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Cytotoxic effects of mycotoxin combinations in mammalian kidney cells
Food and Chemical Toxicology 49 (2011) 2718–2724
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Cytotoxic effects of mycotoxin combinations in mammalian kidney cells María-José Ruiz ⇑, Petra Macáková, Ana Juan-García, Guillermina Font Laboratori de Toxicologia, Facultat de Farmacia, Universitat de Valencia, Av. Vicent Andres Estelles, 46100 Burjassot, Valencia, Spain
a r t i c l e
i n f o
Article history: Received 3 June 2011 Accepted 6 July 2011 Available online 21 July 2011 Keywords: Beauvericin Deoxynivalenol T-2 toxin Vero cells Interaction
a b s t r a c t The cytotoxicity of three Fusarium mycotoxins (beauvericin, deoxynivalenol and T-2 toxin) has been investigated using the NR assay, after 24, 48 and 72 h of incubation. The IC50 values ranged from 6.77 to 11.08, 3.30 to 10.00 and 0.004 to 0.005 for beauvericin, deoxynivalenol and T-2 toxin, respectively. Once the potential interaction has been detected, a quantitative assessment is necessary to ensure and characterize these interactions, that is, each mycotoxin contributes to the toxic effect in accord with its own potency. Combination of mycotoxins was determined in Vero cells after 24, 48 and 72 h of exposure. Isobolograms and median effect method of Chou and Talalay were used to assess the nature and quantitative aspects of interaction observed between studied mycotoxins. Median effect analysis was used to calculate the combination index (CI) with values >1 indicating synergism, 1 additive effect, and <1 antagonism. CI values of BEA + DON (1.22–2.74), BEA + T-2 toxin (1.43–5.89), DON + T-2 toxin (3.13–7.62) and BEA + DON + T-2 toxin (1.32–2.68) for 24, 48 and 72 h produced antagonistic effects in Vero cells. The highest antagonistic effect in Vero cells was observed with binary DON and T-2 toxin mixture. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Several classes of mycotoxins are quite common contaminants that can occur simultaneously in food and raw materials. Particularly species from the genus Fusarium have been identified as important contaminants in foodstuffs for human or animal consumption (Bouaziz et al., 2006; Meca et al., 2010). The trichothecenes deoxynivalenol (DON) and T-2 toxin and the cyclic hexadepsipeptide beauvericin (BEA) belong to a group of mycotoxins synthesised by Fusarium fungi. Mainly due to the diversity of their chemical structures, these mycotoxins elicit a wide range of toxicological effects. DON, when ingested, can induce a decrease in food intake or refusal to eat food, vomiting, and digestive disorders, with subsequent looses of weight gain (Pestka, 2010). In vitro toxicity studies revealed that DON binds to the ribosomal peptidyltransferase site and inhibits protein synthesis, resulting in decreased cell proliferation. Moreover, it has been demonstrated that trichothecenes rapidly activate MAPK which modulates physiological processes including cell growth, differentiation and apoptosis. (Kouadio et al., 2007; Marzocco et al., 2009; Luongo et al., 2010). Among trichothecenes, T-2 toxin is the most toxic compound. It is considered to be a major causative agent in fatal alimentary toxic aleukia in humans affecting the mucosa and the ⇑ Corresponding author. Address: Laboratory of Toxicology, Faculty of Pharmacy, University of Valencia, C/Vicent Andres Estelles, S/N, 46100 Burjassot, Valencia, Spain. Tel.: +34 963543055; fax: +34 963544954. E-mail address: [email protected] (M.-J. Ruiz). 0278-6915/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2011.07.021
immune system. Leucopenia and necrotic lesions of the oral cavity, esophagus and stomach are the main pathological findings (McKean et al., 2006; Bouaziz et al., 2006; Chaudhari et al., 2009). BEA shows antimicrobial, insecticidal, cytotoxic, ionophoric, apoptotic and immunosuppressive activities (Tedjiotsop Feudjio et al., 2010). It is the most potential specific inhibitor of cholesterol acyltransferase. BEA increases ion permeability in biological membranes by forming a complex with essentials cations (Ca2+, Na+, K+) and/or cation-selective channels in lipid membranes, which may affect ionic homeostasis (Klaric et al., 2006; Dornestshuber et al., 2009; Ferrer et al., 2009). Because of the natural co-occurrence in food of mycotoxins, there is increasing concern about the hazard of exposure to mixtures. During last decade, the Scientific Committee on Food from the European Food Safety Authority (EFSA; Directorate General for Consumers) has been considered the general and relevant aspects on some individual and combined Fusarium mycotoxins, although it is acknowledged that there are gaps in the toxicological information available (http://ec.europa.eu/food/food/chemicalsafety/contaminants/fusarium_en.htm). The Committee considers that although Fusarium mycotoxins as T-2 toxin, HT-2 toxin, DON and nivalenol (NIV) appear to cause similar effects at the biochemical and cellular levels, and there are similarities in toxic effects, there are also substantial differences in the toxic effects in vivo. Furthermore, there are very few studies addressing the combined effects of these mycotoxins and at the present the database describing possible effects of combined exposure of trichothecenes is very weak and not sufficient for establishing either the nature of
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combined effect or the relative potencies of the trichothecenes. So, there is a concern that simultaneous exposure of different mycotoxins could be of potential health significance. In such cases a given combined exposure to several mycotoxins, may exceed the threshold dose for toxic effect, although the exposure for each single compound is below their respective No-Observed-Adverse-Effect-Levels (NOAELs). On the basis of current knowledge, which of these Fusarium toxins are of most concern for public health and because of their natural co-occurrence, which mycotoxin combined exposure have higher concern in health? So, to evaluate the toxicology of mycotoxins, most of mycotoxin studies should be focused on mixtures of mycotoxins (Tajima et al., 2002). The resulting effect of a combined exposure can produce (a) effect additive (dissimilar mode of action or independent action), (b) dose additive (similar mode of action), and (c) deviation from additive (synergism or antagonism) (Chou and Talalay, 1984). Effect additive means that the toxicity of one compound is exerted independently from the toxicity of another compound, even if they act on the same target organ. This means that when the doses of the chemicals are below the NOAELs of the individual compounds, the combined action of all chemicals together will also have no-effect. Toxicological interactions between mycotoxins have been evaluated by both in vivo and in vitro assays (Thuvander et al., 1999; Bernhoft et al., 2004; McKean et al., 2006; Kouadio et al., 2007; Klaric et al., 2008; Golli-Bennour et al., 2010; Ruiz et al., 2011). The in vivo assays require a big number of experimental group’s for the evaluation of the two or more than two mycotoxins in the mixture. Considering the importance of mycotoxin combinations, several in vitro bioassays have been proposed to detect cytotoxicity of mycotoxins. For quantitative dose–response assessment of mycotoxin mixtures, formulas are available that only require information on the toxicity and dose–response relationship of the individual mycotoxins and pairs of the individual mycotoxins. However, to evaluate the accuracy of these formulas statistical analysis, based on isobologram method, are being carried out. Isobologram method provides a visual assessment of the interaction between mycotoxins tested in the mixture (Chou and Talalay, 1984; Chou, 2006). In the present study the MTT assay was used to determine the cytotoxicity of individual and combined mycotoxins exposed to Vero cells at 24, 48 and 72 h. The results obtained by the dose– response curve were the basis for formulate the extent and nature of the interactions using the isobologram method of Chou and Talalay (1984).
2. Materials and methods 2.1. Reagents The reagent grade chemicals and cell culture components used, namely culture medium DMEM (Dulbecco’s Modified Eagle’s medium), antibiotics, trypsin/EDTA solution, HEPES, neutral red (NR) and the 3–4,5-dimethylthiazol-2-yl, 2,5-diphenyltetrazolium bromide (MTT) dyes and mycotoxins were from Sigma Chemical Co. (St. Louis, MO, USA). BEA (783.95 g/mol), DON (296.32 g/mol) and T2-toxin (466.58 g/ mol) were up to 98% purity. Fetal calf serum (FCS) was from Cambrex Company (Belgium). All other reagents were of standard laboratory grade.
