Fusariotoxins are frequently existing contaminants in cereal and other plant products. High relationship was

found between the levels of the trichothecene T-2 and HT-2 toxin detected in contaminated commodities. Many in vitro

studies investigated the cytotoxicity of T-2 and HT-2, but the interaction between them has not been thoroughly studied

yet. The aim of the study was to examine the dose dependent cytotoxic effect of T-2 and HT-2 toxins, single and in combination,

using pig lymphocytes in the methyl thiazol tetrazolium (MTT ) assay. The mycotoxins were added at various

concentrations, i.e. 0.5, 0.1, 0.05, 0.01 and 0.001 μM of T-2; 1.0, 0.5, 0.2., 0.1 and 0.05 μM of HT-2; and both mycotoxins

combined. Two exposure times (6 and 24 h) were tested. Both T-2 and HT-2 toxins exerted a dose dependent effect.

After 6 h incubation, the increase in concentration of T-2 from 0.001 to 0.5 μM and HT-2 for 0.05 to 1.0 μM resulted

in lower cell viability by 22 and 17%, respectively. After 24 h cell viability was significantly lower compared to values

obtained at 6 h, except 0.5 μM T-2 and 0.05, 1.0 μM HT-2, respectively. Measured cell viability for combinations of T-2

and HT-2 was higher compared to the calculated expected values. The two toxins caused lower cell survival when applied

together than in single administration after 6 h incubation. After 24 h incubation this tendency was not consistent

Bodnár, Zsófia

Dutton, Michael F.

