Food and Chemical Toxicology 68 (2014) 44–52

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In vivo effects of deoxynivalenol (DON) on innate immune responses of carp (Cyprinus carpio L.) 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, Switzerland Friedrich-Loeffler-Institute (FLI), Federal Research Institute for Animal Health, Institute of Animal Nutrition, Bundesallee 50, 38116 Braunschweig, Germany Gesellschaft für Marine Aquakultur (GMA) mbH, Hafentörn 3, D-25761 Büsum, Germany d Department of Ecophysiology and Aquaculture, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Mueggelseedamm 310, Berlin, Germany b c

a r t i c l e

i n f o

Article history: Received 17 September 2013 Accepted 7 March 2014 Available online 17 March 2014 Keywords: Mycotoxin Haematology Immune system Aquaculture

a b s t r a c t Deoxynivalenol (DON) is one of the most important members of Fusarium toxins since it often can be found in relevant concentrations in animal feeds. The effects of this group of toxins on fish are mostly unknown. The present study shows results from a feeding trial with carp (Cyprinus carpio L.) using three different concentrations of DON (352 lg kg1, 619 lg kg1, and 953 lg kg1 final feed, respectively) which are comparable to levels found in commercial fish feeds. Effects on growth and mass of fish were not observed during this 6 weeks lasting experiment. Only marginal DON concentrations were found in muscle and plasma samples. Blood parameters were not influenced although smaller erythrocytes occurred in fish treated with 352 lg kg1 DON. Analysis of antioxidative enzymes in erythrocytes showed increased superoxid dismutase and catalase activities in fish fed the low-dose feed. Immunosuppressive effects of DON were confirmed whereby cytotoxic effects on immune cells only partly explained the impairment of innate immune responses. Exact polarization of the immune system into pro-inflammatory 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 in aquaculture. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction The occurrence of mycotoxins in feeds for farm animals and their numerous impacts on animal health is an issue that is increasingly addressed (D’Mello et al., 1999; Döll and Dänicke, 2011). At present, a general recommendation on guidance values of 5 mg kg1 DON in complete feedingstuff was established by the European Commission (2006/576/EC). Contamination of feed ingredients can already occur on the native cereals growing on the fields. This contamination is often due to growth of fungi belonging to the genus Fusarium (Yazar and Omurtag, 2008; Foroud and Eudes, 2009) which are responsible for the production of deoxynivalenol (DON). DON is relatively stable even during food processing steps (Neira et al., 1997; Sugita-Konishi et al., 2006). Cereals are increasingly used for production of fish feeds, since the steadily growing aquaculture sector utilizes increasing amounts of feed sources and limited fish meal supply cannot fulfil ⇑ Corresponding author. Tel.: +41 61 267 0405; fax: +41 61 267 0409. E-mail address: [email protected] (C. Pietsch). http://dx.doi.org/10.1016/j.fct.2014.03.012 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.

this demand (FAO, 2012). Thus, cereals replace fish meal in fish feed whereby cyprinids accept up to 70% cereals as feed ingredients and salmonids tolerate less than 30% cereal compounds (Matz, 1991; Berntssen et al., 2010). However, this leads to the introduction of cereal-borne mycotoxins into fish feeds (Pietsch et al., 2013) with up to now mostly unknown consequences. Contamination of feed affects farm animal metabolism and health. For example, exposure to DON often resulted in changes of nutritional status in farm animals (Döll et al., 2009; Ferrari et al., 2009). Especially swine proved to be highly sensitive to DON-contaminated feed and occasionally refused feed (Eriksen and Pettersson, 2004; Dänicke et al., 2006; Gutzwiller, 2010). Immunotoxic effects of DON have already been found in higher vertebrates (Pestka and Bondy, 1990; Pestka, 2008; Awad et al., 2013). Recent investigations on salmonids showed that histo-pathological changes and lesions in the liver and intestinal tract of fish occur upon feeding with DON-contaminated diets (Döll et al., 2011; Hooft et al., 2011). In addition, it has been assumed that the increased usage of plant materials as protein sources in feeds for aquaculture leading to mycotoxin contamination might be