2.2. Cell culture and treatment Mammalian kidney epithelial (Vero) cells were grown in DMEM supplemented with 25 mM HEPES buffer (pH 7.4), 10% FCS, 100 U/ml penicillin and 100 mg/ml streptomycin in a humidified atmosphere of 5% CO2, at 37 °C. The passage number ranger was maintained between 40 and 50. Cell viability was determined by exclusion of trypan blue dye. Numeration of cells was performed with a Beckman Coulter Z1 (Florida, USA). Absence of mycoplasma was checked routinely using the Mycoplasma Stain Kit (Sigma–Aldrich, St. Louis, MO, USA).
The Vero cells were cultured in 9 cm2 polystyrene tissue culture dishes. For experimental purpose, cells were cultured in 96-well microplates. The optimum cell concentration as determined by the growth profile of the cell line was 2 104 cells/ well. Cells were allowed to attach for 24 h before treatment with mycotoxins. Stock solutions of mycotoxins were prepared in methanol. Final concentration of test mycotoxins were achieved by adding the culture medium and the final methanol concentration in the medium was 1% (v/v). Cells were treated with six dilutions (serial dilution factor = 2) of each individual mycotoxins: BEA (from 0.78 to 25 lM), DON (from 0.25 to 8 lM) and T-2 (from 0.0016 to 0.05 lM) at 24, 48 and 72 h of exposure. Solutions of BEA, DON and T-2 prepared as described above were used individual and in two and three combinations. Vero cells were exposed with serial dilutions of each individual mycotoxin with a fixed constant ratio (BEA + DON, ratio = 3:1; BEA + T-2, ratio = 1250:1; DON + T-2, ratio = 160:1; and BEA + DON + T-2, ratio = 1250:400:1), below its individual EC50 value, in their binary and ternary combinations. Six dilutions of each mycotoxin and combination plus a control were tested in three independent experiments with replicate samples.
2.3. Determination of cytotoxicity assays The MTT and NR assays are widely used in in vitro toxicology studies for the determination of cytotoxicity or cell viability following exposure to mycotoxins. Both of them were selected and compared to detecting cytotoxicity of individual mycotoxins. And, they showed similar sensitive effect in detecting cytotoxicity events. Therefore, NR assay has been selected to carrier out the interaction between mycotoxin mixtures based on literature information as well as on preliminary studies performed in our laboratory. The MTT and NR assays were performed according on the protocol described by Ruiz et al. (2006). Briefly for NR assay, for the purposes of the experiments after the incubation time (24, 48 and 72 h) with different concentrations of each mycotoxin, 200 ll of freshly prepared NR solution (40 lg/ml) pre-warmed to 37 °C was added to each well and all plates returned to the incubator at 37 °C for 3 h. The cells were washed twice with phosphate buffer saline (PBS) and fixed with formaldehyde– CaCl2 solution, and then extracted by adding acetic acid–ethanol solution. Plates were gentle shaking for 5 min so that complete dissolution was achieved before measured absorbance at 540 nm with an automatic ELISA reader. Briefly for MTT assay, following exposure to mycotoxins (24, 48 and 72 h) with different concentrations of each mycotoxin, the medium was removed and cells of each well received fresh medium containing 50 ll MTT. After 4 h incubation, the resulting formazan was solubilized in DMSO. Plates were gentle shaking for 5 min so that complete dissolution was achieved. The absorbance was measured at 570 nm by using an automatic ELISA reader. By following the NR assay, cell proliferation was also quantified in the same plate. Initially, cells were lysed with 0.1 N NaOH for 2 h at 37 °C. Then Coomassie brilliant blue G-250 (Bio-Rad Laboratories Richmond, CA) was added and plates were incubated for 30 min at room temperature (http://www.bio-rad.com/LifeScience/pdf/Bulletin_9005.pdf). The absorbance was measured at 620 nm with an automatic ELISA reader. The results were expressed in relative form to cell culture protein content in order to avoid misinterpretation due to the influence of the chemical tested on cell proliferation and detachment.
2.4. Experimental design for isobolograms In isobolographic analyses, a traditional concentration–response relationship is constructed by experimentally measuring the response at a variety of concentrations of the factors both individually and in combination. After, design and run the main experiments for sufficient combinations of the factors which produce the desirable effect (% decreasing viability), the isoeffective points can be interpolated from the results and used to plot the isobologram. The isobologram analysis provides a combination index (CI) value, which is a quantitative measure of the degree of mycotoxin interaction betweeen two or three mycotoxins. The IC index was calculated according to Chou and Talalay (1984) and Chou (2006), as follows:
n
ðCIÞx ¼
n X ðDÞj =ðDx Þj ¼ J¼1
ðDx Þ1n f½Dj
Pn
J¼1 ½Dg
ðDm Þj fðfaxÞj =½1 ðfaxÞj g1=mj
where n(CI)x is the combination index for n compounds (e.g., mycotoxins) at x% inhibition (e.g., proliferation inhibition); (Dx)1n is the sum of the concentration of n P compounds that exerts x% inhibition in combination, f½Dj = n1 ½Dg is the proportionality of the concentration of each of n compounds that exerts x% inhibition in combination; and (Dm)j {(fax)j/[1 (fax)j]}1/mj is the concentration of each compound alone that exerts x% inhibition. A CI near 1 indicates additivity, <1 synergy, and >1 antagonism of the mycotoxin combinations. Synergy between BEA, DON and T-2 toxin was determined by isobologram analysis using CalcuSyn software (Biosoft, Cambridge, UK), which analyzes the data from proliferation assays to determine the interaction between mycotoxin combinations.
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2.5. Statistical analysis Statistical analysis of data was carried out using PSAW Statistic 17.0 (SPSS, Chicago, IL, USA), statistical software package. Data were expressed as mean ± SD of four independent experiments. The statistical analysis of the results was performed by Student’s t-test for paired samples. Differences between groups were analyzed statistically with one-way ANOVA followed by the Tukey HSD post hoc test for multiple comparisons. The level of P 6 0.05 was considered statistically significant.