Horvatovich, Katalin

Kametler, László

Kovács, Melinda

Rajli, Veronika

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20th Int. Symp. “Animal Science Days”, Kranjska gora, Slovenia, Sept. 19th−21st, 2012.Acta argiculturae Slovenica, Supplement 3, 253–257, Ljubljana 2012COBISS: 1.08Agris category code: H01 PreLImInAry InveStIgAtIOnS On tHe SIngLe AnD COmBIneD CytOtOxIC effeCt Of t-2 AnD Ht-2 tOxIn meASureD By metHyL tHIAzOL tetrAzOLIum (mtt) CytOtOxICIty teSt uSIng PIg LymPHOCyteSveronika rAJLI 1, zsófia BODnÁr 1, Katalin HOrvAtOvICH 1, László KAmetLer 1, michael f. DuttOn 2, melinda KOvÁCS 1, 3 1 Kaposvár univ., fac. of Animal Science, guba S. 40, 7400 Kaposvár, Hungary, e-mail: kovacs.melinda@ke.hu2 fac. of Health Sciences, Doornfontein Campus, univ. of Johannesburg, 2028 Doornfontein, gauteng, South Africa3 research group of mycotoxins in the food Chain of the Hungarian Academy of Sciences and Kaposvár univ., guba S. 40, 7400 Kaposvár, HungaryAbStrActfusariotoxins are frequently existing contaminants in cereal and other plant products. High relationship was found between the levels of the trichothecene t-2 and Ht-2 toxin detected in contaminated commodities. many in vitro studies investigated the cytotoxicity of t-2 and Ht-2, but the interaction between them has not been thoroughly studied yet. The aim of the study was to examine the dose dependent cytotoxic effect of t-2 and Ht-2 toxins, single and in com-bination, using pig lymphocytes in the methyl thiazol tetrazolium (mtt) assay. The mycotoxins were added at various concentrations, i.e. 0.5, 0.1, 0.05, 0.01 and 0.001 µm of t-2; 1.0, 0.5, 0.2., 0.1 and 0.05 µm of Ht-2; and both mycotoxins combined. two exposure times (6 and 24 h) were tested. Both t-2 and Ht-2 toxins exerted a dose dependent effect. After 6 h incubation, the increase in concentration of t-2 from 0.001 to 0.5 µm and Ht-2 for 0.05 to 1.0 µm resulted in lower cell viability by 22 and 17%, respectively. After 24 h cell viability was significantly lower compared to values obtained at 6 h, except 0.5 µm t-2 and 0.05, 1.0 µm Ht-2, respectively. measured cell viability for combinations of t-2 and Ht-2 was higher compared to the calculated expected values. The two toxins caused lower cell survival when ap-plied together than in single administration after 6 h incubation. After 24 h incubation this tendency was not consistent. Key words: mycotoxins / cytotoxicity / pig lymphocytes / methyl thiazol tetrazolium (mtt) assay1 IntroductIonIn vitro  methyl Thiazol tetrazolium (mtt) cell culture assay is one of the most frequently used tests for preliminary screening of cytotoxicity of Fusarium  tox-ins. It is a rapid, versatile, quantitative, and highly repro-ducible colorimetric assay for mammalian cell viability/metabolic activity; it is useful on a large scale mycotoxins screening assay (Dombrink-Kurtzman et al., 1994). Al-though a variety of mammalian cell cultures were used to detect Fusarium mycotoxins in contaminated extracts none of them showed high sensitivity to all of the Fusar-ium mycotoxins tested (Cetin and Bullerman, 2005). The use of human lymphocytes has been described in myco-toxin cytotoxicity test by maenetje et al. (2008). In the past bird, mouse, rat and cattle lymphocytes have already been tested, but they have not been proven to be ideal for routine cytotoxicity testing (Dombrink-Kurtzman et al., 1994; Lioi et al., 2004). Pigs are better test animals, may better correlate with human physiology and are more sensitive to most mycotoxins than other domesticated animals. Our preliminary results obtained in a study on the combined effect of fB1 and ochratoxin A have shown the feasibility of using isolated pig lymphocytes as test cells in the mtt assay (mwanza et al., 2009). mycotoxins have significant economic, scientific and public health significance all over the world. They are biologically ac-tive secondary fungal metabolites found as contaminants of food- and feedstuff. Fusaria are moulds predominant-ly producing two types of mycotoxins: the non-oestro-genic trichothecene and the myco-oestrogens including zearalenone and its zearalenol metabolites. These fusa-Acta agriculturae Slovenica, Supplement 3 – 2012254v. rAJLI et al.riotoxins are frequently existing contaminants in cereal and other plant products (Scott, 1990). The t-2 and Ht-2 toxin belong to a group of trichothecenes (De nus et al., 1996). t-2 toxin is the most acute toxic compound among trichothecenes: it inhibits protein, DnA and rnA synthesis, alters cellular membrane functions, stimulates lipid peroxidation, it is cytotoxic and immunotoxic