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

related to feed-induced negative effects on growth and immune parameter and the occurrence of intestinal cancer in salmonid fish (Sagstad et al., 2007; Dale et al., 2009; Sissener et al., 2011). However, direct effects of DON as one of the most relevant Fusarium toxins on the immune system of fish have so far not been investigated. Impaired immune functions of fish due to mycotoxin exposure would implicate a possible contribution to disease problems and may represent an urgent threat to fish in aquaculture. The present study, therefore, aimed to causally determine the effects of experimentally DON-contaminated diets on blood parameters and innate immune responses of carp (Cyprinus carpio) since this fish species is important for aquaculture (FAO, 2012).

45

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

2. Materials and methods

From each fish, blood was drawn from the caudal vein using heparinized syringes immediately after removal from each tank. Haematocrit was measured in heparinized glass capillary tubes (Huber & Co. AG, Reinach, Switzerland) in duplicate after centrifugation at 3000 rpm for 10 min (Haematokrit Typ 2010, Hettich Zentrifugen, Tuttlingen, Germany). Haemoglobin was calculated using the Drabkin method (Drabkin and Austin, 1935). Therefore, 5 ll of freshly drawn blood were added to Drabkin‘s solution containing 30% BrijÒ 35 and absorptions were read at 540 nm (Tecan Infinite M200). A standard curve was established using human haemoglobin (lyophilized powder). Blood smears from each fish were prepared immediately in duplicate on glass slides. From each slide 10 pictures were taken randomly at 400 magnification (Nikon Eclipse E400 equipped with a Nikon Digital Camera DXM1200F). All cells from each picture were counted so that an average number of 7813 cells per fish (mean; maximum = 11,976; minimum = 5684) were used in total for calculating differential blood cell counts from individual fish.

2.1. Chemicals

2.6. Activities of catalase and superoxide dismutase in erythrocytes

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

Lysates from erythrocytes were prepared by centrifugation of 100 ll fresh blood, followed by washing of cells using physiological sodium chloride solution and 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 method described by Aebi (1974) in 20 ll of lysates after addition of 150 ll sodium phosphate buffer (0.1 M, pH 6.5) containing 3.4 micromoles hydrogen peroxide per well. Measurements were conducted in UV-StarÒ 96-well microtitre plates (Greiner Bio-one, HUBERLAB. AG, Aesch, Switzerland) at 240 nm and 20 °C (Infinite M200, Tecan Group Ltd., Männedorf, Switzerland) by recording a kinetic for 20 min. For determination of superoxide dismutase (SOD) activity according to the description of Oberley and Spitz (1984) and Ukeda et al. (1999), 20 ll of lysates were placed in 96-well microtitre plates (RotilaboÒ, Carl Roth AG, Karlsruhe, Germany). Afterwards, 180 ll of a sodium carbonate solution (50 mM, pH 10.2) containing 1 mM ml1 diethylenetriaminepentaacetic acid (DTPA), 1 U ml1 catalase (from bovine liver), 0.177 mM ml1 xanthine and 0.195 mg ml1 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 of 10 ll of xanthine oxidase (1080 mU ml1 in 2 M (NH4)2SO4) whereas only ammonium sulfate was added to blanks. The plates were incubated at 37 °C for 20 min and optical densities were read at 438 nm (Infinite M200, Tecan Group Ltd., Männedorf, Switzerland). Calibration curves were prepared from serial dilutions of a SOD stock solution (9600 U ml1 ammonium sulfate buffer) in phosphate buffered saline. In parallel, aliquots of the cell homogenates were also used for protein determinations using the bicinchoninic acid (BCA) assay (Sigma) according to the manufacturer‘s protocol.

