Food and Chemical Toxicology 68 (2014) 44–52

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Food and Chemical Toxicology

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In vivo effects of deoxynivalenol (DON) on innate immune responsesof carp (Cyprinus carpio L.)

http://dx.doi.org/10.1016/j.fct.2014.03.0120278-6915/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +41 61 267 0405; fax: +41 61 267 0409.E-mail address: constanze.pietsch@unibas.ch (C. Pietsch).

Constanze Pietsch a,⇑, Christian Michel a, Susanne Kersten b, Hana Valenta b, Sven Dänicke b,Carsten Schulz c, Werner Kloas d, Patricia Burkhardt-Holm a

a University Basel, Man–Society–Environment, Department of Environmental Sciences, Vesalgasse 1, CH-4051 Basel, Switzerlandb Friedrich-Loeffler-Institute (FLI), Federal Research Institute for Animal Health, Institute of Animal Nutrition, Bundesallee 50, 38116 Braunschweig, Germanyc Gesellschaft für Marine Aquakultur (GMA) mbH, Hafentörn 3, D-25761 Büsum, Germanyd Department of Ecophysiology and Aquaculture, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Mueggelseedamm 310, Berlin, Germany

a r t i c l e i n f o a b s t r a c t

Article history:Received 17 September 2013Accepted 7 March 2014Available online 17 March 2014

Keywords:MycotoxinHaematologyImmune systemAquaculture

Deoxynivalenol (DON) is one of the most important members of Fusarium toxins since it often can befound in relevant concentrations in animal feeds. The effects of this group of toxins on fish are mostlyunknown. The present study shows results from a feeding trial with carp (Cyprinus carpio L.) using threedifferent concentrations of DON (352 lg kg�1, 619 lg kg�1, and 953 lg kg�1 final feed, respectively)which are comparable to levels found in commercial fish feeds. Effects on growth and mass of fish werenot observed during this 6 weeks lasting experiment. Only marginal DON concentrations were found inmuscle and plasma samples. Blood parameters were not influenced although smaller erythrocytesoccurred in fish treated with 352 lg kg�1 DON. Analysis of antioxidative enzymes in erythrocytes showedincreased superoxid dismutase and catalase activities in fish fed the low-dose feed. Immunosuppressiveeffects of DON were confirmed whereby cytotoxic effects on immune cells only partly explained theimpairment of innate immune responses. Exact polarization of the immune system into pro-inflamma-tory or anti-inflammatory responses due to DON exposure should be clarified in further experiments,especially since the current results raise concern about impaired immune function in fish raised inaquaculture.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The occurrence of mycotoxins in feeds for farm animals andtheir numerous impacts on animal health is an issue that isincreasingly addressed (D’Mello et al., 1999; Döll and Dänicke,2011). At present, a general recommendation on guidance valuesof 5 mg kg�1 DON in complete feedingstuff was established bythe European Commission (2006/576/EC). Contamination of feedingredients can already occur on the native cereals growing onthe fields. This contamination is often due to growth of fungibelonging to the genus Fusarium (Yazar and Omurtag, 2008;Foroud and Eudes, 2009) which are responsible for the productionof deoxynivalenol (DON). DON is relatively stable even during foodprocessing steps (Neira et al., 1997; Sugita-Konishi et al., 2006).Cereals are increasingly used for production of fish feeds, sincethe steadily growing aquaculture sector utilizes increasingamounts of feed sources and limited fish meal supply cannot fulfil

this demand (FAO, 2012). Thus, cereals replace fish meal in fishfeed whereby cyprinids accept up to 70% cereals as feed ingredi-ents and salmonids tolerate less than 30% cereal compounds (Matz,1991; Berntssen et al., 2010). However, this leads to the introduc-tion of cereal-borne mycotoxins into fish feeds (Pietsch et al., 2013)with up to now mostly unknown consequences. Contamination offeed affects farm animal metabolism and health. For example,exposure to DON often resulted in changes of nutritional statusin farm animals (Döll et al., 2009; Ferrari et al., 2009). Especiallyswine proved to be highly sensitive to DON-contaminated feedand occasionally refused feed (Eriksen and Pettersson, 2004;Dänicke et al., 2006; Gutzwiller, 2010). Immunotoxic effects ofDON have already been found in higher vertebrates (Pestka andBondy, 1990; Pestka, 2008; Awad et al., 2013).

Recent investigations on salmonids showed that histo-patho-logical changes and lesions in the liver and intestinal tract of fishoccur upon feeding with DON-contaminated diets (Döll et al.,2011; Hooft et al., 2011). In addition, it has been assumed thatthe increased usage of plant materials as protein sources in feedsfor aquaculture leading to mycotoxin contamination might be

C. Pietsch et al. / Food and Chemical Toxicology 68 (2014) 44–52 45

related to feed-induced negative effects on growth and immuneparameter and the occurrence of intestinal cancer in salmonid fish(Sagstad et al., 2007; Dale et al., 2009; Sissener et al., 2011). How-ever, direct effects of DON as one of the most relevant Fusariumtoxins on the immune system of fish have so far not been investi-gated. Impaired immune functions of fish due to mycotoxin expo-sure would implicate a possible contribution to disease problemsand may represent an urgent threat to fish in aquaculture. Thepresent study, therefore, aimed to causally determine the effectsof experimentally DON-contaminated diets on blood parametersand innate immune responses of carp (Cyprinus carpio) since thisfish species is important for aquaculture (FAO, 2012).

2. Materials and methods

2.1. Chemicals

All chemicals were obtained from Sigma (Buchs, Switzerland) unless indicatedotherwise.

2.2. Preparation of feeds

Experimental diets were designed without cereals in order to excludecereal-based Fusarium toxin contaminations. Therefore, fish meal, blood meal,casein, dextrose, and potato starch were used for feed preparation (at 30%, 12.5%,12.0%, 13.0%, and 21.1%, respectively). Vitamins and minerals were added to dietsto meet the dietary requirements of carp (NRC, 1993). All ingredients were mixedthoroughly. Deoxynivalenol (DON, dissolved in ethanol; purity >98%, lot-no.011M4065V) was added to the fish oil (accounting for 12% of the final feed) at threedifferent concentrations (low dose: 352 lg kg�1, medium dose: 619 lg kg�1 andhigh dose: 954 lg kg�1 final feed, respectively) prior to addition to the otheringredients. Diets were manufactured to 4 mm pellets in a pelletizer (L 14–175,Amandus Kahl, Reinbek, Germany). The diets were formulated to be isonitrogenous(41.36 ± 0.54% crude protein, mean ± SD) and isocaloric (22.41 ± 0.11 MJ kg�1 drymatter, mean ± SD). Pellets were allowed to cool down to room temperature fortwo hours before storage at 4 �C until use.