and induces apoptosis (SCf, 2001). many in vitro studies in-vestigated the cytotoxicity of t-2 and Ht-2 (as reported by van der fels-Klerx and Stratakou, 2010), but the inter-action between t-2 and Ht-2 has not thoroughly studied yet. There are currently no eC limits for t-2 and Ht-2, and there is a lack of a recent overview of the occurrence of these two toxins. In general, a high relationship (ratios between Ht-2 and t-2 varied from 1.75 to 7) was found between the levels of the two toxins detected in contami-nated commodities (van der fels-Klerx and Stratakou, 2010). The aim of the present study was to examine the dose dependent cytotoxic effect of t-2 and Ht-2 toxins, single and in combination, using pig lymphocytes.2 MAterIAlS And MethodSAll chemicals used in this study, including t-2 and Ht-2 mycotoxin standards, were obtained from Sigma-Aldrich (Hungary).The research protocol was reviewed by the Animal use and Care Administrative Advisory Committee and approved by the Agricultural Administrative Authority (Protocol no.23.1/02322/007/2008). venous blood from pigs of landrace-large white f1 genotype (of 4 month age and 24 kg body weight) was taken from the v. cava cra-nialis. The blood was mixed with an equal volume of tis-sue culture medium consisting of rPmI-1640, then over-laid on Histopaque 1077 and centrifuged as described in mwanza et al. (2009). The interface layer consisting of mononuclear cells (referred to here as lymphocytes) were washed with 9 ml rPmI-1640 at room tempera-ture and centrifuged at 700 g for 15 min, the supernatant was poured, and were washed with 12 ml rPmI-1640 at room temperature and centrifuged at 700 g for 5 min. The pelleted cells were resuspended in 10 ml of complete culture media (CCm), placed in a 96-well culture plates and were cultured at 37 °C in 5% CO2 humidified incu-bator for 24h. The CCm was made from 1 ml foetal calf serum (fCS), 100 µl 100u/ml penicillin and 100 µg/ml streptomycin (PenStrep), 150 µl glutamine and made up to a total of 10 ml with rPmI-1640. The paleness of the CCm during the incubation period confirms the growth of cells.Cell enumeration was carried out by blue dye exclu-sion method as described by mwanza et al. (2009). Cell count was carried out and % viability was determined as: % viability = (viable cell counted (dye excluded) / total no. of cells) * 100The cell cultures were subdivided and placed in a 96-well culture plates containing 100 µl/well of culture medium. Phytohaemagglutinin (PHA) was used as a cell stimulant (20 µl/well). The mycotoxins were added at various concentrations (0.5, 0.1, 0.05, 0.01 and 0.001 µm of t-2; 1.0, 0.5, 0.2, 0.1 and 0.05 µm of Ht-2) and both mycotoxins combined (table 1), dissolved in ethanol and distilled water. The solvents were also used as controls. An amount of cell culture medium (rPm-1640) was added to each well to make a total of 400 µl mixture per well. The content was then incubated for 6 and 24 hours and cytotoxicity determined after the incubation time by adding 30 µl mtt solution (mwanza et al., 2009) with mixing. The culture plates were then incubated for 2 h (at 37 °C in 5% CO2) after which 50 µl dimethylsulphoxide (DmSO) was added to each well and left for further 20 min to solubilise the formazan crystals formed. The opti-cal density (OD) was then read using a micro plate reader at 540 and 620 nm. Cell proliferation as influenced by mycotoxin exposure was determined as percentage vi-ability of the control: % viability = (ODm/ODn) * 100; where ODm is the OD value of the mycotoxin-treated cells; ODn is the OD value of the control (no mycotoxin) cells.treatment, concentration and time effect was meas-ured in six replicates.The cell viability results were analysed by using the multiway AnOvA procedure of the SPSS 19 software (SPSS Inc., Chicago, IL, uSA). The significance of dif-treatmentsConcentration categories / toxin concentration (µm)1 2 3 4 5t-2 0.5 0.1 0.05 0.01 0.001Ht-2 1.0 0.5 0.2 0.1 0.05C 0.5 t-2 + 1.0 Ht-2 0.1 t-2 + 0.5 Ht-2 0.05 t-2 + 0.2 Ht-2 0.01 t-2 + 0.1 Ht-2 0.001 t-2 + 0.05 Ht-2Table 1: Toxin treatments used in the MTT assayActa agriculturae Slovenica, Supplement 3 – 2012 255PreLImInAry InveStIgAtIOnS On tHe SIngLe AnD COmBIneD CytOtOxIC effeCt Of t-2 ... uSIng PIg LymPHOCyteSferences was tested by tukey and LSD post hoc tests, the level of signifi cance was set at P < 0.05. measured values of combined exposure were compared with the expected values, and the signifi cance of diff erence between the two values was calculated as described by Šegvić Klarić et al. (2012).3 reSultS And dIScuSSIonIn the experiment 