2.2. Preparation of feeds Experimental diets were designed without cereals in order to exclude cereal-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 diets to meet the dietary requirements of carp (NRC, 1993). All ingredients were mixed thoroughly. Deoxynivalenol (DON, dissolved in ethanol; purity >98%, lot-no. 011M4065V) was added to the fish oil (accounting for 12% of the final feed) at three different concentrations (low dose: 352 lg kg1, medium dose: 619 lg kg1 and high dose: 954 lg kg1 final feed, respectively) prior to addition to the other ingredients. 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 kg1 dry matter, mean ± SD). Pellets were allowed to cool down to room temperature for two 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 at 12–16 cm in total length. Fish were kept at a 16 h light/8 h dark photoperiod at 25 ± 0.2 °C (mean ± SD) in a flow through system. Rearing of fish and experimental procedures has been approved by the Cantonal veterinarian authorities of BaselStadt (Switzerland) under the permission number 2410. Fish were acclimated for 3 weeks to the experimental tanks where all animals received the uncontaminated experimental diet at daily feed administration of two per cent of body mass. Each of the 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 contaminated diets for four weeks while a control group received the uncontaminated feed. Thereafter, two tanks per feeding group were sampled. All remaining tanks per feeding group were fed the uncontaminated diet for additional two weeks before sampling to investigate possible recovery from DON feeding. During the experiments the flow through was adjusted to 6 L conditioned fresh water per h for each of the tanks. Water temperature, pH (WTW pH 315i, Wissenschaftlich-Technische Werkstätten GmbH, Weilheim, Germany), conductivity (WTW cond 315i, Wissenschaftlich-Technische Werkstätten GmbH, Weilheim, Germany) and dissolved oxygen (WTW Oxi 330i, Wissenschaftlich-Technische Werkstätten GmbH, Weilheim, Germany) were recorded for each tank at least five times a week. Every tank was cleaned at least every second day including removal of faeces and scraping of 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 were analyzed by HPLC-DAD (high performance liquid chromatography (consisting of a pump (LC-10ADVP), an autoinjector (SIL-10ADVP), and a column oven (CTO10ACVP) from Shimadzu (Duisburg, Germany) with diode array detection using the detector SPD-M10AVP (Shimadzu, Duisburg, Germany). To evaluate the leaching of DON into water 25 g of the medium dose feed were exposed to 10 L aquarium water for 0, 0.5, 1, 2, 4, 8, 12 h, and 24 h in duplicates. Thereafter, the remaining feed samples 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-affinity columns, DONprep™, R-Biopharm, Darmstadt, Germany) prior to analyses according to manufacturer’s procedure with slight modifications as described previously (Oldenburg et al., 2007). The detection limit was 30 lg kg1, the mean recovery was approximately 90%.

2.7. Analysis of plasma cortisol Plasma was prepared by centrifugation at 3000g for 20 min at 4 °C (Centrifuge 5415R, Eppendorf, Basel, Switzerland). Thereafter, 100 ll of plasma were mixed with the same volume of sterile UltraPure Water (Cayman, Chemie Brunschwig AG, Switzerland), and stored at 80 °C until steroid extraction. All samples were extracted twice with 1 ml diethyl ether, and both extracts were combined and stored at 20 °C after evaporation of diethyl ether at room temperature. Prior to analyses, extracts were re-mobilized in 5% ethanol and cortisol contents were determined using 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 described by Pietsch et al. (2011a). Stimulation of NO production was conducted by addition of 30 lg ml1 bacterial lipopolysaccharide (LPS from E. coli, serotype O111:B4) to wells. After incubation of cells with and without LPS at 25 °C and 5% CO2 for 96 h in the dark NO production was measured using the Griess reagent as described by Pietsch et al. (2008). Arginase activity was measured after 24 h with and without addition of forskolin as described previously (Pietsch et al., 2011a). All experimental incubations 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 the uptake of neutral red (3-amino-7-dimethylamino-2-methyl-phenanzine hydrochloride, NR) to evaluate membrane integrity and lysosomal function based on the method described by Borenfreund and Puerner (1985). Therefore, a stock solution of NR was prepared with 0.05% NR in RPMI medium. Cells were incubated with