3. Results 3.1. Cytotoxicity of individual mycotoxins The cytotoxic effect of BEA, DON and T-2 toxin on Vero cells was evaluated by the NR and MTT assays over 24, 48 and 72 h to determine the molar concentration of mycotoxin that reached 50% inhibition of cellular proliferation (IC50). The results consistently demonstrated that all mycotoxins reduce cell viability in a time and concentration-dependent manner (Fig. 1 and 2). The IC50 values by the MTT assay were from 6.25 to 10.02 lM, 5.05 to 8.02 lM and 0.007 to 0.012 lM for BEA, DON and T2 toxin, respectively (graphic not shown). The IC50 values by the NR assay were from 6.77 to 11.08 lM, 3.30 to 10.00 lM and 0.004 to 0.005 lM for BEA, DON and T2 toxin, respectively (Figs. 1 and 2). 3.2. Cytotoxicity effect of binary and tertiary mycotoxin mixture Considering similar IC50 value for each mycotoxin by both assays tested, and preliminary runs during this interaction study to optimize the concentration ranges, the NR assay was selected to carrier out the interaction study. The IC50 for BEA, DON and T-2 toxin by the NR assay was then determined for each mycotoxin to establish the ratio in the mixtures. The dose–response curves of binary and tertiary mixtures of BEA, DON and T-2 toxin are compared to the dose–response curve of each mycotoxin when applied individually in the same assay (Figs. 1 and 2). NR assay revealed that mycotoxin combinations inhibited Vero cell growth in a dose-dependent manner. The effect of combined mycotoxins was further assessed by the in vitro isobologram method. Typical CI/ fractional effect curves of mycotoxin combinations in Vero cell line are given in Fig. 3. In all cases, mycotoxin combinations presented a CI > 1 which confirmed an antagonistic mode of action between these three mycotoxins on Vero cells (Table 1 and Fig. 3). Data in Table 1 demonstrates that a moderate (CI = 1.22–1.50) to antagonism (CI = 1.73–2.74) effect over a wide range of EC50–EC90 concentrations of BEA and DON in combination. The strongest antagonistic effect on the three time tested was detected when exposed with higher doses of these two mycotoxins in combination. Similar IC values were obtained with the tertiary mixture. The CI values were from moderate (CI = 1.32–1.66) to antagonism (CI = 1.77–2.68) effect with BEA, DON and T-2 toxin in combination. Higher CI values were obtained for T-2 toxin plus BEA or DON. Strong antagonism effect, CI = 1.43–5.89 of BEA + T-2 toxin and CI = 3.13–7.62 of DON + T-2 toxin in combination was observed. The strongest antagonistic effect of all mycotoxin combination tested was detected with the mixture DON plus T-2 toxin. Moreover, for this binary combination, the strongest antagonistic effect was observed at lower doses. 4. Discussion The objective of the present study was to investigate the joint action of three Fusarium mycotoxins and to examine interactions among them. The results obtained shows that Vero cells were more sensitive toward T-2 toxin than toward DON and, DON was more cytotoxic than BEA (Figs. 1 and 2). There are many studies addressing the IC50 values for BEA, DON and T-2 toxin in cell culture. How-
ever, only very few studies were carried out in Vero cells. In a study of Thompson and Wannemacher (1986) the ability of nineteen trichothecenes to inhibit protein synthesis in Vero cells was developed. Similar results than those of this study were obtained about the potential effect of T-2 toxin and DON in Vero cells. They found that T-2 toxin was more than 100-fold higher in inhibiting protein synthesis than DON. Bouaziz et al. (2006) compared the cytotoxicity of T-2 toxin by two different endpoints, the NR and the MTT assays after 24 h of exposure. The results obtained by the NR assay was similar than those obtained in this study (IC50 = 4 nM) after 24 h of exposure. However, lower cytotoxicity (IC50 = 60 lM) value was obtained by the MTT assay. Differences between authors could be attributed to experimental conditions like the culture medium used, supplements added to the culture medium (amino acids or percentage of serum), period of exposure, and concentrations ranging assayed. On the other hand, there are a few studies addressing the effects of combined exposure to several mycotoxins. And, there is need to develop easy and accurate cell tests to assess the hazard of exposure of mixtures of mycotoxins. Combined effect of selected Penicillium (Bernhoft et al., 2004; Heussner et al., 2006; Tammer et al., 2007; Bouslimi et al., 2008), Fusarium (McKean et al., 2006; Severino et al., 2006; Kouadio et al., 2007; Luongo et al., 2008, 2010; Ruiz et al., 2011) and mixtures of Fusarium and Penicillium (Klaric et al., 2006, 2008; GolliBennour et al., 2010) mycotoxins on in vitro proliferation cell types has been carried out. On the basis of our current knowledge, the synergistic effect of emerging BEA mycotoxin has not been investigated in combination with trichothecenes yet. So, in this study the synergistic effect of these mycotoxins in Vero cells has been characterized. BEA, DON and T-2 toxin interaction was analyzed according to Chou and Talalay method, which provides a fundamental basis for assessing whether cytotoxicity induced by combinations of mycotoxins tested is greater, equal to or smaller than would have been expected for the individual mycotoxins. The IC50 values obtained by NR assay are concordant with isobologram analysis in Vero cell line under the culture conditions described. Table 1 shows the dose–effect curve parameters (CI, Dm, m and r) of all mycotoxin tested in combination which resulting in an antagonistic effect in Vero cell line. These findings suggest that the simultaneous presence of mycotoxins in food commodities and diet may be less cytotoxic than the presence of mycotoxins alone. Since, however, toxicity profiles of both or three mycotoxins are different, decreasing overlapping toxicity would like will mild. In general, it is difficult to clarify the mechanisms underlying the cytotoxic effects of mycotoxin combinations. The possibility that the mycotoxin interactions are due to some unknown mechanism related to complex perturbations of biochemical processes cannot be excluded. Similar results were obtained by Thuvander et al. (1999). They examined the combination of DON with T2 toxin which resulted in a toxicity that was significantly lower than, or similar to, the toxicity produced when exposed to T-2 toxin alone indicating an antagonistic interaction. Tajima et al. (2002) studied interactive effects in an inhibition of DNA synthesis between five Fusarium mycotoxins in the mixtures. They found that the interaction between DON and NIV (type B trichothecene) and T-2 toxin and NIV on DNA synthesis inhibition in L929 fibroblasts has been considered as synergistic (Tajima et al., 2002). These data are in agreement with their previous findings (Groten et al., 1998). Thuvander et al. (1999) studied the combined effects of NIV with T-2 toxin or DON. These combinations resulted in additive toxicity, with an inhibition of the proliferative responses that was significantly higher than when the human peripheral mononuclear cells were only exposed to one of the mycotoxins. On the contrary, results obtained by Luongo et al. (2008) showed no interactive effects between NIV and DON on porcine blood immune cell proliferation. Moreover, Marzocco et al. (2009) demonstrated that simultaneously
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M.-J. Ruiz et al. / Food and Chemical Toxicology 49 (2011) 2718–2724
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Fig. 1. Dose–response curves for the individual and all pair mixtures of BEA (. . .s. . .), DON (h), T-2 toxin ( ), and binary combinations (——) in Vero cells. The kidney cells were exposed to indicate concentrations of mycotoxins for 24, 48 and 72 h and then the viability was determined by the NR assay. p 6 0.05 (), p 6 0.001 () and p 6 0.000 () represent significant difference as compared to control values.
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a) 150
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Concentration (µM) Fig. 2. Dose–response curves for the individual and tertiary mixture of BEA (. . .s. . .), DON (h), T-2 toxin ( ), and combination (——) in Vero cells. The kidney cells were exposed to indicate concentrations of mycotoxins for (a) 24, (b) 48 and ⁄c) 72 h and then the viability was determined by the NR assay. p 6 0.05 (), p 6 0.001 () and p 6 0.000 () represent significant difference as compared to control values.
administration of NIV and DON on J774A.1 macrophages did not lead to a synergic cytotoxic effect indicating that their simultaneous presence did not enhance each own singular cytotoxicity. Other authors report antagonism for low concentrations and synergism for high concentration for mixture of DON and zearalenone (ZEA; Kouadio et al., 2007). However, Groten et al. (1998) and Tajima et al. (2002) showed that combined exposure to ZEA and DON or NIV or T-2 toxin results in an additive effect. On the other hand, acute combinative toxicity for T-2 toxin and aflatoxin B1 (AFB1) in HepG2 and BEAS-2B cells was carried out by McKean et al. (2006) showing different response in the interaction type. Whereas, the combination on T-2 toxin and AFB1 resulted in
synergistic interaction in BEAS-2B cells, this combination could be described as additive in HepG2 cells. BEA is an emerging Fusarium mycotoxin, which has led to recent interest in developing toxicity studies over the last few years. Combined effects of BEA, ochratoxin A (OTA) and fumonisin B1 (FB1) on porcine kidney epithelial (PK15) cells survival has been carried (Klaric et al., 2006, 2008). The binary and tertiary combinations versus single- or two toxin exposures of PK15 cells resulted mostly in additive effects. Antagonism was recorded at the highest concentrations tested. In conclusion, T-2 toxin was 100-fold more toxic than DON and BEA. All mycotoxin combinations showed antagonistic interaction, but the higher results were obtained for the binary DON plus T-2
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Fig. 3. Typical combination index (CI)/fractional affected (fa) curve as described by Chou and Talalay model for Vero cell. Each point represents the CI at a dose effect as determined in our experiments. The dotted line indicates additivity, the area under the dotted line synergy, and the area above of the dotted line antagonism. Vero cells were exposed with (a) BEA + DON (molar ratio of 3:1), (b) BEA + T-2 toxin (molar ratio of 1250:1), (c) DON + T-2 (molar ratio of 160:1), and (d) (c) BEA + DON + T-2 (molar ratio of 1250:400:1). Color version of figure is available on line: 24 h of exposure (+), 48 h of exposure ( ), 72 h of exposure ().