3 toxin treatments, 5 concentra-tions of each toxin single and in combination, and 2 ex-posure times were tested, their eff ect on cell viability us-ing pig lymphocytes in mtt assay was detemined. Th e selection of concentrations was based on data available in the literature concerning cytotoxicity of t-2 and Ht-2. In a study of Königs et al. (2009) on two hu-man cell lines in primary culture t-2 toxin showed the strongest cytotoxic eff ect (IC50 values of 0.2 and 0.5 μm) while the metabolites, Ht-2 toxin and neosolaniol, re-vealed weaker cytotoxicity (IC50 values of 0.7–3.0 μm). Similar results were reported by nielsen et al. (2009), ac-cording to which IC50 values were 4.4–10.8 nmol/l for t-2, while 7.5–55.8 nmol/l for Ht-2 toxin. In this study all controls were negative, i.e., their cell viability was taken as 100%.Both t-2 and Ht-2 toxins exerted a dose dependent eff ect (fig. 1). Aft er 6 h incubation, the increase in con-centration of t-2 from 0.001 to 0.5 µm and Ht-2 for 0.05 to 1.0 µm resulted in lower cell viability by 22 and 17%, respectively. Aft er 24 h cell viability was signifi cantly lower compared to values obtained at 6 h, except 0.5 µm t-2 and 0.05, 1.0 µm Ht-2, respectively. Aft er 24 h incubation the increase in t-2 from 0.001 to 0.05 caused 20% decrease in cell viability, thereaft er increasing toxin concentration did not result in higher cell death. In case of Ht-2 0.1 µm was the highest dose causing signifi cant decrease (by 22%) in cell survival.Signifi cant (P < 0.001) interaction was found be-tween concentration × treatment, and concentration × time, while the time × treatment interaction was not sta-tistically proven (P = 0.084). to estimate the interaction of the two toxins expect-ed values were calculated by Šegvić Klarić et al. (2012) and compared to the measured values (fig. 2). Higher cell viability was measured for all combinations of t-2 and Ht-2 except the lowest concentrations applied for 6 h, where the diff erence between the measured and calcu-lated values was not signifi cant. When comparing the single and combined eff ect of the toxins, taking the same concentration categories and incubation time into consideration (fig. 3), the two tox-ins caused consistently lower cell survival when applied together than in single administration aft er 6 h incuba-tion. Aft er 24 h incubation this tendency was not expe-rienced, at concentration 2 the combined application, while at concentration 4 and 5 the single administration of t-2 and Ht-2, respectively, resulted in higher cell sur-vival, i.e. was less cytotoxic. t-2 is rapidly absorbed from the gut, and it is dis-tributed in the organism with little or no accumulation in any specifi c organs. Th ere is little direct information available on the toxicity of Ht-2 toxin alone, according to most data available, t-2 and Ht-2 toxin induce toxic eff ects with similar potency (Joint fAO/WHO …, 2001). many studies determined a lower cytotoxicity of t-2 me-tabolites (Ht-2, t-2 triol and t-2 tetraol) assuming a de-toxifi cation reaction of the organism (Babich and Boren-freund, 1991). As t-2 is rapidly metabolised to Ht-2 in vivo, the harmful eff ects of t-2 might partly be attributed to Ht-2. In our study t-2 and Ht-2 at the concentrations used, decreased cell viability nearly to the same extend (from 80 to 60%) no signifi cant diff erence between val-ues of t-2 and Ht-2 were obtained within the same con-Figure 1: Cell viability of pig lymphocytes aft er 6 and 24 h treat-ment of T-2 (0.001, 0.01, 0.05, 0.1 and 0.5 µM) and HT-2 (0.05, 0.1, 0.2, 0.5 and 1.0 µM) toxin (n = 6; *signifi cant (P < 0.05) diﬀ er-ence between values of 6 and 24 h exposure; a,b,c,d signifi cant (P < 0.05) diﬀ erence between concentrations)Acta agriculturae Slovenica, Supplement 3 – 2012256v. rAJLI et al.Figure 2: Cell viability (mean ± SE) of pig lymphocytes aft er 6 and 24 h of combined treatment with T-2 and HT-2 (grey bars represent the measured values and white bars the expected values. n = 6, *signifi cant (P < 0.05) diﬀ erence between measured and expected values)centration ranges. As Ht-2 was added always in higher concentration then t-2, our data supported the lower toxicity of Ht-2 compared to t-2 reported previously (Babich and Borenfreund, 1991; nielsen et al., 2009) while are contrary to those of Königs et al. (2009) who found similar toxicity of t-2 and Ht-2 on human renal tubule epithelial and human lung fi broblast cells.further investigations are needed to determine the IC50 for t-2 and Ht-2 for pig’s lymphocytes, to clarify the type of interaction between the two toxins in lower concentrations, and also to determine if pig lymphocytes do metabolise t-2 to Ht-2, as was demonstrated in the case of some cell types (Königs et al., 2009). 