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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 in 50 ll ethanol containing 2% acetic acid. Optical densities were read at 540 nm using a 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 °C and 5% CO2 for 96 h in the dark and incubated for 1 h with 1 mg NBT salt ml1 culture medium with and without 0.24 lg ml1 phorbol myristate acetate (PMA) as a stimulant of production of reactive oxygen species by leukocytes. Subsequently, the supernatant was discarded and the cells were fixed using 70% methanol. Dried plates were incubated with 100 ll dimethyl sulfoxide (DMSO) and 100 ll potassium hydroxide (KOH) to solubilize the formazan. The absorbance at 620 nm was measured spectrophotometrically in duplicates with a microplate reader (Infinite M200, 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–Whitney U test. Successful stimulation of immune cells within a treatment group was analyzed using Friedman test followed by Wilcoxon test (SPSS 9.0 for Windows). The coefficients of variation were calculated as standard deviation: mean fish mass * 100. Differences between treatment groups were considered statistically significant when P < 0.05.

3. Results The regular measurement of water parameters showed values of 6.8 ± 0.5 mg L1 for dissolved oxygen, a conductivity of 221 ± 5 lS m1 and a pH value of 7.5 ± 0.1 (mean ± SD) for all tanks during the experimental phase. Contamination of experimental diets was successful. DON values of 352, 635 and 953 lg kg1 feed have been analyzed. No DOM-1 was found in the experimental diets. The remaining mycotoxin concentration in feed after exposure to water was determined which revealed that more than 50% of the DON concentration in feed was leached to the surrounding water when pellets were exposed to water for 2 h (Fig. 1). These measurements also showed that the DON concentrations in feed decreased in a time-dependent manner. However, no refusal of feed by fish was observed at a restricted daily feed administration of two per cent of body mass, although the fish of the high dose group occasionally took more time (approximately 30 min) for the entire intake of the daily feed ration. Since a continuous flow-through for every tank was used, the leaching before ingestion of pellets by fish and exposure to water-borne DON may be insignificant in the present study. Accumulation of DON was not observed in plasma and muscle and only residual DON concentrations were found in these samples (Supplement Table I). Two of sampled three carps showed low

750

DON [µg kg-1]

625

DON concentrations (0.6–0.9 ng ml1) in plasma sampled 8 to 10 h after feed application (Supplement Table II). The mass of fish at the beginning of experiments was not significantly different between feeding groups (Table 1). During the experiment no mortality occurred. The fish mass at the end of the DON feeding for 4 weeks was not significantly different between 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 significant increase to the initial fish masses in the medium dose group and the high dose group (P = 0.041 and P = 0.009, respectively). The fish mass after the recovery phase showed increases in mass that were not significantly different compared to the weight at the start of the 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 fed the low dose, medium dose and high dose feed, respectively). Furthermore, all fish showed no differences in individual weight gain during the experiments and no differences in individual specific growth rates (calculated as [log(m2)/log(m1)]/days of experiment * 100 whereby m1 is the mass at the first sampling date and m2 is the mass at the second sampling date). Fish were not stressed by husbandry conditions or during sampling procedures as can be seen by low blood cortisol levels (Tables 2 and 3). Haematocrit values were not significantly different between groups. Haemoglobin concentrations were not significantly influenced by DON treatment although the fish fed the low dose diet showed higher haemoglobin values by trend (p = 0.065) (Table 2). Differential blood cell counts showed no significant differences between feeding groups although the treatment with the high dose diet lowered the numbers of monocytes in fish by on third compared to fish from the control group (P = 0.132; Table 4). Only low numbers of immature erythrocytes occurred which have consequently not been quantified, while the dimensions of erythrocytes with mature appearance were influenced by feeding DON (Table 5). The length of erythrocytes was reduced by feeding the low dose compared to control fish. Influences on the dimensions of the nuclei of erythrocytes have been observed between fish treated with different DON levels but not compared to control fish. For example, fish fed the low dose diet showed shorter nuclei than fish in the high dose diet, but the width of nuclei was increased in this group compared to fish in the medium dose-treated group. Antioxidative enzymes in erythrocytes showed a significant increase of catalase in the feeding period and SOD activity in the recovery period only in fish treated with the low dose feed compared to control animals but not in the other feeding groups (Fig. 2). LPS significantly increased NO production in trunk kidney and head kidney cells after four weeks of experimental feeding (Figs. 3A and 4A). NO production in fish treated with high dose DON showed lower values produced by LPS-stimulated trunk kidney cells than similar treated cells from control fish after four

500 375 Table 1 Initial and final fish masses of experimental fish after 4 weeks of DON feeding and after two weeks of recovery, mean ± SEM, n = 6.