Table 1 Dose–effect relationship parameters and mean combination index (CI) values (as a function of fractional inhibition of proliferation) for the different mycotoxin combinations. Mycotoxin
BEA + DON
BEA + T2
DON + T2
BEA + DON + T2
Time (h)
24 48 72 24 48 72 24 48 72 24 48 72
Dose–effect parameters
CI values
Dm (lM)
m
r
EC50
4.98 2.89 3.21 7.56 3.18 4.77 4.14 3.04 3.19 2.75 3.58 3.28
1.58 1.52 1.43 1.18 2.39 2.19 1.24 1.58 1.74 1.46 2.88 2.93
0.9082 0.9837 0.9586 0.9851 0.9413 0.9507 0.9900 0.8990 0.9866 0.9011 0.9156 0.9375
1.73 1.22 1.78 3.20 1.81 3.41 5.03 6.82 7.62 1.32 2.38 2.68
EC75 Ant Ant Ant Ant Ant Ant Ant Ant Ant Ant Ant Ant
2.18 1.50 2.18 4.09 1.59 2.96 3.61 6.45 7.05 1.45 1.92 2.10
EC90 Ant Ant Ant Ant Ant Ant Ant Ant Ant Ant Ant Ant
2.74 1.86 2.69 5.89 1.43 2.59 3.13 6.11 6.61 1.77 2.57 1.66
Ant Ant Ant Ant Ant Ant Ant Ant Ant Ant Ant Ant
The parameters m, Dm and r are the antilog of x-intercept, the slope and the linear correlation coefficient of the median-effect plot, which signifies the shape of the dose–effect curve, the potency (EC50), and conformity of the data to the mass–action law, respectively (Chou and Talalay, 1984; Chou, 2006). Dm and m values are used for calculating the CI values (CI < 1, =1, and >1 indicate synergism (Syn), additive effect (Add), and antagonism (Ant), respectively. EC50, EC75 and EC90, are the doses required to inhibit proliferation 50, 75 and 90%, respectively. The ratio of BEA + DON: 3:1; BEA + T-2:1250:1; DON + T-2:160:1; and BEA + DON + T-2, ratio = 1250:400:1 was used for the experimental design. Computer software CalcuSyn was used for automated calculation and simulation.
toxin combination, although the mechanisms of interaction remain unknown. On the other hand, there is a clear discrepancy with results obtained in literature, so additivity, and synergism but also antagonism has been observed for T-2 toxin, DON and BEA combinations in vitro. Although there could be indications for dose additivity in some of the in vitro studies, at present the database describing possible effects of combined exposure of Fusarium mycotoxins is very weak and not sufficient for establishing either the nature of combined effects or the relative potencies of the Fusarium mycotoxins. Moreover, there are large differences depending on combination mycotoxins, assay carried out, type of
cell used, and other factors. However, in term of risk assessment in food and diet, mycotoxins are commonly found on cereals grown in temperate areas, and the presence of several of them could increase the potential toxicity of each of them separately. So, toxic effects of combinative mycotoxins should be determined or revalidated in the light of continuously toxicological data obtained by scientific groups. Conflict of interest The authors declare that there are no conflicts of interest.
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Acknowledgements This study was supported by the Conselleria de Sanitat, Generalitat Valenciana (project n° EVES2010-028). References Bernhoft, A., Keblys, M., Morrison, E., Larse, H.J.S., Flaoyen, A., 2004. Combined effects of selected Penicillium mycotoxins on in vitro proliferation of porcine lymphocytes. Mycopathologia 158, 441–450. Bouaziz, C., Abid Essefi, S., Bouslimi, A., El Golli, E., Bacha, H., 2006. Cytotoxicity and related effects of T-2 toxin on cultured Vero-cells. Toxicon 48, 343–352. Bouslimi, A., Ouannes, Z., Golli, E.E., Bouaziz, Ch., Hassen, W., Bacha, H., 2008. Cytotoxicity and oxidative damage in kidney cells exposed to the mycotoxins ochratoxin A and citrinin: individual and combined effect. Toxicol. Mech. Methods 18, 341–349. Chaudhari, M., Jayaraj, R., Bhaskar, A.S.B., Lakshmana Rao, P.V., 2009. Oxidative stress induction by T-2 toxin causes DNA damage and triggers apoptosis via caspase pathway in human cervical cancer cells. Toxicology 262, 153–161. Chou, T.-Ch., 2006. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol. Rev. 58, 621–681. Chou, T.-Ch., Talalay, P., 1984. Quantitative analysis of dose–effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 22, 27–55. Dornestshuber, R., Heffeter, P., Sulyok, M., Schumacher, R., Chiba, P., Kopp, S., Koellensperger, G., Micksche, M., Lemmens-Gruber, R., Berger, W., 2009. Interactions between ABC-transport proteins and the secondary Fusarium metabolites enniatin and beauvericin. Mol. Nutr. Food Res. 53, 904–920. Ferrer, E., Juan-García, A., Font, G., Ruiz, M.J., 2009. Reactive oxygen species induced by beauvericin, patulin and zearalenone in CHO-K1 cells. Toxicol. In Vitro 23, 1504–1509. Golli-Bennour, E., Kpuidhi, B., Bouslimi, A., Abid-Essefi, S., Hassen, W., Bacha, H., 2010. Cytotoxicity and genotoxicity induced by aflatoxin B1, ochratoxin A, and their combination in cultured Vero cells. J. Biochem. Mol. Toxicol. 24, 42–50. Groten, J.P., Tajima, O., Feron, V.J., Schoen, E.D., 1998. Statistically designed experiments to screen chemical mixtures for possible interactions. Environ. Health Perspect. 106, 1361–1365. Heussner, A.H., Dietrich, D.R., O’Brien, E., 2006. In vitro investigation of individual and combined cytotoxic effects of ochratoxin A and other selected mycotoxins on renal cells. Toxicol. In Vitro 20, 332–341. http://ec.europa.eu/food/food/chemicalsafety/contaminants/fusarium_en.htm. http://www.bio-rad.com/LifeScience/pdf/Bulletin_9005.pdf. Klaric, M.S., Pepeljnjak, S., Domijan, A.-M., Petrik, J., 2006. Lipid peroxidation and glutathione levels in porcine kidney PK15 cells after individual and combined treatment with fumonisin B1, beauvericin and ochratoxin A. Basic Clin. Pharmacol. Toxicol. 100, 157–167.
Klaric, M.S., Rumora, L., Ljubanovic, D., Pepeljnjak, S., 2008. Cytotoxicity and apoptosis induced by fumonisin B1, beauvericin and ochratoxin A in porcine kidney PK15 cells: effects of individual and combined treatment. Arch. Toxicol. 82, 247–255. Kouadio, J.H., Sebastien, D.D., Moukha, S., 2007. Effect of combinations of fusarium mycotoxins on the inhibition of macromolecular synthesis malondialdehyde levels, DNA methylation and fragmentation and viability in Caco-2 cells. Toxicon 49, 306–317. Luongo, D., De Luna, R., Russo, R., Severino, L., 2008. Effects of four Fusarium toxins (fumonisin B1, a-zearalenol, nivalenol and deoxynivalenol) on porcine wholeblood cellular proliferation. Toxicon 52, 156–162. Luongo, D., Severino, L., Bergamo, P., D’Arienzo, R., Rossi, M., 2010. Trichothecenes NIV and DON modulate the maturation of murine dendritic cells. Toxicon 55, 73–80. Marzocco, S., Russo, R., Bianco, G., Autore, G., Severino, L., 2009. Pro-apoptotic effects of nivalenol and deoxynivalenol trichothecenes in J774A.1 murine macrophages. Toxicol. Lett. 189, 21–26. McKean, C., Tang, L., Billam, M., Tang, M., Theodorakis, C.W., Kendall, R.J., Wang, J.-S., 2006. Comparative acute and combinative toxicity of aflatoxin B1 and T-2 toxin in animals and immortalized human cell lines. J. Appl. Toxicol. 26, 139–147. Meca, G., Ruiz, M.J., Soriano, J.M., Ritieni, A., Morettti, A., Font, G., Mañes, J., 2010. Isolation and purification of enniatins A, A1, B, B1, produced by Fusarium tricinctum in solid culture, and cytotoxicity effects on Caco-2 cells. Toxicon 56, 418–424. Pestka, J.J., 2010. Deoxynivalenol: mechanisms of action, human exposure, and toxicological relevance. Arch. Toxicol. 84, 663–679. Ruiz, M.J., Festila, L.E., Fernandez, M., 2006. Comparison of basal cytotoxicity of seven carbamates in CHO-K1 cells. Toxicol. Environ. Chem. 88, 345–354. Ruiz, M.J., Franzova, P., Juan-Garcia, A., Font, G., 2011. Toxicological interactions between the mycotoxins beauvericin, deoxynivalenol and T-2 toxin in CHO-K1 cells in vitro. Toxicon. doi:10.1016/j.toxicon.2011.07.015. Severino, L., Luongo, D., Bergamo, P., Lucisano, A., Rossi, M., 2006. Mycotoxins nivalenol and deoxynivalenol differentially modúlate cytokine mRNA expression in Jurkat T cells. Cytokine 36, 75–82. Tajima, O., Schoen, E.D., Feron, V.J., Groten, J.P., 2002. Statistically designed experiments in a tiered approach to screen mixtures of Fusarium mycotoxins for possible interactions. Food Chem. Toxicol. 40, 685–695. Tammer, B., Lehmann, I., Nieber, K., Altenburger, R., 2007. Combined effects of mycotoxin mixtures on human T cell function. Toxicol. Lett. 170, 124–133. Tedjiotsop Feudjio, F., Dornetshuber, R., Lemmens, M., Hoffmann, O., Lemmens_Gruber, R., Berger, W., 2010. Beauvericin and enniatin: emerging toxins and/or remedies? Word Mycotoxin J. doi:10.3920/WMJ2010.1245. Thompson, W.L., Wannemacher Jr., R.W., 1986. Structure–function relationships of 12,13-epoxytrichothecene mycotoxins in cell culture: comparison to whole animal lethality. Toxicon 24, 985–994. Thuvander, A., Wikman, C., Gadhasson, I., 1999. In vitro exposure of human lymphocytes to trichothecenes: Individual variation in sensitivity and effects of combined exposure on lymphocyte function. Food Chem. Toxicol. 37, 639– 648.