2.3. Exposure of fish

Carp were raised from eggs in our facilities and used for the experiments at12–16 cm in total length. Fish were kept at a 16 h light/8 h dark photoperiod at25 ± 0.2 �C (mean ± SD) in a flow through system. Rearing of fish and experimentalprocedures has been approved by the Cantonal veterinarian authorities of Basel-Stadt (Switzerland) under the permission number 2410. Fish were acclimated for3 weeks to the experimental tanks where all animals received the uncontaminatedexperimental diet at daily feed administration of two per cent of body mass. Each ofthe four different feeding groups (control, low dose, medium dose, and high dose)included four tanks (54 L) containing 6 fish each. Fish were fed the contaminateddiets for four weeks while a control group received the uncontaminated feed.Thereafter, two tanks per feeding group were sampled. All remaining tanks perfeeding group were fed the uncontaminated diet for additional two weeks beforesampling to investigate possible recovery from DON feeding. During the experi-ments the flow through was adjusted to 6 L conditioned fresh water per h for eachof the tanks. Water temperature, pH (WTW pH 315i, Wissenschaftlich-TechnischeWerkstätten GmbH, Weilheim, Germany), conductivity (WTW cond 315i, Wissens-chaftlich-Technische Werkstätten GmbH, Weilheim, Germany) and dissolvedoxygen (WTW Oxi 330i, Wissenschaftlich-Technische Werkstätten GmbH,Weilheim, Germany) were recorded for each tank at least five times a week. Everytank was cleaned at least every second day including removal of faeces and scrapingof the inside walls of the aquaria.

2.4. Analysis of DON in experimental diets

DON and its metabolite de-epoxy-DON (DOM-1) in experimental feed wereanalyzed by HPLC-DAD (high performance liquid chromatography (consisting of apump (LC-10ADVP), an autoinjector (SIL-10ADVP), and a column oven (CTO-10ACVP) from Shimadzu (Duisburg, Germany) with diode array detection usingthe detector SPD-M10AVP (Shimadzu, Duisburg, Germany). To evaluate the leach-ing of DON into water 25 g of the medium dose feed were exposed to 10 L aquariumwater for 0, 0.5, 1, 2, 4, 8, 12 h, and 24 h in duplicates. Thereafter, the remaining feedsamples were collected from the aquaria, dried at 55 �C for 48 h, and stored at�20 �C until analyses. All samples were cleaned-up with IAC (immuno-affinitycolumns, DONprep™, R-Biopharm, Darmstadt, Germany) prior to analyses accord-ing to manufacturer’s procedure with slight modifications as described previously(Oldenburg et al., 2007). The detection limit was 30 lg kg�1, the mean recoverywas approximately 90%.

2.5. Preparation of blood smears, differential blood cell counts, haematocrit andhaemoglobin determination

From each fish, blood was drawn from the caudal vein using heparinizedsyringes immediately after removal from each tank. Haematocrit was measuredin heparinized glass capillary tubes (Huber & Co. AG, Reinach, Switzerland) in dupli-cate after centrifugation at 3000 rpm for 10 min (Haematokrit Typ 2010, HettichZentrifugen, Tuttlingen, Germany). Haemoglobin was calculated using the Drabkinmethod (Drabkin and Austin, 1935). Therefore, 5 ll of freshly drawn blood wereadded to Drabkin‘s solution containing 30% Brij� 35 and absorptions were read at540 nm (Tecan Infinite M200). A standard curve was established using humanhaemoglobin (lyophilized powder). Blood smears from each fish were preparedimmediately in duplicate on glass slides. From each slide 10 pictures were takenrandomly at 400�magnification (Nikon Eclipse E400 equipped with a Nikon DigitalCamera DXM1200F). All cells from each picture were counted so that an averagenumber of 7813 cells per fish (mean; maximum = 11,976; minimum = 5684) wereused in total for calculating differential blood cell counts from individual fish.

2.6. Activities of catalase and superoxide dismutase in erythrocytes

Lysates from erythrocytes were prepared by centrifugation of 100 ll freshblood, followed by washing of cells using physiological sodium chloride solutionand addition of 500 ll homogenizing buffer (50 mM phosphate buffer, pH 7.4,containing 150 mM potassium chloride). Samples were sonificated for 10 min(Bandelin Sonorex Typ RK 255 H, Bandelin electronic – GmbH & Co. KG, Berlin,Germany) and centrifuged at 3000g for 20 min at 4 �C (Centrifuge 5415R, Eppendorf,Basel, Switzerland). The supernatant was stored at �80 �C until analyses.

Measurement of catalase (CAT) activity was conducted according to the methoddescribed by Aebi (1974) in 20 ll of lysates after addition of 150 ll sodiumphosphate buffer (0.1 M, pH 6.5) containing 3.4 micromoles hydrogen peroxideper well. Measurements were conducted in UV-Star� 96-well microtitre plates(Greiner Bio-one, HUBERLAB. AG, Aesch, Switzerland) at 240 nm and 20 �C (InfiniteM200, Tecan Group Ltd., Männedorf, Switzerland) by recording a kinetic for 20 min.

For determination of superoxide dismutase (SOD) activity according to thedescription of Oberley and Spitz (1984) and Ukeda et al. (1999), 20 ll of lysateswere placed in 96-well microtitre plates (Rotilabo�, Carl Roth AG, Karlsruhe,Germany). Afterwards, 180 ll of a sodium carbonate solution (50 mM, pH 10.2)containing 1 mM ml�1 diethylenetriaminepentaacetic acid (DTPA), 1 U ml�1

catalase (from bovine liver), 0.177 mM ml�1 xanthine and 0.195 mg ml�1 WST-1(2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,Na-salt;Dojindo Laboratories, Japan) were added. The reaction was started by addition of10 ll of xanthine oxidase (1080 mU ml�1 in 2 M (NH4)2SO4) whereas only ammo-nium sulfate was added to blanks. The plates were incubated at 37 �C for 20 minand optical densities were read at 438 nm (Infinite M200, Tecan Group Ltd., Männe-dorf, Switzerland). Calibration curves were prepared from serial dilutions of a SODstock solution (9600 U ml�1 ammonium sulfate buffer) in phosphate buffered saline.

In parallel, aliquots of the cell homogenates were also used for protein determi-nations using the bicinchoninic acid (BCA) assay (Sigma) according to the manufac-turer‘s protocol.

2.7. Analysis of plasma cortisol

Plasma was prepared by centrifugation at 3000g for 20 min at 4 �C (Centrifuge5415R, Eppendorf, Basel, Switzerland). Thereafter, 100 ll of plasma were mixedwith the same volume of sterile UltraPure Water (Cayman, Chemie BrunschwigAG, Switzerland), and stored at�80 �C until steroid extraction. All samples were ex-tracted twice with 1 ml diethyl ether, and both extracts were combined and storedat �20 �C after evaporation of diethyl ether at room temperature. Prior to analyses,extracts were re-mobilized in 5% ethanol and cortisol contents were determinedusing an ELISA kit (IBL International GmbH, Hamburg, Germany).

2.8. Culture of immune cells and exposure to stimulants

Primary cell cultures from head and trunk kidneys were prepared as describedby Pietsch et al. (2011a). Stimulation of NO production was conducted by additionof 30 lg ml�1 bacterial lipopolysaccharide (LPS from E. coli, serotype O111:B4) towells. After incubation of cells with and without LPS at 25 �C and 5% CO2 for 96 hin the dark NO production was measured using the Griess reagent as describedby Pietsch et al. (2008). Arginase activity was measured after 24 h with and withoutaddition of forskolin as described previously (Pietsch et al., 2011a). All experimentalincubations were run in 3 independent replicates.

