chemicals HPLC, France) with HPLC grade. For the clean-up step, ZearaStar LC immunoaffinity columns (IAC) were purchased from Vitalab (ROMER Labs). 2.2. Standard solutions A stock solution of ZEN was prepared by dissolving 1 mg of ZEN in 1 ml of methanol. The ZEN concentration was checked using its specific extinction coefficient (3 ¼ 13,900 at 274 nm). The standard curve solutions were prepared from appropriate dilutions of the stock solution with methanol (400, 300, 200, 100 and 50 ng/ml). All the solutions were stored at þ4  C.

were collected from different areas in the North of Tunisia including Jendouba (50), Beja (65), El Kef (25), Siliana (51) and Bizerte (14). These regions are the most productive of cereals in Tunisia. These harvest samples were taken immediately after the harvest with a minimum of 500 g of each sample. The samples were packed in plastic bags and stored at þ4  C until their analysis.

2.4. Instruments The determination of ZEN was carried out using the HPLC Agilent 1100 system equipped with a quaternary isocratic pump (Quart-Pump G1311A), auto-injector (Injection Valve Assembly G1313A) and a fluorometric detector (Varian Prostar) (l excitation ¼ 274 nm, l emission ¼ 450 nm). The separation was carried out on a C18 reversed-phase column (250  4 mm, 5 mm) (Spherisorb ODII, Leonberg). Data were exploited using Chemstation 3D software (Agilent Technologies ChemStation Family Software products) coupled with the HPLC system.

2.5. Extraction procedure ZEN analyses were performed by HPLC-FLD according to the procedure described by Manova and Mladenova (2009) with modifications. Twenty-five grams (25 g) of each ground sample were extracted with 100 ml acetonitrile:water (90:10, v/v) by high speed blending for 30 min. After shaking, the samples were centrifuged for 15 min at 4200 rpm. Twenty milliliters (20 ml) of filtrate was collected and mixed with 20 ml of distilled water. The diluted extract was purified with immunoaffinity column prior to HPLC analysis.

2.6. Clean-up with immunoaffinity column The diluted extract was cleaned-up through the immunoaffinity column (IAC ZearaStarÒ, Romer Labs, USA) at a flow rate of 1e2 drops/s. The column was washed twice with 5 ml of distilled water and ZEN was then slowly eluted from the column with 3 ml of methanol HPLC grade. The eluted sample was evaporated to dryness at 70  C and the dry residue was reconstituted in 250 ml of mobile phase and stored at þ4  C till HPLC analysis.

2.7. HPLC quantification HPLC analyses of ZEN were performed with an 1100 series HPLC system from Agilent Technologies equipped with a fluorescence detector (l excitation ¼ 274 nm, l emission ¼ 450 nm). The mobile phase consisted of a mixture of acetonitrile:water:methanol (48:50:3, v/v/v). A 50 ml of the reconstituted extract was injected onto the column at a flow rate of 1.0 ml/min. The quantification of ZEN was performed by the measurement of the peak area at ZEN retention time and the comparison with the relevant calibration curve (400, 300, 200, 100 and 50 ng/ml, r ¼ 0.9983).

2.8. Statistical analysis 2.3. Sample collection Two hundred and five (205) samples of wheat grains collected during the crop year 2010 were analyzed for ZEN. One hundred fifty-five (155) durum wheat and fifty (50) tender wheat samples

The statistical analysis was performed using the SPSS software program (SPSS Institute, 2007, version 17.0) and the differences in ZEN levels between regions were analyzed by ANOVA test. p < 0.05 was considered to be statistically significant.

C. Zaied et al. / Food Control 25 (2012) 773e777 Table 1 Average recoveries for ZEN in wheat. Concentrations of ZEN (ng/ml)

400 ng/ml

200 ng/ml

100 ng/ml

Recoveries  RSDa (%)

80  7

75  6

74  3

Average recovery ± RSDa (%)

76 ± 3

a

3. Results and discussion 3.1. Method validation

3.2. Occurrence of ZEN in durum wheat The results of ZEN occurrence in these samples are presented in Table 2. As shown in this table, the levels of contamination ranged between 0 and 560 mg/kg with a median (35 mg/kg). The average of the total samples is 58 mg/kg and the average of the positives samples is 110 mg/kg. According to the obtained results, Bizerte presented the highest average of contamination by ZEN which is 176 mg/kg compared to the other regions. Siliana, Jendouba, El Kef and Beja have nearly the same level of contamination by ZEN, 56, 61, 31 and 36 mg/kg, respectively. Twenty three percent (23%) of the total samples of wheat collected in 2010 exceeded the tolerable limit (100 mg/kg) as recommended by the European Union (European Commission, 2007). The statistical analysis showed