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Cytotoxic effects of mycotoxin combinations in mammalian kidney cells María-José Ruiz ⇑, Petra Macáková, Ana Juan-García, Guillermina Font Laboratori de Toxicologia, Facultat de Farmacia, Universitat de Valencia, Av. Vicent Andres Estelles, 46100 Burjassot, Valencia, Spain
a r t i c l e
i n f o
Article history: Received 3 June 2011 Accepted 6 July 2011 Available online 21 July 2011 Keywords: Beauvericin Deoxynivalenol T-2 toxin Vero cells Interaction
a b s t r a c t The cytotoxicity of three Fusarium mycotoxins (beauvericin, deoxynivalenol and T-2 toxin) has been investigated using the NR assay, after 24, 48 and 72 h of incubation. The IC50 values ranged from 6.77 to 11.08, 3.30 to 10.00 and 0.004 to 0.005 for beauvericin, deoxynivalenol and T-2 toxin, respectively. Once the potential interaction has been detected, a quantitative assessment is necessary to ensure and characterize these interactions, that is, each mycotoxin contributes to the toxic effect in accord with its own potency. Combination of mycotoxins was determined in Vero cells after 24, 48 and 72 h of exposure. Isobolograms and median effect method of Chou and Talalay were used to assess the nature and quantitative aspects of interaction observed between studied mycotoxins. Median effect analysis was used to calculate the combination index (CI) with values >1 indicating synergism, 1 additive effect, and <1 antagonism. CI values of BEA + DON (1.22–2.74), BEA + T-2 toxin (1.43–5.89), DON + T-2 toxin (3.13–7.62) and BEA + DON + T-2 toxin (1.32–2.68) for 24, 48 and 72 h produced antagonistic effects in Vero cells. The highest antagonistic effect in Vero cells was observed with binary DON and T-2 toxin mixture. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Several classes of mycotoxins are quite common contaminants that can occur simultaneously in food and raw materials. Particularly species from the genus Fusarium have been identified as important contaminants in foodstuffs for human or animal consumption (Bouaziz et al., 2006; Meca et al., 2010). The trichothecenes deoxynivalenol (DON) and T-2 toxin and the cyclic hexadepsipeptide beauvericin (BEA) belong to a group of mycotoxins synthesised by Fusarium fungi. Mainly due to the diversity of their chemical structures, these mycotoxins elicit a wide range of toxicological effects. DON, when ingested, can induce a decrease in food intake or refusal to eat food, vomiting, and digestive disorders, with subsequent looses of weight gain (Pestka, 2010). In vitro toxicity studies revealed that DON binds to the ribosomal peptidyltransferase site and inhibits protein synthesis, resulting in decreased cell proliferation. Moreover, it has been demonstrated that trichothecenes rapidly activate MAPK which modulates physiological processes including cell growth, differentiation and apoptosis. (Kouadio et al., 2007; Marzocco et al., 2009; Luongo et al., 2010). Among trichothecenes, T-2 toxin is the most toxic compound. It is considered to be a major causative agent in fatal alimentary toxic aleukia in humans affecting the mucosa and the ⇑ Corresponding author. Address: Laboratory of Toxicology, Faculty of Pharmacy, University of Valencia, C/Vicent Andres Estelles, S/N, 46100 Burjassot, Valencia, Spain. Tel.: +34 963543055; fax: +34 963544954. E-mail address: [email protected] (M.-J. Ruiz). 0278-6915/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2011.07.021
immune system. Leucopenia and necrotic lesions of the oral cavity, esophagus and stomach are the main pathological findings (McKean et al., 2006; Bouaziz et al., 2006; Chaudhari et al., 2009). BEA shows antimicrobial, insecticidal, cytotoxic, ionophoric, apoptotic and immunosuppressive activities (Tedjiotsop Feudjio et al., 2010). It is the most potential specific inhibitor of cholesterol acyltransferase. BEA increases ion permeability in biological membranes by forming a complex with essentials cations (Ca2+, Na+, K+) and/or cation-selective channels in lipid membranes, which may affect ionic homeostasis (Klaric et al., 2006; Dornestshuber et al., 2009; Ferrer et al., 2009). Because of the natural co-occurrence in food of mycotoxins, there is increasing concern about the hazard of exposure to mixtures. During last decade, the Scientific Committee on Food from the European Food Safety Authority (EFSA; Directorate General for Consumers) has been considered the general and relevant aspects on some individual and combined Fusarium mycotoxins, although it is acknowledged that there are gaps in the toxicological information available (http://ec.europa.eu/food/food/chemicalsafety/contaminants/fusarium_en.htm). The Committee considers that although Fusarium mycotoxins as T-2 toxin, HT-2 toxin, DON and nivalenol (NIV) appear to cause similar effects at the biochemical and cellular levels, and there are similarities in toxic effects, there are also substantial differences in the toxic effects in vivo. Furthermore, there are very few studies addressing the combined effects of these mycotoxins and at the present the database describing possible effects of combined exposure of trichothecenes is very weak and not sufficient for establishing either the nature of
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combined effect or the relative potencies of the trichothecenes. So, there is a concern that simultaneous exposure of different mycotoxins could be of potential health significance. In such cases a given combined exposure to several mycotoxins, may exceed the threshold dose for toxic effect, although the exposure for each single compound is below their respective No-Observed-Adverse-Effect-Levels (NOAELs). On the basis of current knowledge, which of these Fusarium toxins are of most concern for public health and because of their natural co-occurrence, which mycotoxin combined exposure have higher concern in health? So, to evaluate the toxicology of mycotoxins, most of mycotoxin studies should be focused on mixtures of mycotoxins (Tajima et al., 2002). The resulting effect of a combined exposure can produce (a) effect additive (dissimilar mode of action or independent action), (b) dose additive (similar mode of action), and (c) deviation from additive (synergism or antagonism) (Chou and Talalay, 1984). Effect additive means that the toxicity of one compound is exerted independently from the toxicity of another compound, even if they act on the same target organ. This means that when the doses of the chemicals are below the NOAELs of the individual compounds, the combined action of all chemicals together will also have no-effect. Toxicological interactions between mycotoxins have been evaluated by both in vivo and in vitro assays (Thuvander et al., 1999; Bernhoft et al., 2004; McKean et al., 2006; Kouadio et al., 2007; Klaric et al., 2008; Golli-Bennour et al., 2010; Ruiz et al., 2011). The in vivo assays require a big number of experimental group’s for the evaluation of the two or more than two mycotoxins in the mixture. Considering the importance of mycotoxin combinations, several in vitro bioassays have been proposed to detect cytotoxicity of mycotoxins. For quantitative dose–response assessment of mycotoxin mixtures, formulas are available that only require information on the toxicity and dose–response relationship of the individual mycotoxins and pairs of the individual mycotoxins. However, to evaluate the accuracy of these formulas statistical analysis, based on isobologram method, are being carried out. Isobologram method provides a visual assessment of the interaction between mycotoxins tested in the mixture (Chou and Talalay, 1984; Chou, 2006). In the present study the MTT assay was used to determine the cytotoxicity of individual and combined mycotoxins exposed to Vero cells at 24, 48 and 72 h. The results obtained by the dose– response curve were the basis for formulate the extent and nature of the interactions using the isobologram method of Chou and Talalay (1984).