2.9. Measurement of cell viability and respiratory burst activity

In parallel, cell viability after exposure to DON was measured by assessing theuptake of neutral red (3-amino-7-dimethylamino-2-methyl-phenanzine hydrochlo-ride, NR) to evaluate membrane integrity and lysosomal function based on themethod described by Borenfreund and Puerner (1985). Therefore, a stock solutionof NR was prepared with 0.05% NR in RPMI medium. Cells were incubated with

46 C. Pietsch et al. / Food and Chemical Toxicology 68 (2014) 44–52

working solution prepared of 45 ll stock solution per ml RPMI medium for 3 h.Afterwards, cells were washed twice with sterile Earle‘s medium and lysed in50 ll ethanol containing 2% acetic acid. Optical densities were read at 540 nm usinga plate reader (Infinite M200, Tecan Instruments).

Respiratory burst activity was analysed with the nitroblue tetrazolium (NBT)assay (Chung and Secombes, 1988). Therefore, leukocytes were cultured at 25 �Cand 5% CO2 for 96 h in the dark and incubated for 1 h with 1 mg NBT salt ml�1 cul-ture medium with and without 0.24 lg ml�1 phorbol myristate acetate (PMA) as astimulant of production of reactive oxygen species by leukocytes. Subsequently, thesupernatant was discarded and the cells were fixed using 70% methanol. Driedplates were incubated with 100 ll dimethyl sulfoxide (DMSO) and 100 ll potas-sium hydroxide (KOH) to solubilize the formazan. The absorbance at 620 nm wasmeasured spectrophotometrically in duplicates with a microplate reader (InfiniteM200, Tecan Instruments) using DMSO/KOH alone as blank.

2.10. Statistics

Effects of treatments were determined by comparison of treatment groups(n = 6) to controls using the Kruskal–Wallis test followed by the Mann–WhitneyU test. Successful stimulation of immune cells within a treatment group wasanalyzed using Friedman test followed by Wilcoxon test (SPSS 9.0 for Windows).The coefficients of variation were calculated as standard deviation: mean fishmass * 100. Differences between treatment groups were considered statisticallysignificant when P < 0.05.

3. Results

The regular measurement of water parameters showedvalues of 6.8 ± 0.5 mg L�1 for dissolved oxygen, a conductivity of221 ± 5 lS m�1 and a pH value of 7.5 ± 0.1 (mean ± SD) for all tanksduring the experimental phase.

Contamination of experimental diets was successful. DONvalues of 352, 635 and 953 lg kg�1 feed have been analyzed. NoDOM-1 was found in the experimental diets. The remainingmycotoxin concentration in feed after exposure to water wasdetermined which revealed that more than 50% of the DONconcentration in feed was leached to the surrounding water whenpellets were exposed to water for 2 h (Fig. 1). These measurementsalso showed that the DON concentrations in feed decreased in atime-dependent manner. However, no refusal of feed by fish wasobserved at a restricted daily feed administration of two per centof body mass, although the fish of the high dose group occasionallytook more time (approximately 30 min) for the entire intake of thedaily feed ration. Since a continuous flow-through for every tankwas used, the leaching before ingestion of pellets by fish and expo-sure to water-borne DON may be insignificant in the present study.Accumulation of DON was not observed in plasma and muscle andonly residual DON concentrations were found in these samples(Supplement Table I). Two of sampled three carps showed low

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Fig. 1. Time-dependent leaching of DON from pellets of the medium dose, n = 2,mean ± SD.

DON concentrations (0.6–0.9 ng ml�1) in plasma sampled 8 to10 h after feed application (Supplement Table II).

The mass of fish at the beginning of experiments was not signif-icantly different between feeding groups (Table 1). During theexperiment no mortality occurred. The fish mass at the end ofthe DON feeding for 4 weeks was not significantly different be-tween feeding groups due to high variation in the control group(coefficient of variation for the control group, CV = 42.3%),CV = 46.6% for low dose group, CV = 22.4% for medium dose group,and CV = 17.8% for high dose group, but represented a significantincrease to the initial fish masses in the medium dose group andthe high dose group (P = 0.041 and P = 0.009, respectively). The fishmass after the recovery phase showed increases in mass that werenot significantly different compared to the weight at the start ofthe experiment due to high variation of fish masses (CVs = 46.7%,40.1%, 46.9%, and 45.2% for the control group and the groups fedthe low dose, medium dose and high dose feed, respectively).

Furthermore, all fish showed no differences in individual weightgain during the experiments and no differences in individualspecific growth rates (calculated as [log(m2)/log(m1)]/days ofexperiment * 100 whereby m1 is the mass at the first sampling dateand m2 is the mass at the second sampling date).

Fish were not stressed by husbandry conditions or duringsampling procedures as can be seen by low blood cortisol levels(Tables 2 and 3). Haematocrit values were not significantlydifferent between groups. Haemoglobin concentrations were notsignificantly influenced by DON treatment although the fish fedthe low dose diet showed higher haemoglobin values by trend(p = 0.065) (Table 2).

Differential blood cell counts showed no significant differencesbetween feeding groups although the treatment with the high dosediet lowered the numbers of monocytes in fish by on third com-pared to fish from the control group (P = 0.132; Table 4). Onlylow numbers of immature erythrocytes occurred which haveconsequently not been quantified, while the dimensions of eryth-rocytes with mature appearance were influenced by feeding DON(Table 5). The length of erythrocytes was reduced by feeding thelow dose compared to control fish. Influences on the dimensionsof the nuclei of erythrocytes have been observed between fishtreated with different DON levels but not compared to control fish.For example, fish fed the low dose diet showed shorter nuclei thanfish in the high dose diet, but the width of nuclei was increased inthis group compared to fish in the medium dose-treated group.

Antioxidative enzymes in erythrocytes showed a significant in-crease of catalase in the feeding period and SOD activity in therecovery period only in fish treated with the low dose feed com-pared to control animals but not in the other feeding groups(Fig. 2).

LPS significantly increased NO production in trunk kidney andhead kidney cells after four weeks of experimental feeding(Figs. 3A and 4A). NO production in fish treated with high doseDON showed lower values produced by LPS-stimulated trunkkidney cells than similar treated cells from control fish after four

Table 1Initial and final fish masses of experimental fish after 4 weeks of DON feeding andafter two weeks of recovery, mean ± SEM, n = 6.

Basal feed Low DON Medium DON High DON

DON-treated:Initial mass (g) 53.4 ± 7.4 49.7 ± 11.1 45.3 ± 5.1 46.6 ± 3.4Final mass (g) 80.9 ± 14.0 63.7 ± 12.1 65.9 ± 6.0 71.8 ± 5.2

RecoveryInitial mass (g) 51.4 ± 7.9 43.8 ± 6.3 48.7 ± 6.9 41.1 ± 6.7Final mass (g) 87.2 ± 16.6 65.3 ± 10.7 68.1 ± 13.0 73.3 ± 13.5

Table 2Haematocrit (Ht), haemoglobin (Hb), splenosomatic indices (SSI) and plasma cortisol levels after 4 weeks of DON feeding, n = 6 each, mean ± SEM.