250 125 0 0

0.5

1

2

4

8

12

24

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

Basal feed

Low DON

Medium DON

High DON

DON-treated: Initial mass (g) Final mass (g)

53.4 ± 7.4 80.9 ± 14.0

49.7 ± 11.1 63.7 ± 12.1

45.3 ± 5.1 65.9 ± 6.0

46.6 ± 3.4 71.8 ± 5.2

Recovery Initial mass (g) Final mass (g)

51.4 ± 7.9 87.2 ± 16.6

43.8 ± 6.3 65.3 ± 10.7

48.7 ± 6.9 68.1 ± 13.0

41.1 ± 6.7 73.3 ± 13.5

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C. Pietsch et al. / Food and Chemical Toxicology 68 (2014) 44–52 Table 2 Haematocrit (Ht), haemoglobin (Hb), splenosomatic indices (SSI) and plasma cortisol levels after 4 weeks of DON feeding, n = 6 each, mean ± SEM.

Ht (%) Hb (mg dL1) SSI (% body weight) Cortisol (ng ml1 plasma)

Basal feed

Low DON

Medium DON

High DON

31.47 ± 0.97 7.6 ± 0.5 0.152 ± 0.020 11.53 ± 2.83

34.20 ± 1.82 10.3 ± 0.9 0.149 ± 0.022 17.76 ± 6.69

32.52 ± 1.02 8.7 ± 0.6 0.146 ± 0.025 11.26 ± 3.38

32.73 ± 1.32 8.2 ± 0.3 0.133 ± 0.032 12.27 ± 2.27

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

Ht (%) Hb (mg dL1) SSI (% body weight) Cortisol (ng/ml plasma)

Basal feed

Low DON

Medium DON

High DON

33.00 ± 1.19 9.5 ± 1.1 0.157 ± 0.023 12.67 ± 3.04

32.40 ± 0.83 9.0 ± 0.4 0.132 ± 0.020 14.70 ± 3.53

34.18 ± 1.37 8.9 ± 0.4 0.148 ± 0.053 19.53 ± 4.80

31.02 ± 1.64 8.0 ± 0.5 0.166 ± 0.031 13.47 ± 2.07

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

Leukocytes (% total blood cells) Lymphocytes (% all white blood cells) Thrombocytes (% all white blood cells) Monocytes (% all white blood cells) Granulocytes (% all white blood cells)

Basal feed

Low DON

Medium DON

High DON

4.4 ± 0.3 57.8 ± 4.5 36.1 ± 4.0 2.6 ± 0.5 3.5 ± 1.0

4.1 ± 0.5 55.0 ± 3.3 38.4 ± 3.4 3.2 ± 0.9 3.5 ± 0.9

4.3 ± 0.3 59.4 ± 1.2 35.6 ± 1.8 2.3 ± 0.3 2.8 ± 1.0

4.5 ± 0.3 60.6 ± 2.1 33.9 ± 1.2 1.8 ± 0.4 3.7 ± 0.9

Table 5 Size 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 same letter (a and/or b) are not significantly different from each other (Wilcoxon test, P < 0.05).

Erythrocyte length (lm) Erythrocyte width (lm) Nucleus length (lm) Nucleus width (lm) Shape factor

