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Effects of deoxynivalenol and zearalenone on oxidative stress and bloodphagocytic activity in broilersRadka Borutova a; Stefan Faix a; Iveta Placha a; Lubomira Gresakova a; Klaudia Cobanova a; Lubomir Leng a

a Institute of Animal Physiology, Slovak Academy of Sciences, Kosice, Slovak Republic

Online Publication Date: 01 August 2008

To cite this Article Borutova, Radka, Faix, Stefan, Placha, Iveta, Gresakova, Lubomira, Cobanova, Klaudia and Leng,Lubomir(2008)'Effects of deoxynivalenol and zearalenone on oxidative stress and blood phagocytic activity in broilers',Archives ofAnimal Nutrition,62:4,303 — 312

To link to this Article: DOI: 10.1080/17450390802190292

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Effects of deoxynivalenol and zearalenone on oxidative stress and blood

phagocytic activity in broilers

Radka Borutova*, Stefan Faix, Iveta Placha, Lubomira Gresakova, Klaudia Cobanovaand Lubomir Leng

Institute of Animal Physiology, Slovak Academy of Sciences, Kosice, Slovak Republic

(Received 3 January 2008; accepted 28 April 2008)

Effects of dietary contamination with various levels of deoxynivalenol (DON) andzearalenone (ZEA) were investigated on Ross 308 hybrid broilers of both sexes. Afterhatching, all chickens were fed an identical control diet for two weeks. Then chickens ofGroup 1 received a diet contaminated with DON and ZEA, both being 3.4 mg � kg71,while Group 2 received DON and ZEA at 8.2 and 8.3 mg � kg71, respectively. The dietof the control group contained background levels of mycotoxins. Samples of blood andtissues were collected after two weeks. Intake of both contaminated diets resulted in asignificantly decreased activity of glutathione peroxidase (GPx) and increased level ofmalondialdehyde (MDA) in liver tissue, while in kidneys the concentration of MDA wassignificantly increased only in Group 1. On the other hand, activities of blood GPx andplasma g-glutamyltransferase (GGT) were elevated in Group 2 only. Activities ofthioredoxin reductase in liver and GPx in duodenal mucosa tissues, superoxidedismutase (SOD) in erythrocytes as well as levels of MDA in duodenal mucosa and a-tocopherol in plasma were not affected by dietary mycotoxins. Blood phagocytic activitywas significantly depressed in Group 1 and 2. These results demonstrate that dietscontaminated with DON and ZEA at medium levels are already able to induce oxidativestress and compromise the blood phagocytic activity in fattening chickens.

Keywords: poultry; fusarium mycotoxins; antioxidative enzymes; lipid peroxidation;phagocytosis

1. Introduction

Trichothecene mycotoxins are a group of structurally similar fungal metabolites that arecapable of producing a wide range of toxic effects. Deoxynivalenol (DON, vomitoxin), is atrichothecene mycotoxin produced by several Fusarium species, primarily F. graminearum,and appears as a contaminant with other mycotoxins in many commodities. DON is awell-known inhibitor of protein synthesis. The toxin binds to peptidyl transferase(Feinberg and McLaughlin 1989), inhibits the synthesis of RNA and DNA via binding tothe ribosome, and also affects cell membranes. It is assumed that cells and tissues with highprotein turnover rates such as the small intestine, liver and the immune system are mostseverely affected by DON intoxication (Doll et al. 2003).