4 concluSIonSt-2 and Ht-2 toxins had time and dose dependent cytotoxic eff ect in pigs’ lymphocytes measured by mtt assay, Ht-2 showing similar cytotoxicity to t-2 but in 2–50-fold higher concentrations. Th e two toxins caused consistently lower cell survival when applied together than in single administration aft er 6 h incubation, but for the synergistic eff ect additional investigations are needed, because the diff erences in cell viability are not consistent. 5 AcKnoWledGeMentresearch was funded by the Hungarian Scientifi c research fund (project no.: 100810), the Hungarian Academy of Sciences (within the frame of the mtA-Ke mycotoxins in the food Chain research group) and the tÁmOP 4.2.1.B-10/2/KOnv-2010-0002 project. We are grateful to András Szabó for his help in the statistical analyses. 6 reFerenceSBabich H., Borenfreund e. 1991. Cytotoxicity of t-2 toxin and its metabolites determined with the neutral red cell viabil-ity assay. Applied and environmental microbiology, 57: 2101–2103Cetin y., Bullerman L.B. 2005. Cytotoxicity of Fusarium my-cotoxins to mammalian cell cultures as determined by the mtt bioassay. food and Chemical toxicology, 43: 755–764De nus m., rombouts f., notermans S. 1996. Fusarium molds and their mycotoxins. Journal of food Safety, 16: 15–58Dombrink-Kurtzman m.A., Bennett g.A., richard J.L. 1994. An optimized mtt bioassay for determination of cyto-toxicity of fumonisins in turkey lymphocytes. Journal of AOAC International, 77, 2: 512–516Joint fAO/WHO expert Committee on food Additives (JeC-Acta agriculturae Slovenica, Supplement 3 – 2012 257PreLImInAry InveStIgAtIOnS On tHe SIngLe AnD COmBIneD CytOtOxIC effeCt Of t-2 ... uSIng PIg LymPHOCyteSfA) 2001. Safety evaluation of certain mycotoxins in food. WHO food Additives Series, no. 47/fAO food and nu-trition Paper 74. http://www.inchem.org/documents/jecfa/jecmono/v74je01.htmKönigs m., mulac D., Schwerdt g., gekleb m., Humpf H.u. 2009. metabolism and cytotoxic eff ects of t-2 toxin and its metabolites on human cells in primary culture. toxicology, 258: 106–115Lioi m.B., Santoro A., Barbieri r., Salzano S., ursini m.v. 2004. Ochratoxin A and zearalenone: a comparative study on genotoxic eff ects and cell death induced in bovine lympho-cytes. mutation research, 557: 19–27maenetje P.W., de villiers n., Dutton m.f. 2008. Th e use of iso-lated human lymphocytes in mycotoxin cytotoxicity testing. International Journal of molecular Sciences, 9: 1515–1526mwanza m., Kametler L., Bonai A., rajli v., Kovacs m., Dut-ton m.f. 2009. Th e cytotoxic eff ects of fumonisin B1  and ochratoxin A on human and pig lymphocytes using methyl thiazol tetrazolium (mtt) assay. mycotoxin research, 25, 4: 233–238nielsen C., Casteel m., Dider A., Dietrich r., märtlbauer e. 2009. trichothecene-induced cytotoxicity on human cell lines. mycotoxin research, 25: 77–84Šegvić Klarić, m., zeljezic, D., rumora, L., Peraica, m., Pepeljnjek, S., Domijan, A.m. 2012. A potential role of calcium in apoptosis and aberrant chromatin forms in porcine kidney PK15 cells induced by individual and combined ochratoxin A and citrinin. Archives of toxicology, 86, 1: 97–107 SCf – Scientifi c Committee on food 2001. Opinion of the Scientifi c Committee on food on fusarium toxins. Part 5: t-2 toxin and Ht-2 toxin. european Commission, Brussels, Belgium.     http://ec.europa.eu/food/fs/sc/scf/out88_en.pdfScott P.m. 1990. trichothecenes in grains. Cereal foods World, 35: 661–666van der fels-Klerx H.J., Stratakou I. 2010. t-2 toxin and Ht-toxin in grain and grain-based commodities in europe: occurrence, factors aff ecting occurrence, co-occurrence and toxicological eff ects. World mycotoxin Journal, 3, 4: 349–367Figure 3: Comparison of single and combined eﬀ ect of T-2 and HT-2 toxins (1 fi ve decreasing toxin concentrations were applied both single and in combination (see Materials and methods); C = combined application of T-2 and HT-2; a,b signifi cant diﬀ erence between treatments (T-2, HT-2 and C) with the same concentration category (from 1 to 5) and incubation time (P < 0.05)

Preliminary investigations on the single and combined cytotoxic effect of T-2 and HT-2 measured by Methyl Thiazol Tetrazolium (MTT) cytotoxicity test using pig lymphocytes

Preliminary investigations on the single and combined cytotoxic effect of T-2 and HT-2 measured by Methyl Thiazol Tetrazolium (MTT) cytotoxicity test using pig lymphocytes

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