ADC1 A, ADC1 CHANNEL A (ZENPOS.D) 4.268 - ZEN

mAU 47.2

a b

Range of ZEN (mg/kg)

Average of total samples (mg/kg)a

Average of contamination (mg/kg)b

Median

37/50 32/37 34/40 14/14 6/14

0e560 0e148 0e204 16e525 0e120

61 36 56 176 31

68 50 70 176 58

35 26 35 64 84

123/155

0e560

58

110

35

Contaminated over total

Average contamination of total samples. Average contamination of positive samples.

a significant difference in ZEN levels between these regions (ANOVA test, p < 0.05). 3.3. Occurrence of ZEN in tender wheat Fifty (50) samples of tender wheat were collected from fields located in Beja, El Kef and Siliana during the harvest of 2010. The results of ZEN occurrence in tender wheat samples are presented in Table 2. The statistical study showed a significant difference in ZEN levels between the wheat samples analyzed (ANOVA test, p < 0.05). As shown in Table 3, ZEN was found in 32 out of the 50 analyzed samples of tender wheat. The levels of contamination in the total samples ranged from 0 to 260 mg/kg; the average of ZEN in the positive samples was 50 mg/kg with a median (26 mg/kg). The samples taken from El Kef contained the highest level of ZEN contamination with an average of 148 mg/kg compared to the other regions. Beja and Siliana have nearly the same average of contamination by ZEN in the total samples: 30 and 22 mg/kg, respectively. Eighteen percent (18%) of the positive samples of tender wheat exceed the tolerable levels (100 mg/kg). Cereals, especially wheat, represent a staple food for the Tunisian population; it therefore has a high social, economic and nutritional relevance. According to the obtained results, positive samples of wheat were contaminated with ZEN at levels which are 2e5 times higher than the maximum limit of 100 mg/kg established in Europe (Commission Regulation, 2007). These levels are lower than those obtained in previous studies in the temperate regions in the world. Indeed, Placinta, D’Mello, and Macdonald (1999) reported the contamination of cereals samples, especially in wheat from many European countries. In Germany, ZEN was found at high levels in wheat samples, up to 8000 mg/kg (Muller, Reimann, Schumacher, & Schwadorf, 1997; Schollenberger et al., 2006). In Poland, the contamination of wheat by ZEN was confirmed by Perkowski, Plattner, Golinski, and Vesonder (1990) with levels up to 2000 mg/kg. In France, a high incidence of ZEN was found in wheat samples (16% of the samples with a maximum level of 1820 mg/kg) (SCOOP, 2003). Most African countries present a climate characterized by high humidity and high temperature which favor growth of molds. In fact, in Morocco, 15% of Table 3 Levels of ZEN contamination of the tender wheat grains, collected in 2010.

1.272

46.8

Regions

Total

In order to carry out a study on ZEN contamination level in wheat, we successfully validated in our laboratory conditions a chromatographic analytical method by the adaptation of the method given by Manova and Mladenova (2009) including a purification step using immunoaffinity columns. The validation of this method was based on the criteria of linearity, selectivity, reproductibility, repeatability, detection limit, quantification limit and recovery. Under the chromatographic conditions described above, the retention time of ZEN was found about 4.3  0.5 min. Linearity was confirmed using the calibration curve for each ZEN concentration. It was linear in the range of 50e400 ng/ml (50, 100, 200, 300 and 400 ng/ml) with a coefficient of correlation r ¼ 0.9983, indicating a good calibration curve. The limit of detection (LOD) (signal-to-noise ratio ¼ 3) was calculated to be 0.15 mg/kg and the limit of quantification (LOQ) (signal-to-noise ratio ¼ 10) was 0.4 mg/kg of ZEN in wheat. Recovery experiments were determined by spiking ZEN-free samples of wheat with ZEN at concentrations of 400, 200 and 100 ng/ml in the same day, by the same operator and with the same HPLC system. The recoveries were 80  7, 75  6 and 74  3%, respectively, for 400, 200 and 100 ng/ml (Table 1). The average recovery of the extraction method was 76  3%. A chromatogram for a positive sample of wheat is given in Fig. 1.

47

Table 2 Levels of ZEN contamination of the durum wheat grains, collected in 2010, from the major cropping areas in Tunisia.

Jendouba Beja Siliana Bizerte EL Kef

RSD: relative standard deviation.