Basal feed Low DON Medium DON High DON

Ht (%) 31.47 ± 0.97 34.20 ± 1.82 32.52 ± 1.02 32.73 ± 1.32Hb (mg dL�1) 7.6 ± 0.5 10.3 ± 0.9 8.7 ± 0.6 8.2 ± 0.3SSI (% body weight) 0.152 ± 0.020 0.149 ± 0.022 0.146 ± 0.025 0.133 ± 0.032Cortisol (ng ml�1 plasma) 11.53 ± 2.83 17.76 ± 6.69 11.26 ± 3.38 12.27 ± 2.27

Table 3Haematocrit (Ht), splenosomatic indices (SSI) and plasma cortisol levels 2 weeks after DON feeding (recovery phase), n = 6 each, mean ± SEM.

Basal feed Low DON Medium DON High DON

Ht (%) 33.00 ± 1.19 32.40 ± 0.83 34.18 ± 1.37 31.02 ± 1.64Hb (mg dL�1) 9.5 ± 1.1 9.0 ± 0.4 8.9 ± 0.4 8.0 ± 0.5SSI (% body weight) 0.157 ± 0.023 0.132 ± 0.020 0.148 ± 0.053 0.166 ± 0.031Cortisol (ng/ml plasma) 12.67 ± 3.04 14.70 ± 3.53 19.53 ± 4.80 13.47 ± 2.07

Table 4Differential blood cell counts of experimental fish after 4 weeks of DON feeding, mean ± SEM, n = 6.

Basal feed Low DON Medium DON High DON

Leukocytes (% total blood cells) 4.4 ± 0.3 4.1 ± 0.5 4.3 ± 0.3 4.5 ± 0.3Lymphocytes (% all white blood cells) 57.8 ± 4.5 55.0 ± 3.3 59.4 ± 1.2 60.6 ± 2.1Thrombocytes (% all white blood cells) 36.1 ± 4.0 38.4 ± 3.4 35.6 ± 1.8 33.9 ± 1.2Monocytes (% all white blood cells) 2.6 ± 0.5 3.2 ± 0.9 2.3 ± 0.3 1.8 ± 0.4Granulocytes (% all white blood cells) 3.5 ± 1.0 3.5 ± 0.9 2.8 ± 1.0 3.7 ± 0.9

Table 5Size of erythrocytes, nucleus and shape factors (calculated as length: width of individual erythrocytes) after 4 weeks of DON feeding, mean ± SEM, n = 6; means with the sameletter (a and/or b) are not significantly different from each other (Wilcoxon test, P < 0.05).

Basal feed Low DON Medium DON High DON

Erythrocyte length (lm) 11.9 ± 0.1a 11.1 ± 0.3b 11.5 ± 0.1a,b 11.8 ± 0.1a

Erythrocyte width (lm) 8.0 ± 0.2 8.0 ± 0.1 7.7 ± 0.1 8.0 ± 0.1Nucleus length (lm) 5.0 ± 0.1a,b 4.9 ± 0.1a 4.9 ± 0.1a 5.4 ± 0.1b

Nucleus width (lm) 2.7 ± 0.1a,b 3.0 ± 0.1a 2.6 ± 0.1b 2.9 ± 0.1a,b

Shape factor 1.5 ± 0.0 1.5 ± 0.0 1.5 ± 0.0 1.5 ± 0.0

C. Pietsch et al. / Food and Chemical Toxicology 68 (2014) 44–52 47

weeks of feeding. In head kidney cells from the same fish thisdifference was observed in medium dose-treated fish.

Significant effects of LPS stimulation on NO production in headkidney and trunk kidney was also observed after two weeks ofrecovery following the experimental feeding of DON (P < 0.05;Figs. 3B and 4B) with the exception of fish treated with high doseDON diet in the recovery phase. In addition, differences inLPS-stimulated leukocytes due to DON treatment of fish were notobserved after the recovery phase of two weeks.

Cell viability of leukocyte cultures was measured in test platesused previously for the assay of NO production in order to investi-gate whether effects on NO production are related to altered cellviability. Effects of DON feeding on cell viability were not observedin leukocytes isolated from trunk kidneys at both sampling datesafter 96 h of in vitro culture (Fig. 5). Cell viability was reduced inun-stimulated head kidney cells from fish of all DON-treatedgroups and in LPS-treated leukocytes of fish fed high dose DON dietfor four weeks compared to control fish (Fig. 6A). This was notfound in DON-treated fish which received control feed for addi-tional two weeks (Fig. 6B). Moreover, cell viability was significantlylower in LPS-treated head kidney cells in all fish from the first sam-pling date compared to un-stimulated cells from the same fish(Fig. 6A) which was not observed in cells similarly derived fromtrunk kidneys (Fig. 5A). In addition, in all cell cultures derived fromhead kidneys and trunk kidneys cell viability was not significantly

influenced by treatment with bacterial LPS at the second sampling(after feeding of uncontaminated feed for additional two weeks).

Although a significant effect of PMA stimulation in the respira-tory burst assay was not observed in trunk kidney-derived cells offish fed the experimental diets for 4 weeks (Fig. 7), stimulationwith PMA increased NBT conversion in all head kidney leukocyteswith the exception of the fish that received the high dose feed(Fig. 8). Respiratory burst measured as increased NBT conversionwas not significantly influenced in trunk kidney cells after fourweeks of DON application to fish (Fig. 7A). However, after twoweeks of recovery stimulation with PMA showed less reactivityin leukocytes of fish that received the medium and the high dosefeed (Fig. 7B). The respiratory burst of un-stimulated head kidneycells was reduced in fish fed the medium dose feed whereas theNBT conversion in the other groups was only slightly influenced(Fig. 8A). Moreover, PMA-stimulated head kidney cells of allDON-fed fish showed less reaction to PMA than cells from controlfish after four weeks of feeding (Fig. 8A) which was not observedafter two weeks of recovery (Fig. 8B).

Arginase activity of leukocytes was not significantly influencedby DON treatment of fish when treatment groups were comparedto control fish (Fig. 9). However, the reduction of NO productioncorrelates with the response of the arginase assays in head kidneycells (Pearson correlation coefficient: �0.480, significance: 0.018for untreated leukocytes and Pearson correlation coefficient:

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Fig. 2. Activities of catalase (CAT) and superoxide dismutase (SOD) in lysates oferythrocytes from DON-treated and of fish with additional 2 weeks of recovery,n = 6; mean ± SE; means with the same letter are not significantly different fromeach other (Wilcoxon test, P < 0.05).

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Fig. 3. NO production in trunk kidney cells after ex vivo incubation with andwithout LPS for 96 h, cells were isolated from experimental fish after 4 weeks offeeding (A) and with additional 2 weeks of recovery (B), n = 6; mean ± SE; meanswith the same letter are not significantly different from each other which applies tobars of the same colour (Wilcoxon test, P < 0.05).