2. Materials and methods 2.1. Reagents The reagent grade chemicals and cell culture components used, namely culture medium DMEM (Dulbecco’s Modified Eagle’s medium), antibiotics, trypsin/EDTA solution, HEPES, neutral red (NR) and the 3–4,5-dimethylthiazol-2-yl, 2,5-diphenyltetrazolium bromide (MTT) dyes and mycotoxins were from Sigma Chemical Co. (St. Louis, MO, USA). BEA (783.95 g/mol), DON (296.32 g/mol) and T2-toxin (466.58 g/ mol) were up to 98% purity. Fetal calf serum (FCS) was from Cambrex Company (Belgium). All other reagents were of standard laboratory grade.
2.2. Cell culture and treatment Mammalian kidney epithelial (Vero) cells were grown in DMEM supplemented with 25 mM HEPES buffer (pH 7.4), 10% FCS, 100 U/ml penicillin and 100 mg/ml streptomycin in a humidified atmosphere of 5% CO2, at 37 °C. The passage number ranger was maintained between 40 and 50. Cell viability was determined by exclusion of trypan blue dye. Numeration of cells was performed with a Beckman Coulter Z1 (Florida, USA). Absence of mycoplasma was checked routinely using the Mycoplasma Stain Kit (Sigma–Aldrich, St. Louis, MO, USA).
The Vero cells were cultured in 9 cm2 polystyrene tissue culture dishes. For experimental purpose, cells were cultured in 96-well microplates. The optimum cell concentration as determined by the growth profile of the cell line was 2 104 cells/ well. Cells were allowed to attach for 24 h before treatment with mycotoxins. Stock solutions of mycotoxins were prepared in methanol. Final concentration of test mycotoxins were achieved by adding the culture medium and the final methanol concentration in the medium was 1% (v/v). Cells were treated with six dilutions (serial dilution factor = 2) of each individual mycotoxins: BEA (from 0.78 to 25 lM), DON (from 0.25 to 8 lM) and T-2 (from 0.0016 to 0.05 lM) at 24, 48 and 72 h of exposure. Solutions of BEA, DON and T-2 prepared as described above were used individual and in two and three combinations. Vero cells were exposed with serial dilutions of each individual mycotoxin with a fixed constant ratio (BEA + DON, ratio = 3:1; BEA + T-2, ratio = 1250:1; DON + T-2, ratio = 160:1; and BEA + DON + T-2, ratio = 1250:400:1), below its individual EC50 value, in their binary and ternary combinations. Six dilutions of each mycotoxin and combination plus a control were tested in three independent experiments with replicate samples.
2.3. Determination of cytotoxicity assays The MTT and NR assays are widely used in in vitro toxicology studies for the determination of cytotoxicity or cell viability following exposure to mycotoxins. Both of them were selected and compared to detecting cytotoxicity of individual mycotoxins. And, they showed similar sensitive effect in detecting cytotoxicity events. Therefore, NR assay has been selected to carrier out the interaction between mycotoxin mixtures based on literature information as well as on preliminary studies performed in our laboratory. The MTT and NR assays were performed according on the protocol described by Ruiz et al. (2006). Briefly for NR assay, for the purposes of the experiments after the incubation time (24, 48 and 72 h) with different concentrations of each mycotoxin, 200 ll of freshly prepared NR solution (40 lg/ml) pre-warmed to 37 °C was added to each well and all plates returned to the incubator at 37 °C for 3 h. The cells were washed twice with phosphate buffer saline (PBS) and fixed with formaldehyde– CaCl2 solution, and then extracted by adding acetic acid–ethanol solution. Plates were gentle shaking for 5 min so that complete dissolution was achieved before measured absorbance at 540 nm with an automatic ELISA reader. Briefly for MTT assay, following exposure to mycotoxins (24, 48 and 72 h) with different concentrations of each mycotoxin, the medium was removed and cells of each well received fresh medium containing 50 ll MTT. After 4 h incubation, the resulting formazan was solubilized in DMSO. Plates were gentle shaking for 5 min so that complete dissolution was achieved. The absorbance was measured at 570 nm by using an automatic ELISA reader. By following the NR assay, cell proliferation was also quantified in the same plate. Initially, cells were lysed with 0.1 N NaOH for 2 h at 37 °C. Then Coomassie brilliant blue G-250 (Bio-Rad Laboratories Richmond, CA) was added and plates were incubated for 30 min at room temperature (http://www.bio-rad.com/LifeScience/pdf/Bulletin_9005.pdf). The absorbance was measured at 620 nm with an automatic ELISA reader. The results were expressed in relative form to cell culture protein content in order to avoid misinterpretation due to the influence of the chemical tested on cell proliferation and detachment.
2.4. Experimental design for isobolograms In isobolographic analyses, a traditional concentration–response relationship is constructed by experimentally measuring the response at a variety of concentrations of the factors both individually and in combination. After, design and run the main experiments for sufficient combinations of the factors which produce the desirable effect (% decreasing viability), the isoeffective points can be interpolated from the results and used to plot the isobologram. The isobologram analysis provides a combination index (CI) value, which is a quantitative measure of the degree of mycotoxin interaction betweeen two or three mycotoxins. The IC index was calculated according to Chou and Talalay (1984) and Chou (2006), as follows:
n
ðCIÞx ¼
n X ðDÞj =ðDx Þj ¼ J¼1
ðDx Þ1n f½Dj
Pn
J¼1 ½Dg
ðDm Þj fðfaxÞj =½1 ðfaxÞj g1=mj
where n(CI)x is the combination index for n compounds (e.g., mycotoxins) at x% inhibition (e.g., proliferation inhibition); (Dx)1n is the sum of the concentration of n P compounds that exerts x% inhibition in combination, f½Dj = n1 ½Dg is the proportionality of the concentration of each of n compounds that exerts x% inhibition in combination; and (Dm)j {(fax)j/[1 (fax)j]}1/mj is the concentration of each compound alone that exerts x% inhibition. A CI near 1 indicates additivity, <1 synergy, and >1 antagonism of the mycotoxin combinations. Synergy between BEA, DON and T-2 toxin was determined by isobologram analysis using CalcuSyn software (Biosoft, Cambridge, UK), which analyzes the data from proliferation assays to determine the interaction between mycotoxin combinations.
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2.5. Statistical analysis Statistical analysis of data was carried out using PSAW Statistic 17.0 (SPSS, Chicago, IL, USA), statistical software package. Data were expressed as mean ± SD of four independent experiments. The statistical analysis of the results was performed by Student’s t-test for paired samples. Differences between groups were analyzed statistically with one-way ANOVA followed by the Tukey HSD post hoc test for multiple comparisons. The level of P 6 0.05 was considered statistically significant.