Poultry is known to be relatively resistant to the effects of DON in comparison to pigsand other species. Prelusky et al. (1986) estimated systemic absorption of DON tobe 51% of the dose. DON undergoes de-epoxidation and glucuronidation resulting in

*Corresponding author. Email: borutova@saske.sk

Archives of Animal Nutrition

Vol. 62, No. 4, August 2008, 303–312

ISSN 1745-039X print/ISSN 1477-2817 online

� 2008 Taylor & Francis

DOI: 10.1080/17450390802190292

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less toxic metabolite DOM-1 (Beasley et al. 1986). In general, DON has the ability toinduce toxicologic and immunomodulating effects (Oswald et al. 2005). DON can beimmunostimulatory or immunosuppressive depending on dose and exposure frequency(Bondy and Pestka 2000). High-dose trichothecene injures the spleen, thymus, bonemarrow and intestinal mucosa, which can result in immunosuppression and potentiallyincreased susceptibility to several pathogens. The greatest effect of DON on immunity isprobably related to cell-mediated processes, thymic and bursal involution in poultry,impaired phagocytosis and suppression of lymphoblastogenesis (Pestka and Smolinski2005).

Zearalenone (ZEA) is known to induce reproducible disorders due to its estrogenicproperties when it appears in high concentrations in animal feeds. Poultry has been shownto be relatively resistant to this mycotoxin (Danicke et al. 2001; Doll et al. 2003).

It has been suggested that ergosterol per se at high feed concentration can have similarharmful effects on animals as mycotoxins (Bailly et al. 2005). It is known to be acomponent of fungus cell membranes, where it has properties like cholesterol in animalcells. Ergosterol in high doses is toxic after ingestion, inhalation or absorption through theskin. Ergosterol is known as a biologic precursor of vitamin D2.

Adverse effects of mycotoxins on cells are also associated with the increasedproduction of free radicals and reactive oxygen species resulting in oxidative damage oftarget tissues (Dvorska et al. 2007). There is a very delicate balance between antioxidantsand pro-oxidants in the body of young chickens. Nutritional stress factors can act asbreakers of the antioxidant/pro-oxidant balance (Surai 2002).

The aim of this experiment was to investigate the effects of diets contaminated withDON and ZEA up to levels 8.3 mg � kg71 complete feed on parameters of tissue oxidativestress, antioxidant status and blood phagocytic activity in fattening chickens.

2. Materials and methods

2.1. Animals, diets and treatments

Sixty Ross 308 hybrid broilers of both sexes were randomly divided at day of hatching intothree groups (n ¼ 20 in each) and all were fed an uncontaminated diet for two weeks.After this time, broilers of Groups 1 and 2 started being fed diets contaminated withdifferent concentrations of mycotoxins. The control group of birds continued to be fed theuncontaminated diet.

The broilers were placed in large pens with wood shavings. Rearing of the chickens wasdone with a lighting regimen of 23 h light to 1 h dark and lasted for four weeks. The initialroom temperature of 32–338C was reduced weekly by 18C to a final temperature of 288C.All birds had free access to water and feed. The experiment was carried out in accordancewith established standards for use of experimental animals and birds. The protocol wasapproved by the local ethical and scientific authorities.

To provide stable dietary contents of mycotoxins during the entire experimentalperiod, the chickens were fed only one type of diet (HYD-01). The composition of this dietis given in Table 1.

The final diets were obtained by mixing the basal diet (BD, the part of complete dietbefore addition of 40% portion of control or contaminated maize) supplied byAgrokonzult s.r.o., Nove Zamky, Slovakia, with control or contaminated maize batches.The contents of deoxynivalenol (DON), zearalenone (ZEA), total aflatoxins, ochratoxin Aand ergosterol in control maize, two batches of contaminated maize and in BD aresummarised in Table 2. Contaminated batches of maize were obtained by their cultivation

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with Fusarium graminearum for four weeks at the Slovak Agriculture University in Nitra(Labuda et al. 2003). The final concentrations of mycotoxins and ergosterol in theexperimental diets are shown in Table 3.

2.2. Sample collections

At four weeks of age, six randomly chosen chickens from each group were anaesthetisedby intraperitoneal injection of xylazine (Rometar 2%, SPOFA, Czech Republic) and

Table 1. Composition of diet HYD-01* fed to broilers during the entire experiment (4 weeks).