48 C. Pietsch et al. / Food and Chemical Toxicology 68 (2014) 44–52

�0.454, significance: 0.018 for LPS- versus forskolin-treatedimmune cells).

4. Discussion

Addition of DON to experimental feed was successful. Contam-ination of experimental diets was comparable to DON values thatcan be found in commercially available feeding stuffs reachingvalues of up to 825 lg kg�1 DON (Pietsch et al., 2013). These valuesare far below the recommended levels by the EuropeanCommission (2006/576/EC). In other vertebrate species DON israpidly absorbed into the systemic circulation (tmax � 0.2–4.1 h)and is further characterized by a marked species-dependent varia-tion in bioavailability (6–139%) (Dänicke and Brezina, 2013). How-ever, only very low DON and no DOM-1 concentrations could bedetected in biological samples of carp (data can be found in theSupplement). On the one hand, this was probably due to the lim-ited amount of tissue or plasma samples that were available foranalyses. On the other hand, retention and accumulation of DONin animal tissues is generally low due to its rapid metabolization(Prelusky and Trenholm, 1991; Eriksen and Pettersson, 2004;Setyabudi et al., 2012). The leaching of DON from experimentaldiets, although it is fast and time-dependent, probably did notinfluence the application of DON because fish in all groups took-up the pellets at least within 30 min. Exposure of fish to DON inwater was negligible due to the high flow-through during theexperiments. Intake of DON-contaminated feed in mice was reduced(Gouze et al., 2006) resulting in effects on weight gain. The latterwas also found at concentrations of 3.7 mg DON per kg feed inAtlantic salmon after 15 weeks of feeding and at concentrations

of 0.3–2.6 mg DON per kg feed in rainbow trout after 56 days offeeding (Döll et al., 2011; Hooft et al., 2011). However, cyprinidsappear to be less sensitive because weight gain of carp was notinfluenced by DON at concentrations ranging from 352 to953 lg kg�1 which was also observed in zebrafish in a feeding trialfor 45 and 260 days using DON concentrations ranging from to 0.1to 3 mg DON per kg feed (Sanden et al., 2012).

However, blood haematology was influenced. DON has not beenshown to affect red blood cells previously. Erythrocytes are re-leased into circulation at an early stage of development and elon-gation and flattening occur in the blood stream (Sekhon andBeams, 1969; Hardig, 1978). A reduced length of circulating eryth-rocytes in fish fed the low dose diet indicates an increased abun-dance of juvenile erythrocytes which are smaller and moreround-shaped (Houston, 1997). In carp head kidney and spleencontribute to the release of erythrocytes into circulation (Ken-ichiand Yasuo, 1989; Kondera et al., 2012). Whether juvenile cells havebeen released from storage in the spleen or head kidney or resultedfrom de novo formation could not be distinguished. The occurrenceof more juvenile cells in circulation can have several reasons sincealtered respiratory circumstances in fish may lead to increasederythropoiesis, increased division of circulating cells, karyorrhexisor cell breakdown (Houston and Murad, 1992). The increased nu-cleus size in erythrocytes of fish in the high-dose group suggeststhat karyorrhexis took place which is accompanied by occurrenceof enlarged, more round-shaped nuclei and a subsequent reductionof cytoplasmic volume (Murad et al., 1990). However, this was notaccompanied by significant effects on the abundance and size ofmelano-macrophage centers in spleen of DON-treated fish (data

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Fig. 4. NO production by isolated leukocytes from head kidney of experimental fishafter 4 weeks of feeding (A) and with additional 2 weeks of recovery (B), n = 6; afterex vivo incubation with and without LPS for 96 h, mean ± SE; means with the sameletter are not significantly different from each other which applies to bars of thesame colour (Wilcoxon test, P < 0.05).

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Fig. 5. Cell viability after 96 h of incubation with and without LPS in in vitrocultures of trunk kidney cells of fish after 4 weeks of DON feeding (A) and additional2 weeks of recovery (B), n = 6, mean ± SE.

C. Pietsch et al. / Food and Chemical Toxicology 68 (2014) 44–52 49

not shown) which would support the hypothesis of increasedelimination of senescent and damaged erythrocytes. However,there was no evidence of anemia in any of the experimental fish.

How DON leads to an increased red blood cell formation with-out significantly affecting spleen histology remains obscure, butthe involvement of the kidneys has not been investigated. How-ever, it may be assumed that the cytotoxicity of this toxin in theblood stream contributed to this effect. Since cytotoxicity of DONoften also involves oxidative stress (Kouadio et al., 2005; Pietschet al., 2011b), this might also have occurred in DON-treated carp.Similar to all aerobic organisms, fish have regularly to deal withoxidative stress and antioxidative enzymes have been detected inmost fishes (Martínez-Álvarez et al., 2005). Effects of DON on anti-oxidative enzymes have been observed in erythrocyte samples offish fed the low-contaminated diet. Increased expression of hepaticCuZn SOD in liver has also been shown in zebrafish treated withmuch higher DON concentrations (2 or 3 mg DON per kg feed)(Sanden et al., 2012). The reason for the lack of effects on theantioxidative enzymes in erythrocytes of carp treated with themedium and the high dose DON diet remains obscure. It may beassumed that toxicity of DON in the blood stream prevented aresponse of the antioxidative enzymes in erythrocytes. DecreasedSOD activity in DON-treated rat liver cells has been reported whichshows that induction of SOD is not a general response to DON(Sahu et al., 2008). The involvement of oxidative stress in DONtoxicity in carp has already been shown previously, wherebyincreased lipid peroxidation was observed in head kidney, spleenand liver of carp treated with the high dose DON feed (Pietschet al., 2014).

Since acute toxicity of DON has often been shown in activelydividing cells, the immune system is an important target. Depend-ing on dose and exposure regime, DON has been shown to be bothimmunosuppressive and immunostimulatory in mammals (Pestkaet al., 2004; Pestka, 2008). In the present study both, pro-inflam-matory (NO and ROS production) and anti-inflammatory (arginaseactivity) immune reactions, have been determined which revealedthat pro-inflammatory immune responses were affected by DON.The effects on pro-inflammatory reactions of immune cells showedsome differences between leukocytes isolated from head kidneysand trunk kidneys. Since isolated cell cultures from both organsdo not show substantial different cell populations (Pietsch et al.,2008), it can be assumed that the reactivity of the immune cellsfrom both organs and ability to be stimulated by intrinsic or extrin-sic signals are different. However, there is no explanation for thatat the moment.

The effects on the anti-inflammatory enzyme arginase paral-leled the effects on the pro-inflammatory immune responses inhead kidney cells although no differences in arginase activitieshave been observed between the treatment groups. Thus, a distinctpolarization of the immune responses into pro-inflammatory oranti-inflammatory directions is not obvious. An involvement ofglucocorticoids in the resulting immune responses can be ex-cluded, because the measured cortisol levels indicated no generalstress response in fish. It is concluded that a mixture of activatingand suppressing reactions occurs upon DON exposure. Mixed im-mune responses have already been observed in the mammalianimmune system (Benoit et al., 2008; Porta et al., 2009). The possi-ble mechanisms by which DON can both suppress and stimulateimmune functions have been suggested in mammalian systems.