3. Results 3.1. Cytotoxicity of individual mycotoxins The cytotoxic effect of BEA, DON and T-2 toxin on Vero cells was evaluated by the NR and MTT assays over 24, 48 and 72 h to determine the molar concentration of mycotoxin that reached 50% inhibition of cellular proliferation (IC50). The results consistently demonstrated that all mycotoxins reduce cell viability in a time and concentration-dependent manner (Fig. 1 and 2). The IC50 values by the MTT assay were from 6.25 to 10.02 lM, 5.05 to 8.02 lM and 0.007 to 0.012 lM for BEA, DON and T2 toxin, respectively (graphic not shown). The IC50 values by the NR assay were from 6.77 to 11.08 lM, 3.30 to 10.00 lM and 0.004 to 0.005 lM for BEA, DON and T2 toxin, respectively (Figs. 1 and 2). 3.2. Cytotoxicity effect of binary and tertiary mycotoxin mixture Considering similar IC50 value for each mycotoxin by both assays tested, and preliminary runs during this interaction study to optimize the concentration ranges, the NR assay was selected to carrier out the interaction study. The IC50 for BEA, DON and T-2 toxin by the NR assay was then determined for each mycotoxin to establish the ratio in the mixtures. The dose–response curves of binary and tertiary mixtures of BEA, DON and T-2 toxin are compared to the dose–response curve of each mycotoxin when applied individually in the same assay (Figs. 1 and 2). NR assay revealed that mycotoxin combinations inhibited Vero cell growth in a dose-dependent manner. The effect of combined mycotoxins was further assessed by the in vitro isobologram method. Typical CI/ fractional effect curves of mycotoxin combinations in Vero cell line are given in Fig. 3. In all cases, mycotoxin combinations presented a CI > 1 which confirmed an antagonistic mode of action between these three mycotoxins on Vero cells (Table 1 and Fig. 3). Data in Table 1 demonstrates that a moderate (CI = 1.22–1.50) to antagonism (CI = 1.73–2.74) effect over a wide range of EC50–EC90 concentrations of BEA and DON in combination. The strongest antagonistic effect on the three time tested was detected when exposed with higher doses of these two mycotoxins in combination. Similar IC values were obtained with the tertiary mixture. The CI values were from moderate (CI = 1.32–1.66) to antagonism (CI = 1.77–2.68) effect with BEA, DON and T-2 toxin in combination. Higher CI values were obtained for T-2 toxin plus BEA or DON. Strong antagonism effect, CI = 1.43–5.89 of BEA + T-2 toxin and CI = 3.13–7.62 of DON + T-2 toxin in combination was observed. The strongest antagonistic effect of all mycotoxin combination tested was detected with the mixture DON plus T-2 toxin. Moreover, for this binary combination, the strongest antagonistic effect was observed at lower doses. 4. Discussion The objective of the present study was to investigate the joint action of three Fusarium mycotoxins and to examine interactions among them. The results obtained shows that Vero cells were more sensitive toward T-2 toxin than toward DON and, DON was more cytotoxic than BEA (Figs. 1 and 2). There are many studies addressing the IC50 values for BEA, DON and T-2 toxin in cell culture. How-
ever, only very few studies were carried out in Vero cells. In a study of Thompson and Wannemacher (1986) the ability of nineteen trichothecenes to inhibit protein synthesis in Vero cells was developed. Similar results than those of this study were obtained about the potential effect of T-2 toxin and DON in Vero cells. They found that T-2 toxin was more than 100-fold higher in inhibiting protein synthesis than DON. Bouaziz et al. (2006) compared the cytotoxicity of T-2 toxin by two different endpoints, the NR and the MTT assays after 24 h of exposure. The results obtained by the NR assay was similar than those obtained in this study (IC50 = 4 nM) after 24 h of exposure. However, lower cytotoxicity (IC50 = 60 lM) value was obtained by the MTT assay. Differences between authors could be attributed to experimental conditions like the culture medium used, supplements added to the culture medium (amino acids or percentage of serum), period of exposure, and concentrations ranging assayed. On the other hand, there are a few studies addressing the effects of combined exposure to several mycotoxins. And, there is need to develop easy and accurate cell tests to assess the hazard of exposure of mixtures of mycotoxins. Combined effect of selected Penicillium (Bernhoft et al., 2004; Heussner et al., 2006; Tammer et al., 2007; Bouslimi et al., 2008), Fusarium (McKean et al., 2006; Severino et al., 2006; Kouadio et al., 2007; Luongo et al., 2008, 2010; Ruiz et al., 2011) and mixtures of Fusarium and Penicillium (Klaric et al., 2006, 2008; GolliBennour et al., 2010) mycotoxins on in vitro proliferation cell types has been carried out. On the basis of our current knowledge, the synergistic effect of emerging BEA mycotoxin has not been investigated in combination with trichothecenes yet. So, in this study the synergistic effect of these mycotoxins in Vero cells has been characterized. BEA, DON and T-2 toxin interaction was analyzed according to Chou and Talalay method, which provides a fundamental basis for assessing whether cytotoxicity induced by combinations of mycotoxins tested is greater, equal to or smaller than would have been expected for the individual mycotoxins. The IC50 values obtained by NR assay are concordant with isobologram analysis in Vero cell line under the culture conditions described. Table 1 shows the dose–effect curve parameters (CI, Dm, m and r) of all mycotoxin tested in combination which resulting in an antagonistic effect in Vero cell line. These findings suggest that the simultaneous presence of mycotoxins in food commodities and diet may be less cytotoxic than the presence of mycotoxins alone. Since, however, toxicity profiles of both or three mycotoxins are different, decreasing overlapping toxicity would like will mild. In general, it is difficult to clarify the mechanisms underlying the cytotoxic effects of mycotoxin combinations. The possibility that the mycotoxin interactions are due to some unknown mechanism related to complex perturbations of biochemical processes cannot be excluded. Similar results were obtained by Thuvander et al. (1999). They examined the combination of DON with T2 toxin which resulted in a toxicity that was significantly lower than, or similar to, the toxicity produced when exposed to T-2 toxin alone indicating an antagonistic interaction. Tajima et al. (2002) studied interactive effects in an inhibition of DNA synthesis between five Fusarium mycotoxins in the mixtures. They found that the interaction between DON and NIV (type B trichothecene) and T-2 toxin and NIV on DNA synthesis inhibition in L929 fibroblasts has been considered as synergistic (Tajima et al., 2002). These data are in agreement with their previous findings (Groten et al., 1998). Thuvander et al. (1999) studied the combined effects of NIV with T-2 toxin or DON. These combinations resulted in additive toxicity, with an inhibition of the proliferative responses that was significantly higher than when the human peripheral mononuclear cells were only exposed to one of the mycotoxins. On the contrary, results obtained by Luongo et al. (2008) showed no interactive effects between NIV and DON on porcine blood immune cell proliferation. Moreover, Marzocco et al. (2009) demonstrated that simultaneously
*
125
***
* ***
50
* * ***
*** ***
***
25
150
*
125
100 75
a) DON+T-2, 24 h
***
75
*** 50
***
***
***
0.25
0.5
1
2
4
8
DON
0
0.78
1.56
3.125
6.25
12.5
25
BEA
125
0
0.25
0.5
1
2
4
8
0
0.001
0.003
0.006
0.01
0.025
0.05
*** **
**
**
50
1.56
3.125
6.25
12.5
25
0.001
0.003
0.006
0.01
0.025
0.05
*** ***
***
***
**
**
***
***
2
4
8
DON
0
0.25
0.5
1
2
4
8
0
0.78
1.56
3.125
6.25
12.5
25
BEA
0
0.001
0.003
0.006
0.01
0.025
0.05
0
Viability (%)
*
75
* ***
50
***
**
25
*** ***
0
50
T-2
c) DON+T-2, 72 h
150
0
0.25
0.5
1
2
4
8
0
0.78
1.56
3.125
6.25
12.5
25
* **
50
***
** *** *** ***
**
0
0.78
1.56
3.125
6.25
12.5
25
0
0.001
0.003
0.006
0.01
0.025
0.05
BEA T-2
c) BEA+T-2, 72 h
125
75
25
*** **
Concentration (µM)
100
*** 0
Concentration (µM)
***
0
DON
125
*
***
*
75
Concentration (µM) 150
T-2
b) BEA+T-2, 48 h
25
1
c) DON+BEA, 72 h
BEA
Concentration (µM)
***
Concentration (µM)
Viability (%)
0.78
0
*** ***
0.5
125
*** ***
100
75
0.25
100
*** 0
125
0
0
***
T-2
b) DON+T-2, 48 h
25
25
*** ***
DON
Viability (%)
*** **
Viability (%)
Viability (%)
* ***
50
150
25
100
100
*** ***
***
50
Concentration (µM)
b) DON+BEA, 48 h
75
75
0
0
Concentration (µM)
125
***
*** *** ***
100
* ** **
** **
**
0
0.25
0.5
1
2
4
8
BEA
0
0.001
0.003
0.006
0.01
0.025
0.05
DON T-2
**
50 25
0
***
75
**
DON
Concentration (µM)
100
***
** ** ***
***
M.-J. Ruiz et al. / Food and Chemical Toxicology 49 (2011) 2718–2724
0
*** ***
25
0
a) BEA+T-2, 24 h
125
100
Viability (%)
Viability (%)
150
Viability (%)
a) DON+BEA, 24 h
Viability (%)
150
**
0
0.78
1.56
3.125
6.25
12.5
25
0
0.001
0.003
0.006
0.01
0.025
0.05
BEA T-2
Concentration (µM)
Fig. 1. Dose–response curves for the individual and all pair mixtures of BEA (. . .s. . .), DON (h), T-2 toxin ( ), and binary combinations (——) in Vero cells. The kidney cells were exposed to indicate concentrations of mycotoxins for 24, 48 and 72 h and then the viability was determined by the NR assay. p 6 0.05 (), p 6 0.001 () and p 6 0.000 () represent significant difference as compared to control values.