Component Contents

Wheat (ground, 11% CP) [g � kg71] 204.2Maize (ground 8.3%CP) [g � kg71] 400.0Oil-rape seed [g � kg71] 10.0Soybean extracted ground meal (46% CP) [g � kg71] 330.0Fish meal (62% CP) [g � kg71] 20.0Monocalciumphosphate [g � kg71] 7.0Limestone [g � kg71] 16.0Feed salt [g � kg71] 2.8Premix HYD 01 ARO# [g � kg71] 10.0Dry matter [g � kg71] 883Metabolizable energy [MJ � kg71] 12.75Crude protein (analysed) [g � kg71] 210.6

Notes: #Provided premix per kg complete diet: vitamin A, 15 400 IU; vitamin D3, 5000 IU; vitamin K, 3.8 mg;vitamin E, 71.1 mg; lysine, 13.4 g; methionine, 5.81 g; Se, 0.37 mg; riboflavin, 9.28 mg; thiamin, 6.36 mg;pyridoxin, 9.43 mg; cobalamin, 5.81 mg; Zn, 88.47 mg; I, 1.05 mg; Co, 0.12 mg; Mn, 109.97 mg; Cu, 17.12 mg; Fe,145.93 mg; Ca, 9.2 g; P, 6.06 g; Na, 1.52 g; K, 8.16 g; Cl, 2.23 g; Mg, 1.70 g; *HYD-type of feed category,01-chicken broiler starter.

Table 2. Mycotoxins and ergosterol contents in maize batches and in the basal diet (BD, part of thecomplete diet to which 40% portion of control or contaminated maize was later added).

Concentration [mg � kg 7 1]

DON* ZEA# Total aflatoxin Ochratoxin A Ergosterol

MaizeControl group 1.24 0.17 0.004 0.0009 7.4Experimental group 1 8.44 8.40 0.012 0.0072 58.0Experimental group 2 20.22 20.70 0.002 0.0034 103.0

Basal diet 0.17 0.05 0.005 0.0007 6.7

Notes: *Deoxynivalenol; #Zearalenone.

Table 3. Final concentrations of mycotoxins and ergosterol in control and experimental diets.

Concentration [mg � kg 7 1]

DON* ZEA# Total aflatoxin Ochratoxin A Ergosterol

Control group 0.60 0.07 0.004 0.001 6.98Group 1 (lower mycotoxin levels) 3.44 3.36 0.008 0.003 27.22Group 2 (higher mycotoxin levels) 8.19 8.28 0.004 0.001 45.22

Notes: *Deoxynivalenol; #Zearalenone.

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ketamine (Narkamon 5%, Czech Republic) at 0.6 and 0.7 ml � kg71 BW, respectively.After laparotomy, blood was collected into heparinised tubes by intracardial punction andcentrifuged for plasma specimens at 1180 g for 15 min. Samples of blood and plasma foranalysis were frozen and stored at 7658C. Following euthanasia, samples of liver, kidneyand duodenal mucosa tissues were collected and stored also at 7658C until analysis.

2.3. Sample analysis

Mycotoxins in all three maize batches used and in the basal diet were detected using thecommercial competitive enzyme-linked immunosorbent assay-based Veratox 5/5 kit(Neogen Corp., Lansing, Michigan, USA). Concentrations of ergosterol in maizebatches and the basal diet were analysed by the fluorodensitometric method (Baillyet al. 1999).

Activity of blood glutathione peroxidase (GPx, EC 1.11.1.9) was determined using themethod of Paglia and Valentine (1967) with a Ransel kit (Randox, UK). To analyse theactivities of GPx in liver, kidney and duodenal mucosa, pieces of tissue were homogenisedin phosphate buffer saline with pH 7.4 and containing 1 mM Na2 EDTA � 2H2O.Homogenates were centrifuged at 13,680 g at 48C for 20 min. Enzyme activity in thesupernatant was measured by monitoring oxidation of NADPHþH þ at 340 nm, asdescribed by Paglia and Valentine (1967).