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Fig. 6. Cell viability after 96 h of in vitro culture of head kidney cells of fish after4 weeks of feeding DON (A) and with additional 2 weeks of recovery (B) assayed bymeans of uptake of neutral red measured by absorption at 540 nm, n = 6;mean ± SE; means with the same letter are not significantly different from eachother which applies to bars of the same colour (Wilcoxon test, P < 0.05).

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Fig. 7. Respiratory burst after 24 h of in vitro culture of trunk kidney cells of fishafter 4 weeks of feeding DON (A) and with additional 2 weeks of recovery (B)assayed by means of conversion of nitroblue tetrazolium (NBT) salt with andwithout stimulation by phorbol myristate acetate (PMA) for 90 min measured as anabsorbance at 620 nm, n = 6; mean ± SE; * = difference to controls at P < 0.05 whichapplies to bars of the same colour (Wilcoxon test).

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Fig. 8. Respiratory burst after 24 h of in vitro culture of head kidney cells of fishafter 4 weeks of feeding DON (A) and with additional 2 weeks of recovery (B)assayed by means of conversion of NBT with and without additional stimulation byPMA for 90 min measured by absorbance at 620 nm, n = 6; mean ± SE; * = differenceto controls at P < 0.05 which applies to bars of the same colour (Wilcoxon test).

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Fig. 9. Arginase activity in leukocytes derived from head (A) and trunk kidney (B)after 4 weeks of feeding DON at different concentration levels, stimulated for 24with 1 lM forskolin compared to unstimulated cells, n = 6; mean ± SE.

50 C. Pietsch et al. / Food and Chemical Toxicology 68 (2014) 44–52

At high doses a rapid onset of apoptotic events occurs in leukocyteswhich will undoubtedly be manifested as immunosuppression(Bondy and Pestka, 2000). Dose-dependent impairment of mitogen

C. Pietsch et al. / Food and Chemical Toxicology 68 (2014) 44–52 51

responses in DON-treated lymphocyte cultures from mammals hasbeen reported and apoptosis of macrophages was observed (Millerand Atkinson, 1986; Yang et al., 2000; Zhou et al., 2005). Effects ofDON on cell viability were observed in the LPS-treated leukocytesderived from head kidneys of carp. The cytotoxic effects of LPS onimmune cells have been reported to be strain-specific in mice(Peavy et al., 1978). In addition, LPS stimulation has been showto strongly increase pro-inflammatory cytokines and apoptosis ofimmune cells (Islam and Pestka, 2006) which further emphasizesthe influence of DON on immunological functions. In contrast toapoptosis, low concentrations of DON appear to promote expres-sion of a diverse array of cytokines in vitro and in vivo (Azcona-Oli-vera et al., 1995a,b; Dong et al., 1994; Ji et al., 1998; Warner et al.,1994; Wong et al., 1998; Zhou et al., 1997, 1999, 2000; Amuzieet al., 2009; Becker et al., 2011). It has been shown that increasedcytokine expression leads to up-regulation of suppressors ofcytokine signaling which, in turn, minimizes the inflammatory re-sponse and consequently possible damage to tissues. We observeddecreased pro-inflammatory responses in carp leukocytes. Thisprobably is meaningful for fish raised in aquaculture especiallywhen the susceptibility to diseases is concerned. However, furtherdetails on the mode of action of DON on immune cells of fish re-main unknown and especially cytokine signaling in DON-treatedfish needs to be investigated in the future.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Transparency Document

The Transparency document associated with this article can befound in the online version.

Acknowledgements

The authors like to thank Heidi Schiffer, Simon Herzog andCatherine Fehlmann for additional help in the laboratory work.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.fct.2014.03.012.

References

Aebi, M., 1974. Catalase. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis,vol. 2. Academic, New York, NY, USA, pp. 673–684.

Amuzie, C.J., Shinozuka, J., Pestka, J.J., 2009. Induction of suppressors of cytokinesignaling by the trichothecene deoxynivalenol in the mouse. Toxicol. Sci. 111,277–287.

Awad, W., Ghareeb, K., Böhm, J., Zentek, J., 2013. The toxicological impacts of theFusarium mycotoxin, deoxynivalenol, in poultry flocks with special reference toimmunotoxicity. Toxins 5, 912–925.

Azcona-Olivera, J.I., Ouyang, Y., Murtha, J., Chu, F.S., Pestka, J.J., 1995a. Induction ofcytokine mRNAs in mice after oral exposure to the trichothecene vomitoxindeoxynivalenol: Relationship to toxin distribution and protein synthesisinhibition. Toxicol. Appl. Pharmacol. 133, 109–120.

Azcona-Olivera, J.I., Ouyang, Y.L., Warner, R.L., Linz, J.E., Pestka, J.J., 1995b. Effects ofvomitoxin deoxynivalenol and cycloheximide on IL-2, 4, 5 and 6 secretion andmRNA levels in murine CD4+ cells. Food Chem. Toxicol. 35, 433–441.

Becker, C., Reiter, M., Pfaffl, M., Meyer, H., Bauer, J., Meyer, K., 2011. Expression ofimmune relevant genes in pigs under the influence of low doses ofdeoxynivalenol (DON). Mycotox. Res. 27, 287–293.

Benoit, M., Desnues, B., Mege, J.-L., 2008. Macrophage polarization in bacterialinfections. J. Immunol. 181, 3733–3739.

Berntssen, M.H.G., Julshamn, K., Lundebye, A.K., 2010. Chemical contaminants inaquafeeds and Atlantic salmon (Salmo salar) following the use of traditionalversus alternative feed ingredients. Chemosphere 78, 637–646.

Bondy, G.S., Pestka, J.J., 2000. Immunology by fungal toxins. J. Toxicol. Environ.Health, Part B 3, 109–143.

Borenfreund, E., Puerner, J.A., 1985. Toxicity determined in vitro by morphologicalalterations and neutral red absorption. Toxicol. Lett. 24, 119–124.

Chung, S., Secombes, C.J., 1988. Analysis of events occurring within teleostmacrophages during the respiratory burst. Comp. Biochem. Physiol. B 89, 39–54.

Dale, O.B., Tørud, B., Kvellestad, A., Koppang, H.S., Koppang, E.O., 2009. From chronicfeed-induced intestinal inflammation to adenocarcinoma with metastases insalmonid fish. Cancer Res. 69, 4355–4362.

Dänicke, S., Goyarts, T., Döll, S., Grove, N., Spolders, M., Flachowsky, G., 2006. Effectsof the Fusarium toxin deoxynivalenol on tissue protein synthesis in pigs.Toxicol. Lett. 165, 297–311.

Dänicke, S., Brezina, U., 2013. Kinetics and metabolism of the Fusarium toxindeoxynivalenol in farm animals: consequences for diagnosis of exposure andintoxication and carry over. Food Chem. Toxicol. 60, 58–75.

D’Mello, J.P.F., Placinta, C.M., Macdonald, A.M.C., 1999. Fusarium mycotoxins: areview of global implications for animal health, welfare and productivity. Anim.Feed Sci. Technol. 80, 183–205.