46.6

Regions

Total of samples

Range of ZEN (mg/kg)

Average of total samples (mg/kg)a

Average of contamination (mg/kg)b

Median

Beja El Kef Siliana

14/28 11/11 7/11

0e140 52e260 0e90

30 148 22

36 148 27

21 135 17

Total

32/50

0e260

49

50

26

46.4 1.972

46.2 46 45.8 1

2

3

4

775

5

6

7

8

9

min

a

Fig. 1. HPLC chromatogram of one positive wheat sample (as an example).

b

Average contamination of total samples. Average contamination of positive samples.

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C. Zaied et al. / Food Control 25 (2012) 773e777

cereals samples were contaminated by ZEN with an average of 14 mg/ kg and ranged from 11 to 16.5 mg/kg (Zinedine et al., 2006; Zinedine, Miguel, Carlos, & Jordi, 2007). In Egypt, several cereal samples were reported to contain ZEN, especially wheat, corn and rice with the levels up to 45 mg/kg (Abd Alla, 1997). The observed high incidence and levels of ZEN contamination of wheat in Tunisia can be explained; firstly by the fact that as a Mediterranean country, the Tunisian climate favor fungal growth and mycotoxins accumulation. Warm temperature and prolonged wetness are the consistent weather factors increasing Fusarium growth and mycotoxins production during the cultivation and the final ripening period of wheat grains (Beyer, Klix, & Verreet, 2007; Kammoun et al., 2010). Secondly, several farming practices such as the use of sensitive varieties of wheat, no crop rotation; no-till or reduced tillage enhance fungal attacks and therefore increase mycotoxin accumulation in grains (Edwards, 2004). Unfortunately, Tunisian farmers do not practice a suitable crop rotation to prevent the accumulation of Fusarium in the fields. Finally, the social and economic characteristics of the Tunisian population such as the cooking methods, home-made food storage and the eating habits can probably increase the exposure to ZEN (Bensassi et al., 2010). 4. Conclusion Mycotoxins and related pathologies have become a worldwide preoccupation and they raise serious economic and sanitary problems (FAO, 1997). These are also of some concern in Tunisia because of climate and geographic situation in addition to the social and economic conditions. In our previous survey on Tunisia, we have shown that cereals were contaminated by ZEN and other mycotoxins such as aflatoxins, ochratoxin A, trichothecens and citrinin (Bacha, Hadidane, Regnault, Ellouz, & Dirheimer, 1986; Bacha et al., 1988; Hadidane et al., 1985). Our present study presents the first report evaluating the situation of wheat-contamination by ZEN in Tunisia. Our findings suggest that ZEN found at such high levels in wheat may cause serious health problems for both humans and animals. These levels exceed the ZEN limit fixed by the European Commission Regulation. The limit intended for human consumption is 100 mg/kg (European Commission, 2007). Furthermore, these findings revealed that wheat produced under the Tunisian agro-climatic conditions is contaminated by ZEN. This situation should spur the Tunisian authorities to set regulatory limits for ZEN and other Fusarium toxins in cereals, foods and feeds to ensure food safety. A similar study is in preparation to evaluate the levels of other mycotoxins that contaminate cereals in addition to ZEN. Acknowledgments This research was supported by «Le Ministère Tunisien de l’Enseignement Supérieur, de la Recherche Scientifique (Laboratoire de Recherche sur les Substances Biologiquement Compatibles: LRSBC)». References Abbes, S., Ben Salah-Abbes, J., Ouanes, Z., Houas, Z., Othman, O., Bacha, H., et al. (2006). Preventive role of phyllosilicate clay on the immunological and biochemical toxicity of zearalenone in Balb/c mice. International Immunopharmacology, 6, 1251e1258. Abbes, S., Ouanes, Z., Ben Salah-Abbes, J., Abdel-Wahhab, M., Oueslati, R., & Bacha, H. (2007). Preventive role of aluminosilicate clay against induction of micronuclei and chromosome aberrations in bone-marrow cells of Balb/c mice treated with zearalenone. Mutation Research, 631, 85e92. Abd Alla, E. S. (1997). Zearalenone: toxigenic fungi and chemical decontamination in Egyptian cereals. Nahrung, 41, 362e365.