Basal feed

Low DON

Medium DON

High DON

11.9 ± 0.1a 8.0 ± 0.2 5.0 ± 0.1a,b 2.7 ± 0.1a,b 1.5 ± 0.0

11.1 ± 0.3b 8.0 ± 0.1 4.9 ± 0.1a 3.0 ± 0.1a 1.5 ± 0.0

11.5 ± 0.1a,b 7.7 ± 0.1 4.9 ± 0.1a 2.6 ± 0.1b 1.5 ± 0.0

11.8 ± 0.1a 8.0 ± 0.1 5.4 ± 0.1b 2.9 ± 0.1a,b 1.5 ± 0.0

weeks of feeding. In head kidney cells from the same fish this difference was observed in medium dose-treated fish. Significant effects of LPS stimulation on NO production in head kidney and trunk kidney was also observed after two weeks of recovery following the experimental feeding of DON (P < 0.05; Figs. 3B and 4B) with the exception of fish treated with high dose DON diet in the recovery phase. In addition, differences in LPS-stimulated leukocytes due to DON treatment of fish were not observed after the recovery phase of two weeks. Cell viability of leukocyte cultures was measured in test plates used previously for the assay of NO production in order to investigate whether effects on NO production are related to altered cell viability. Effects of DON feeding on cell viability were not observed in leukocytes isolated from trunk kidneys at both sampling dates after 96 h of in vitro culture (Fig. 5). Cell viability was reduced in un-stimulated head kidney cells from fish of all DON-treated groups and in LPS-treated leukocytes of fish fed high dose DON diet for four weeks compared to control fish (Fig. 6A). This was not found in DON-treated fish which received control feed for additional two weeks (Fig. 6B). Moreover, cell viability was significantly lower in LPS-treated head kidney cells in all fish from the first sampling date compared to un-stimulated cells from the same fish (Fig. 6A) which was not observed in cells similarly derived from trunk kidneys (Fig. 5A). In addition, in all cell cultures derived from head 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 respiratory burst assay was not observed in trunk kidney-derived cells of fish fed the experimental diets for 4 weeks (Fig. 7), stimulation with PMA increased NBT conversion in all head kidney leukocytes with the exception of the fish that received the high dose feed (Fig. 8). Respiratory burst measured as increased NBT conversion was not significantly influenced in trunk kidney cells after four weeks of DON application to fish (Fig. 7A). However, after two weeks of recovery stimulation with PMA showed less reactivity in leukocytes of fish that received the medium and the high dose feed (Fig. 7B). The respiratory burst of un-stimulated head kidney cells was reduced in fish fed the medium dose feed whereas the NBT conversion in the other groups was only slightly influenced (Fig. 8A). Moreover, PMA-stimulated head kidney cells of all DON-fed fish showed less reaction to PMA than cells from control fish after four weeks of feeding (Fig. 8A) which was not observed after two weeks of recovery (Fig. 8B). Arginase activity of leukocytes was not significantly influenced by DON treatment of fish when treatment groups were compared to control fish (Fig. 9). However, the reduction of NO production correlates with the response of the arginase assays in head kidney cells (Pearson correlation coefficient: 0.480, significance: 0.018 for untreated leukocytes and Pearson correlation coefficient:

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

b

200

DON-treated recovery

SOD [U mg protein-1]

180 160 140 120 100 80

a

60

a

40

A

40

trunk kidney

- LPS + LPS

35

Nitrite [µmol * L-1]

48

a

30

a

25 20

a,b

a,b

15 10

20

b

5

0 control

low dose

0

medium dose high dose

control

treatment group

low dose

medium dose

high dose

treatment group 2000

40 - LPS + LPS

35

1500 1250 1000 750 500

B Nitrite [µmol * L-1]

CAT [U mg protein-1]

DON-treated recovery

b

1750

a,b

a

250

a

30 25 20 15 10 5

0 control

low dose

medium dose

high dose

treatment group Fig. 2. Activities of catalase (CAT) and superoxide dismutase (SOD) in lysates of erythrocytes 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 from each other (Wilcoxon test, P < 0.05).