Döll, S., Schrickx, J.A., Danicke, S., Fink-Gremmels, J., 2009. Interactions ofdeoxynivalenol and lipopolysaccharides on cytokine excretion and mRNAexpression in porcine hepatocytes and Kupffer cell enriched hepatocytecultures. Toxicol. Lett. 190, 96–105.

Döll, S., Dänicke, S., 2011. The Fusarium toxins deoxynivalenol (DON) andzearalenone (ZON) in animal feeding. Prev. Vet. Med. 102, 132–145.

Döll, S., Valenta, H., Baardsen, G., Möller, P., Koppe, W., Stubhaug, I., Dänicke, S.,2011. Effects of increasing concentrations of deoxynivalenol, zearalenone andochratoxin A in diets for Atlantic salmon (Salmo salar) on performance, healthand toxin residues. In: Proceedings, 33rd Mycotoxin Workshop. Freising,Germany, 30.05.-01.06.2011, pp. 25.

Dong, W., Azcona-Olivera, J.I., Brooks, K.H., Linz, J.E., Pestka, J.J., 1994. Elevated geneexpression and production of interleukins 2, 4, 5, and 6 and cycloheximide inthe EL-4 thymoma. Toxicol. Appl. Pharmacol. 127, 282–290.

Drabkin, D.L., Austin, J.H., 1935. Spectrophotometric studies. II. Preparations fromwashed blood cells; nitric oxide hemoglobin and sulfhemoglobin. J. Biol. Chem.112, 51–65.

Eriksen, G., Pettersson, H., 2004. Toxicological evaluation of trichothecenes inanimal feed. Anim. Feed. Sci. Technol. 114, 205–239.

European Commission, Commission Recommendation (EC) 576 of 17 August 2006on the presence of deoxynivalenol, zearalenone, ochratoxin A, T-2 and HT-2 andfumonisins in products intended for animal feeding. Off. J. Eur. Un. 2006, L229/7–L229/9.

FAO, 2012. The State of World Fisheries and Aquaculture; Food and AgricultureOrganization of the United Nations. Rome, Italy.

Ferrari, L., Cantoni, A.M., Borghetti, P., De Angelis, E., Corradi, A., 2009. Cellularimmune response and immunotoxicity induced by DON (deoxynivalenol) inpiglets. Vet. Res. Commun. 33, S133–S135.

Foroud, N.A., Eudes, F., 2009. Trichothecenes in cereal grains. Int. J. Mol. Sci. 10, 147–173.

Gouze, M.E., Laffitte, J., Rouimi, P., Loiseau, N., Oswald, I.P., Galtier, P., 2006. Effect ofvarious doses of deoxynivalenol on liver xenobiotic metabolizing enzymes inmice. Food Chem. Toxicol. 44, 476–483.

Gutzwiller, A., 2010. Effects of deoxynivalenol (DON) in the lactation diet on thefeed intake and fertility of sows. Mycotox. Res 26, 211–215.

Hardig, J., 1978. Maturation of circulating red blood cells in young Baltic salmon(Salmo salar L.). Acta Physiol. Scand. 102, 290–300.

Hooft, J.M., Elmor, A.E.H.I., Encarnação, P., Bureau, D.P., 2011. Rainbow trout(Oncorhynchus mykiss) is extremely sensitive to the feed-borne Fusariummycotoxin deoxynivalenol (DON). Aquaculture 311, 224–232.

Houston, A.H., Murad, A., 1992. Erythrodynamics in goldfish, Carassius auratus L.:temperature effects. Physiol. Zool. 65 (1), 55–76.

Houston, A.H., 1997. Review: Are the classical hematological variables acceptableindicators of fish health? Transact. Am. Fish. Soc. 126, 879–894.

Islam, Z., Pestka, J.J., 2006. LPS priming potentiates and prolongs proinflammatorycytokine response to the trichothecene deoxynivalenol in the mouse. Toxicol.Appl. Pharmacol 211, 53–63.

Ji, G.E., Park, S.Y., Wong, S.S., Pestka, J.J., 1998. Modulation of nitric oxide, hydrogenperoxide and cytokine production in clonal macrophage model by thetrichothecene vomitoxin deoxynivalenol. Toxicology 125, 203–214.

Ken-ichi, Y., Yasuo, I., 1989. Erythrocyte supply from the spleen of exercised carp.Comp. Biochem. Physiol. – Part A: Physiol. 92 (1), 139–144.

Kondera, E., Dmowska, A., Rosa, M., Witeska, M., 2012. The effect of bleeding onperipheral blood and head kidney hematopoietic tissue in common carp(Cyprinus carpio). Turk. J. Vet. Anim. Sci. 36 (2), 169–175.

Kouadio, J.H., Mobio, T.A., Baudrimont, I., Moukha, S., Dano, S.D., Creppy, E.E., 2005.Comparative study of cytotoxicity and oxidative stress induced bydeoxynivalenol, zearalenone or fumonisin B1 in human intestinal cell lineCaco-2. Toxicology 213, 56–65.

Martínez-Álvarez, R.M., Morales, A.E., Sanz, A., 2005. Antioxidant defenses in fish:biotic and abiotic factors. Rev. Fish Biol. Fisheries 15, 75–88.

Matz, S.A., 1991. The Chemistry and Technology of Cereals as Food and Feed. KluwerAcademic Publishers Group, Dordrecht, The Netherlands, pp. 319.

Miller, K., Atkinson, H.A.C., 1986. The in vitro effects of trichothecenes on theimmune system. Food Chem. Toxicol. 24, 545–549.

Murad, A., Houston, A.H., Samson, L., 1990. Hematological response to reduced O2-carrying capacity, increased temperature and hypoxia in goldfish, Carassiusauratus L. J. Fish Biol. 36, 289–305.

52 C. Pietsch et al. / Food and Chemical Toxicology 68 (2014) 44–52

Neira, M.S., Pacin, A.M., Martinez, E.J., Moltó, G., Resnik, S.L., 1997. The effects ofbakery processing on natural deoxynivalenol contamination. Int. J. FoodMicrobiol. 37, 21–25.

NRC (National Research Council), 1993. Nutrient Requirements of Fish. NationalAcademy, Press, Washington, DC.

Oberley, L.W., Spitz, D.R., 1984. Assay of superoxide dismutase activity in tumortissue. Methods Enzymol. 105, 457–464.

Oldenburg, E., Bramm, A., Valenta, H., 2007. Influence of nitrogen fertilization ondeoxynivalenol contamination of winter wheat – experimental field trials andevaluation of analytical methods. Mycotox. Res. 23, 7–12.

Peavy, D.L., Baughn, R.E., Musher, D.M., 1978. Strain-dependent cytotoxiceffects of endotoxin for mouse peritoneal macrophages. Infect. Immun. 21(1), 310–319.

Pestka, J.J., Bondy, G.S., 1990. Alteration of immune function following dietarymycotoxin exposure. Can. J. Physiol. Pharmacol. 68, 1009–1016.