Abid-Essefi, S., Baudrimont, I., Hassen, W., Ouanes, Z., Mobio, T. A., Anane, R., et al. (2003). DNA fragmentation, apoptosis and cell cycle arrest induced by zearalenone in cultured DOK, Vero and Caco-2 cells: prevention by vitamin E. Toxicology, 192, 237e248. Abid-Essefi, S., Ouanes, Z., Hassen, W., Baudrimont, I., Creppy, E. E., & Bacha, H. (2004). Cytotoxicity, inhibition of DNA and protein syntheses and oxidative damage in cultured cells exposed to zearalenone. Toxicology in Vitro, 18, 467e474. Bacha, H., Hadidane, R., Regnault, C., Ellouz, F., Creppy, E. E., & Dirheimer, G. (1988). Monitoring and identification of fungal toxins in food products, animals feed and cereals in Tunisia. Journal Stored Production Research, 4, 199e206. Bacha, H., Hadidane, R., Regnault, C., Ellouz, F., & Dirheimer, G. (1986). Mycotoxines et Mycotoxicoses en Tunisie. Cahiers Médicaux de Tunisie-Nutrition et santé, 49, 34e35. Bennett, J. W., & Klich, M. (2003). Mycotoxins. Clinical Microbiology Reviews, 16(3), 497e516. Bensassi, F., Zaied, C., Abid, A., Hajlaoui, M., & Bacha, H. (2010). Occurrence of deoxynivalenol in durum wheat in Tunisia. Food Control, 21, 281e285. Beyer, M., Klix, M. B., & Verreet, J. A. (2007). Estimating mycotoxin contents of Fusarium-damaged winter wheat kernels. International Journal of Food Microbiology, 119, 153e158. Bottalico, A. (1998). Fusarium disease of cereals: species complex and related mycotoxin profiles, in Europe. Food Science and Technology Research, 80, 85e103. Boussema-Ayed, I., Bouaziz, C., Rjiba, K., Valenti, K., Laporte, F., Bacha, H., et al. (2008). The mycotoxin zearalenone induces apoptosis in human hepatocytes (HepG2) via p53-dependent mitochondrial signaling pathway. Toxicology in Vitro, 22, 1671e1680. Boussema-Ayed, I., Ouanes, Z., Bacha, H., & Abid, S. (2007). Toxicities induced in cultured cells exposed to zearalenone: apoptosis or mutagenesis? Journal of Biochemical Molecular Toxicology, 21, 136e144. Chekir-Ghedira, L., Maaroufi, K., Zakhama, A., Ellouz, F., Dhouib, S., Creppy, E. E., et al. (1998). Induction of a SOS repairs system in lysogenic bacteria by zearalenone and its prevention by vitamin E. Chemical and Biology Interact, 113, 15e25. Conkova, E., Laciakova, A., Pastorova, B., Seidel, H., & Kovac, G. (2001). The effect of zearalenone on some enzymatic parameters in rabbits. Toxicology Letters, 121, 145e149. Devegowda, G., Raju, M., & Swang, H. (1998). Mycotoxins: novel solutions for their counteraction. Feedstuffs, 70, 12e15. Edwards, S. G. (2004). Influence of agricultural practices on Fusarium infection of cereals and subsequent contamination of grain by trichothecene mycotoxins. Toxicology Letters, 153, 29e35. European Commission (EC) 1126/2007 of 28 September 2007 setting maximum levels for certain contaminants in foodstuffs as regards Fusarium toxins in maize and maize products. (2007). Official Journal of EU, L 255, 14e17. FAO. (1997). Sampling plan for aflatoxins analysis in peanuts and corn (p. 64). FAO food nutrition paper. Rome, Italy: FAO. Ghali, R., Hmaissia-khlifa, K., Ghorbel, H., Maaroufi, K., & Hedili, A. (2008). Incidence of aflatoxins, ochratoxin A and zearalenone in tunisian foods. Food Control, 19, 921e924. Hadidane, R., Roger-Regnault, C., Ellouz, F., Bacha, H., Creppy, E. E., & Dirheimer, G. (1985). Correlation between alimentary mycotoxin contamination and specific diseases. Human Toxicology, 4, 491e501. Hassen, W., Ayed-Boussema, I., Azqueta, A., De Cerain Lopez, O. A., & Bacha, H. (2007). The role of oxidative stress in zearalenone-mediated toxicity in HepG2 cells: oxidative DNA damage, gluthatione depletion and stress proteins induction. Toxicology, 232, 294e302. Hassen, W., El Golli, E., Baudrimont, I., Ladjimi, M., Creppy, E., & Bacha, H. (2005). Cytotoxicity, inhibition and Hsp 70 induction in HepG2 cells in response to zearalenone and cytoprotection by sub-lethal heat shock. Toxicology, 25(2), 203e209. IARC. (1993). IARC monographs on the evaluation of the carcinogenic risk of chemicals to humans, some naturally occurring substances: Heterocyclic aromatic amines and mycotoxins. (pp. 397e444). Lyon, 56. Kammoun, L., Gargouri, S., Barreau, C., Richard-Forget, F., & Hajlaoui, M. (2010). Trichothecene chemotypes of Fusarium culmorum infecting wheat in Tunisia. International Journal of Food Microbiology, 140, 84e89. Logrieco, A., Mule, G., Moretti, A., & Bottalico, A. (2002). Toxigenic Fusarium species and mycotoxins associated with maize ear rot in Europe. European Journal of Plant Pathology, 108, 597e609. Maaroufi, K., Chekir, L., Creppy, E. E., Ellouz, F., & Bacha, H. (1996). Zearalenone induces modifications of haematological and biochemical parameters in rats. Toxicon, 34, 535e540. Manova, R., & Mladenova, R. (2009). Incidence of zearalenone and fumonisins in Bulgarian cereal production. Food Control, 20, 362e365. Muller, H. M., Reimann, J., Schumacher, U., & Schwadorf, K. (1997). Fusarium toxins in wheat harvested during six years in an area of southwest Germany. Natural Toxins, 5, 24e30. Murata, H., Sultana, P., Shimada, N., & Yashioka, M. (2003). Structureeactivity relationships among zearalenone and its derivatives based on bovine neutrophil chemiluminescence. Veterinary Human Toxicology, 45(1), 18e20. Obremski, K., Zielonka, L., Zaluska, G., Zwierzchowski, W., Pirus, K., Gajecki, M. (1999). The influence of low doses of zearalenone on liver enzyme activities in gilts. In Proceedings of the X conference microscopi fungi o plant pathogens and their metabolites (p. 66), Poznan.