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M.-J. Ruiz et al. / Food and Chemical Toxicology 49 (2011) 2718–2724
a) 150
*
Viability (%)
125 100 75
***
*** ***
***
50
***
*** ***
25 0
***
***
*** T-2
0
0.001
0.003
0.006
0.01
0.025
0.05
0
0.25
0.5
1
2
4
8
DON
0
0.78
1.56
3.125
6.25
12.5
25
BEA
Concentration (µM)
b) 150 Viability (%)
125 100
*
75
***
***
***
50
***
***
25
***
***
***
0
***
*** T-2
0
0.001
0.003
0.006
0.01
0.025
0.05
0
0.25
0.5
1
2
4
8
DON
0
0.78
1.56
3.125
6.25
12.5
25
BEA
*** ***
***
Concentration (µM)
c) 150 Viability (%)
125 100 75
*** ***
50
***
25
***
***
0
***
*** T-2
0
0.001
0.003
0.006
0.01
0.025
0.05
0
0.25
0.5
1
2
4
8
DON
0
0.78
1.56
3.125
6.25
12.5
25
BEA
Concentration (µM) Fig. 2. Dose–response curves for the individual and tertiary mixture of BEA (. . .s. . .), DON (h), T-2 toxin ( ), and combination (——) in Vero cells. The kidney cells were exposed to indicate concentrations of mycotoxins for (a) 24, (b) 48 and ⁄c) 72 h and then the viability was determined by the NR assay. p 6 0.05 (), p 6 0.001 () and p 6 0.000 () represent significant difference as compared to control values.
administration of NIV and DON on J774A.1 macrophages did not lead to a synergic cytotoxic effect indicating that their simultaneous presence did not enhance each own singular cytotoxicity. Other authors report antagonism for low concentrations and synergism for high concentration for mixture of DON and zearalenone (ZEA; Kouadio et al., 2007). However, Groten et al. (1998) and Tajima et al. (2002) showed that combined exposure to ZEA and DON or NIV or T-2 toxin results in an additive effect. On the other hand, acute combinative toxicity for T-2 toxin and aflatoxin B1 (AFB1) in HepG2 and BEAS-2B cells was carried out by McKean et al. (2006) showing different response in the interaction type. Whereas, the combination on T-2 toxin and AFB1 resulted in
synergistic interaction in BEAS-2B cells, this combination could be described as additive in HepG2 cells. BEA is an emerging Fusarium mycotoxin, which has led to recent interest in developing toxicity studies over the last few years. Combined effects of BEA, ochratoxin A (OTA) and fumonisin B1 (FB1) on porcine kidney epithelial (PK15) cells survival has been carried (Klaric et al., 2006, 2008). The binary and tertiary combinations versus single- or two toxin exposures of PK15 cells resulted mostly in additive effects. Antagonism was recorded at the highest concentrations tested. In conclusion, T-2 toxin was 100-fold more toxic than DON and BEA. All mycotoxin combinations showed antagonistic interaction, but the higher results were obtained for the binary DON plus T-2
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Fig. 3. Typical combination index (CI)/fractional affected (fa) curve as described by Chou and Talalay model for Vero cell. Each point represents the CI at a dose effect as determined in our experiments. The dotted line indicates additivity, the area under the dotted line synergy, and the area above of the dotted line antagonism. Vero cells were exposed with (a) BEA + DON (molar ratio of 3:1), (b) BEA + T-2 toxin (molar ratio of 1250:1), (c) DON + T-2 (molar ratio of 160:1), and (d) (c) BEA + DON + T-2 (molar ratio of 1250:400:1). Color version of figure is available on line: 24 h of exposure (+), 48 h of exposure ( ), 72 h of exposure ().
Table 1 Dose–effect relationship parameters and mean combination index (CI) values (as a function of fractional inhibition of proliferation) for the different mycotoxin combinations. Mycotoxin
BEA + DON
BEA + T2
DON + T2
BEA + DON + T2
Time (h)
24 48 72 24 48 72 24 48 72 24 48 72
Dose–effect parameters
CI values
Dm (lM)
m
r
EC50
4.98 2.89 3.21 7.56 3.18 4.77 4.14 3.04 3.19 2.75 3.58 3.28
1.58 1.52 1.43 1.18 2.39 2.19 1.24 1.58 1.74 1.46 2.88 2.93
0.9082 0.9837 0.9586 0.9851 0.9413 0.9507 0.9900 0.8990 0.9866 0.9011 0.9156 0.9375
1.73 1.22 1.78 3.20 1.81 3.41 5.03 6.82 7.62 1.32 2.38 2.68
EC75 Ant Ant Ant Ant Ant Ant Ant Ant Ant Ant Ant Ant
2.18 1.50 2.18 4.09 1.59 2.96 3.61 6.45 7.05 1.45 1.92 2.10
EC90 Ant Ant Ant Ant Ant Ant Ant Ant Ant Ant Ant Ant
2.74 1.86 2.69 5.89 1.43 2.59 3.13 6.11 6.61 1.77 2.57 1.66
Ant Ant Ant Ant Ant Ant Ant Ant Ant Ant Ant Ant
The parameters m, Dm and r are the antilog of x-intercept, the slope and the linear correlation coefficient of the median-effect plot, which signifies the shape of the dose–effect curve, the potency (EC50), and conformity of the data to the mass–action law, respectively (Chou and Talalay, 1984; Chou, 2006). Dm and m values are used for calculating the CI values (CI < 1, =1, and >1 indicate synergism (Syn), additive effect (Add), and antagonism (Ant), respectively. EC50, EC75 and EC90, are the doses required to inhibit proliferation 50, 75 and 90%, respectively. The ratio of BEA + DON: 3:1; BEA + T-2:1250:1; DON + T-2:160:1; and BEA + DON + T-2, ratio = 1250:400:1 was used for the experimental design. Computer software CalcuSyn was used for automated calculation and simulation.
toxin combination, although the mechanisms of interaction remain unknown. On the other hand, there is a clear discrepancy with results obtained in literature, so additivity, and synergism but also antagonism has been observed for T-2 toxin, DON and BEA combinations in vitro. Although there could be indications for dose additivity in some of the in vitro studies, at present the database describing possible effects of combined exposure of Fusarium mycotoxins is very weak and not sufficient for establishing either the nature of combined effects or the relative potencies of the Fusarium mycotoxins. Moreover, there are large differences depending on combination mycotoxins, assay carried out, type of
cell used, and other factors. However, in term of risk assessment in food and diet, mycotoxins are commonly found on cereals grown in temperate areas, and the presence of several of them could increase the potential toxicity of each of them separately. So, toxic effects of combinative mycotoxins should be determined or revalidated in the light of continuously toxicological data obtained by scientific groups. Conflict of interest The authors declare that there are no conflicts of interest.
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