Pestka, J.J., Zhou, H.R., Moon, Y., Chung, Y.J., 2004. Cellular and molecularmechanisms for immune modulation by deoxynivalenol and othertrichothecenes: unraveling a paradox. Toxicol. Lett. 153 (1), 61–73.

Pestka, J.J., 2008. Mechanisms of deoxynivalenol-induced gene expression andapoptosis. Food Addit. Contam. Part a-Chem. Anal. Control Expo. Risk Assess. 25,1128–1140.

Pietsch, C., Vogt, R., Neumann, N., Kloas, W., 2008. Production of nitric oxide by carpkidney leukocytes is regulated by cyclic adenosine 30 ,50monophosphate. Comp.Biochem. Physiol. Part A Physiol. 150, 58–65.

Pietsch, C., Neumann, N., Preuer, T., Kloas, W., 2011a. In vivo-treatment withprogestogens causes immunosuppression of carp leukocytes (Cyprinus carpio L.)by affecting nitric oxide production and arginase activities. J. Fish Biol. 79, 53–69.

Pietsch, C., Bucheli, T., Wettstein, F., Burkhardt-Holm, P., 2011b. Frequent biphasiccellular responses of permanent fish cell cultures to deoxynivalenol (DON).Toxicol. Appl. Pharmacol. 256, 24–34.

Pietsch, C., Kersten, S., Burkhardt-Holm, P., Valenta, H., Dänicke, S., 2013.Occurrence of deoxynivalenol and zearalenone in commercial fish feed – aninitial study. Toxins 5, 184–192.

Pietsch, C., Schulz, C., Rovira, P., Kloas, W., Burkhardt-Holm, P., 2014. Organ damageand hepatic lipid accumulation in carp (Cyprinus carpio L.) after feed-borneexposure to the mycotoxin deoxynivalenol (DON). Toxins, accepted.

Porta, C., Rimoldi, M., Raes, G., Brys, L., Ghezzi, P., DiLiberto, D., Dieli, F., Ghisletti, S.,Natoli, G., De Baerselier, P., Mantovani, A., Sica, A., 2009. Tolerance and M2(alternative) macrophage polarization are related processes orchestrated byp50 nuclear factor kB. PNAS 106 (35), 14978–14983.

Prelusky, D.B., Trenholm, H.L., 1991. Tissue distribution of deoxynivalenol in swinedosed intravenously. J. Agric. Food Chem. 39, 748–751.

Sagstad, A., Sanden, M., Haugland, Ø., Hansen, A.C., Olsvik, P.A., Hemre, G.I., 2007.Evaluation of stress- and immune-response biomarkers in Atlantic salmon,Salmo salar L., fed different levels of genetically modified maize (Bt maize),compared with its near-isogenic parental line and a commercial suprex maize. JFish Dis. 30 (4), 201–212.

Sahu, S.C., Garthoff, L.H., Robl, M.G., Chirtel, S.J., Ruggles, D.I., Flynn, T.J., Sobotka, T.J.,2008. Rat liver clone-9 cells in culture as a model for screening hepatotoxic

potential of food-related products: hepatotoxicity of deoxynivalenol. J. Appl.Toxicol. 28, 765–772.

Sanden, M., Jørgensen, S., Hemre, G.-I., Ørnsrud, R., Sissener, N.H., 2012. Zebrafish(Danio rerio) as a model for investigating dietary toxic effects of deoxynivalenolcontamination in aquaculture feeds. Food Chem. Toxicol. 50, 4441–4448.

Sekhon, S.S., Beams, H.W., 1969. Fine structure of developing trout erythrocytes andthrombocytes with special reference to the marginal band and the cytoplasmicorganelles. Am. J. Anat. 125, 353–374.

Setyabudi, F.M.C.S., Böhm, J., Mayer, H.K., Razazzi, F.E., 2012. Analysis ofdeoxynivalenol and de-epoxy- deoxynivalenol in horse blood through liquidchromatography after clean-up with immunoaffinity column. J. Vet. Anim. Sci.2, 21–31.

Sissener, N.H., Hemre, G.I., Lall, S.P., Sagstad, A., Petersen, K., Williams, J., Rohloff, J.,Sanden, M., 2011. Are apparent negative effects of feeding GM MON810 maizeto Atlantic salmon, Salmo salar, caused by confounding factors? Br. J. Nutr. 106(1), 42–56.

Sugita-Konishi, Y., Park, B.J., Kobayashi-Hattori, K., Tanaka, T., Chonan, T.,Yoshikawa, K., Kumagai, S., 2006. Effect of cooking process on thedeoxynivalenol content and its subsequent cytotoxicity in wheat products.Biosci. Biotechnol. Biochem. 70, 1764–1768.

Ukeda, H., Kawana, D., Maeda, S., Sawamura, M., 1999. Spectrophotometric assay forsuperoxide dismutase based on the reduction of highly water-solubletetrazolium salts by xanthine-xanthine oxidase. Biosci. Biotechnol. Biochem.63, 485–488.

Warner, R.L., Brooks, K., Pestka, J.J., 1994. In vitro effects of vomitoxindeoxynivalenol on lymphocyte function: enhanced interleukin production andhelp for IgA secretion. Food Chem. Toxicol. 32, 617–625.

Wong, S.S., Zhou, H.R., Marin-Martinez, M.L., Brooks, K., Pestka, J.J., 1998.Modulation of IL-1b, IL-6 and TNF-a secretion and mRNA expression by thetrichothecene vomitoxin in the RAW 264.7 murine macrophage cell line. FoodChem. Toxicol. 36, 409–419.

Yang, G., Jarvis, B.B., Chung, Y., Pestka, J.J., 2000. Apoptosis induction by thesatratoxins and other trichothecene mycotoxins: relationship to ERK, p38 MAPKand SAPK/JNK activation. Toxicol. Appl. Pharmacol. 164 (2), 149–160.

Yazar, S., Omurtag, G.Z., 2008. Fumonisins, trichothecenes and zearalenone incereals. Int. J. Mol. Sci. 9, 2062–2090.

Zhou, H.R., Yan, D., Pestka, J.J., 1997. Differential cytokine mRNA expression in miceafter oral exposure to the trichothecene vomitoxin deoxynivalenol: doseresponse and time course. Toxicol. Appl. Pharmacol. 144, 294–305.

Zhou, H.R., Harkema, J.F., Yan, D., Pestka, J.J., 1999. Amplified proinflammatorycytokine gene expression and toxicity in mice coexposed to lipopolysaccharideand the trichothecene vomitoxin deoxynivalenol. J. Toxicol. Environ. Health 56,115–136.

Zhou, H.-R., Harkema, J.R., Hotchkiss, J.A., Yan, D., Roth, R.A., Pestka, J.J., 2000.Lipopolysaccharide and the trichothecene vomitoxin (deoxynivalenol)synergistically induce apoptosis in murine lymphoid organs. Toxicol. Sci. 53,253–263.

Zhou, H.R., Jia, Q., Pestka, J.J., 2005. Ribotoxic stress response to the trichothecenedeoxynivalenol in the macrophage involves the SRC family kinase Hck. Toxicol.Sci. 85 (2), 916–926.