C. Zaied et al. / Food Control 25 (2012) 773e777 Ouanes, Z., Abid, S., Ayed, I., Anane, R., Mobio, T., Creppy, E. E., et al. (2003). Induction of micronuclei by zearalenone in Vero monkey kidney cells and in bone marrow cells of mice: protective effect of vitamin E. Mutation Research, 538, 63e70. Ouanes, Z., Ayed-Boussema, I., Baati, T., Creppy, E. E., & Bacha, H. (2005). Zearalenone induces chromosome aberrations in mouse bone marrow: preventive effect of 17b oestradiol, progesterone and vitamin E. Mutation Research, 565, 139e149. Perkowski, J., Plattner, R. D., Golinski, P., & Vesonder, R. F. (1990). Natural occurrence of deoxynivalenol, 3-acetyl-deoxynivalenol, 15-acetyldeoxynivalenol, nivalenol, 4,7-dideoxynivalenol and zearalenone in Polish wheat. Mycotoxin Research, 6, 7e12. Placinta, C. M., D’Mello, J. F., & Macdonald, A. M. C. (1999). A review of worldwide contamination of cereal grains and animal feed with Fusarium mycotoxins. Animal Feed and Science Technology, 78, 21e37. Ryu, D., Jackson, L. S., & Bullerman, L. B. (2002). Effect of processing on zearalenone. Advances in Experimental Medicine and Biology, 504, 204e216. Schollenberger, M., Muller, H. M., Rufle, M., Suchy, S., Plank, S., & Drochner, W. (2006). Natural occurrence of 16 Fusarium toxins in grains and feedstuffs of plant origin from Germany. Mycopathologia, 161, 43e52.

777

SCOOP. (2003). Collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU member states. In A. Vidnes, C. Bergsten, & B. Paulsen (Eds.), Sub-task zearalenone. SCOOP European project, task 3.2.10 (pp. 241e481). Shier, W. T., Sier, A. C., Xie, W., & Mirocha, C. J. (2001). Structureeactivity relationships for human oestrogenic activity in zearalenone mycotoxins. Toxicon, 39, 1435e1438. Torrado, E., Blesa, J., Molto, J., Urraca, J. L., Marazuela, M. D., & Moreno-Bondi, M. C. (2010). Pressurized liquid extraction followed by liquid chromatographyemass spectrometry for determination of zearalenone in cereal flours. Food Control, 21, 399e402. Zaied, C., Abid, S., Zorgui, L., Bouaziz, C., Chouchane, S., Jomaa, M., et al. (2009). Natural occurrence of ochratoxin A in Tunisian cereals. Food Control, 20, 218e222. Zinedine, A., Brera, C., Elakhdari, S., Catano, C., Debegnach, F., Angelini, S., et al. (2006). Natural occurrence of mycotoxins in cereals and spices commercialized in Morocco. Food Control, 17, 868e874. Zinedine, A., Miguel, S., Carlos, M., & Jordi, M. (2007). Review on the toxicity, occurrence, metabolism, detoxification, regulations and intake of zearalenone: an oestrogenic mycotoxin. Food Chemical Toxicology, 45, 1e18.