0.454, significance: 0.018 for LPS- versus forskolin-treated immune cells). 4. Discussion Addition of DON to experimental feed was successful. Contamination of experimental diets was comparable to DON values that can be found in commercially available feeding stuffs reaching values of up to 825 lg kg1 DON (Pietsch et al., 2013). These values are far below the recommended levels by the European Commission (2006/576/EC). In other vertebrate species DON is rapidly absorbed into the systemic circulation (tmax  0.2–4.1 h) and is further characterized by a marked species-dependent variation in bioavailability (6–139%) (Dänicke and Brezina, 2013). However, only very low DON and no DOM-1 concentrations could be detected in biological samples of carp (data can be found in the Supplement). On the one hand, this was probably due to the limited amount of tissue or plasma samples that were available for analyses. On the other hand, retention and accumulation of DON in 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 experimental diets, although it is fast and time-dependent, probably did not influence the application of DON because fish in all groups tookup the pellets at least within 30 min. Exposure of fish to DON in water was negligible due to the high flow-through during the experiments. Intake of DON-contaminated feed in mice was reduced (Gouze et al., 2006) resulting in effects on weight gain. The latter was also found at concentrations of 3.7 mg DON per kg feed in Atlantic salmon after 15 weeks of feeding and at concentrations

0 control

low dose

medium dose

high dose

treatment group Fig. 3. NO production in trunk kidney cells after ex vivo incubation with and without LPS for 96 h, cells were isolated from experimental fish after 4 weeks of feeding (A) and with additional 2 weeks of recovery (B), n = 6; mean ± SE; means with the same letter are not significantly different from each other which applies to bars of the same colour (Wilcoxon test, P < 0.05).

of 0.3–2.6 mg DON per kg feed in rainbow trout after 56 days of feeding (Döll et al., 2011; Hooft et al., 2011). However, cyprinids appear to be less sensitive because weight gain of carp was not influenced by DON at concentrations ranging from 352 to 953 lg kg1 which was also observed in zebrafish in a feeding trial for 45 and 260 days using DON concentrations ranging from to 0.1 to 3 mg DON per kg feed (Sanden et al., 2012). However, blood haematology was influenced. DON has not been shown to affect red blood cells previously. Erythrocytes are released into circulation at an early stage of development and elongation and flattening occur in the blood stream (Sekhon and Beams, 1969; Hardig, 1978). A reduced length of circulating erythrocytes in fish fed the low dose diet indicates an increased abundance of juvenile erythrocytes which are smaller and more round-shaped (Houston, 1997). In carp head kidney and spleen contribute to the release of erythrocytes into circulation (Ken-ichi and Yasuo, 1989; Kondera et al., 2012). Whether juvenile cells have been released from storage in the spleen or head kidney or resulted from de novo formation could not be distinguished. The occurrence of more juvenile cells in circulation can have several reasons since altered respiratory circumstances in fish may lead to increased erythropoiesis, increased division of circulating cells, karyorrhexis or cell breakdown (Houston and Murad, 1992). The increased nucleus size in erythrocytes of fish in the high-dose group suggests that karyorrhexis took place which is accompanied by occurrence of enlarged, more round-shaped nuclei and a subsequent reduction of cytoplasmic volume (Murad et al., 1990). However, this was not accompanied by significant effects on the abundance and size of melano-macrophage centers in spleen of DON-treated fish (data

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

headkidney

A

100

80

- LPS + LPS

a

a,b

60

40

a,b 20

absorbance [540nm]

Nitrite [µmol * L-1]

A

b

0.30

trunk kidney 0.25

- LPS + LPS

0.20 0.15 0.10 0.05 0.00

0 control

low dose

medium dose

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

not shown) which would support the hypothesis of increased elimination 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 without significantly affecting spleen histology remains obscure, but the involvement of the kidneys has not been investigated. However, it may be assumed that the cytotoxicity of this toxin in the blood stream contributed to this effect. Since cytotoxicity of DON often also involves oxidative stress (Kouadio et al., 2005; Pietsch et al., 2011b), this might also have occurred in DON-treated carp. Similar to all aerobic organisms, fish have regularly to deal with oxidative stress and antioxidative enzymes have been detected in most fishes (Martínez-Álvarez et al., 2005). Effects of DON on antioxidative enzymes have been observed in erythrocyte samples of fish fed the low-contaminated diet. Increased expression of hepatic CuZn SOD in liver has also been shown in zebrafish treated with much higher DON concentrations (2 or 3 mg DON per kg feed) (Sanden et al., 2012). The reason for the lack of effects on the antioxidative enzymes in erythrocytes of carp treated with the medium and the high dose DON diet remains obscure. It may be assumed that toxicity of DON in the blood stream prevented a response of the antioxidative enzymes in erythrocytes. Decreased SOD activity in DON-treated rat liver cells has been reported which shows that induction of SOD is not a general response to DON (Sahu et al., 2008). The involvement of oxidative stress in DON toxicity in carp has already been shown previously, whereby increased lipid peroxidation was observed in head kidney, spleen and liver of carp treated with the high dose DON feed (Pietsch et al., 2014).

Fig. 5. Cell viability after 96 h of incubation with and without LPS in in vitro cultures of trunk kidney cells of fish after 4 weeks of DON feeding (A) and additional 2 weeks of recovery (B), n = 6, mean ± SE.

Since acute toxicity of DON has often been shown in actively dividing cells, the immune system is an important target. Depending on dose and exposure regime, DON has been shown to be both immunosuppressive and immunostimulatory in mammals (Pestka et al., 2004; Pestka, 2008). In the present study both, pro-inflammatory (NO and ROS production) and anti-inflammatory (arginase activity) immune reactions, have been determined which revealed that pro-inflammatory immune responses were affected by DON. The effects on pro-inflammatory reactions of immune cells showed some differences between leukocytes isolated from head kidneys and trunk kidneys. Since isolated cell cultures from both organs do not show substantial different cell populations (Pietsch et al., 2008), it can be assumed that the reactivity of the immune cells from both organs and ability to be stimulated by intrinsic or extrinsic signals are different. However, there is no explanation for that at the moment. The effects on the anti-inflammatory enzyme arginase paralleled the effects on the pro-inflammatory immune responses in head kidney cells although no differences in arginase activities have been observed between the treatment groups. Thus, a distinct polarization of the immune responses into pro-inflammatory or anti-inflammatory directions is not obvious. An involvement of glucocorticoids in the resulting immune responses can be excluded, because the measured cortisol levels indicated no general stress response in fish. It is concluded that a mixture of activating and suppressing reactions occurs upon DON exposure. Mixed immune responses have already been observed in the mammalian immune system (Benoit et al., 2008; Porta et al., 2009). The possible mechanisms by which DON can both suppress and stimulate immune functions have been suggested in mammalian systems.

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

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Fig. 8. Respiratory burst after 24 h of in vitro culture of head kidney cells of fish after 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 by PMA for 90 min measured by absorbance at 620 nm, n = 6; mean ± SE; * = difference to controls at P < 0.05 which applies to bars of the same colour (Wilcoxon test).

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low dose

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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 24 with 1 lM forskolin compared to unstimulated cells, n = 6; mean ± SE.

At high doses a rapid onset of apoptotic events occurs in leukocytes which 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

responses in DON-treated lymphocyte cultures from mammals has been reported and apoptosis of macrophages was observed (Miller and Atkinson, 1986; Yang et al., 2000; Zhou et al., 2005). Effects of DON on cell viability were observed in the LPS-treated leukocytes derived from head kidneys of carp. The cytotoxic effects of LPS on immune cells have been reported to be strain-specific in mice (Peavy et al., 1978). In addition, LPS stimulation has been show to strongly increase pro-inflammatory cytokines and apoptosis of immune cells (Islam and Pestka, 2006) which further emphasizes the influence of DON on immunological functions. In contrast to apoptosis, low concentrations of DON appear to promote expression of a diverse array of cytokines in vitro and in vivo (Azcona-Olivera 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; Amuzie et al., 2009; Becker et al., 2011). It has been shown that increased cytokine expression leads to up-regulation of suppressors of cytokine signaling which, in turn, minimizes the inflammatory response and consequently possible damage to tissues. We observed decreased pro-inflammatory responses in carp leukocytes. This probably is meaningful for fish raised in aquaculture especially when the susceptibility to diseases is concerned. However, further details on the mode of action of DON on immune cells of fish remain unknown and especially cytokine signaling in DON-treated fish 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 be found in the online version.

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

Deoxynivalenol (DON) is one of the most important members of Fusarium toxins since it often can be found in relevant concentrations in animal feeds. T...
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