Tissue samples of liver, kidney and duodenal mucosa for malondialdehyde (MDA)determination were homogenised with deionised distilled water and 50 ml of butylatedhydroxytoluene. The MDA concentrations in homogenates were measured by themodified fluorometric method in accordance with Jo and Ahn (1998).

Haemoglobin (Hb) content of blood and superoxide dismutase (SOD, EC 1.15.1.1)activities (Arthur and Boyne 1985) in erythrocytes were analysed using kits from Randox,UK. The protein concentrations in the tissues examined were measured by thespectrophotometric method of Bradford (1976).

Spectrophotometric determination of thioredoxin reductase (TrxR, EC 1.8.1.9)activity was done with a Thioredoxin Reductase Assay Kit (Sigma-Aldrich Inc., StLouis, MO, USA) by the method of Holmgren and Bjornstedt (1995). It is basedon the reduction of 5,50-dithiobis (2-nitrobenzoic) acid (DTNB) with NADPH to 5-thio-2-nitrobenzoic acid (TNB), which produces a strong yellow colour that is measured at412 nm.

The g-glutamyltransferase (GGT, EC 2.6.1.2) activity was measured using the kit fromRandox, UK, by the method of Szasz (1969).

The a-tocopherol concentrations in plasma were determined by the HPLC method ofTuckova and Kastel (1999).

The phagocytic activity was measured by direct counting procedure using microspherichydrophilic particles (MSHP). Ingestion of MSHP particles by polymorphonuclear cells(PMNs) was determined with a modified test described by Vetvicka et al. (1982).Briefly, 50 ml of MSHP particle suspension (ARTIM, Prague, Czech Republic) was mixedwith 100 ml of blood in an Eppendorf-type test tube and incubated at 378C for 1 h. Thenblood smears were prepared and stained in accordance with May-Grunwald and Giemsa-Romanowski. In each smear, 100 cells were examined to determine the relative number ofwhite cells containing at least three engulfed particles (phagocytic activity) and the indexof phagocytic activity (number of engulfed particles per total number of neutrophils andmonocytes observed). The percentage of phagocytic cells was evaluated using an opticalmicroscope, by counting PMN up to 100.

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2.4. Statistical analysis

Statistical analysis was done by one-way analysis of variance (ANOVA) with the post hocTukey multiple comparison test using GraphPad Software (USA). The results are given asmeans + SEM.

3. Results

Chickens for fattening did not show any visible clinical signs of toxicosis during the wholeof our experiment. However, two weeks of intake of diets contaminated with mycotoxinsresulted in almost 3-fold increased levels of MDA in liver tissue in groups with either lowerand higher concentrations of mycotoxins while its concentration in kidneys wassignificantly elevated only in Group 1, fed on the diet with lower contents of DON andZEA. Neither the levels of MDA nor activities of GPx in the tissue of duodenal mucosawere influenced by feeding on diets with different concentrations of Fusarium mycotoxins.Dietary mycotoxin contamination led to significantly decreased GPx activity in liver tissueof broilers, while no change of TrxR activity in this tissue was noticed (Table 4).

The GPx activity in blood and GGT activity in plasma were significantly increasedonly in the group of birds fed on a diet with higher concentration of mycotoxins. Noeffects of mycotoxin contaminated diet on plasma levels of a-tocopherol and SOD activityin erythrocytes were observed in the experimental groups of birds.

Blood phagocytic activity was significantly reduced in both groups fed contaminateddiets and seemed to show a dose dependent pattern. The index of phygocytic activity wasaffected by intake of diets with higher content of mycotoxins only (Table 5).

4. Discussion and conclusions

It is well established now that DON is mostly metabolised already in the digestive tractcontents and mucosa. In case of higher mycotoxin delivery by feed, DON undergoes de-epoxidation and glucuronidation predominantly in the liver, where it is delivered by the

Table 4. Concentration of malondialdehyde (MDA), activity of glutathione peroxidase (GPx) andthioredoxin reductase (TrxR) in tissues of broilers fed diets contaminated with differentconcentrations of mycotoxins (Means + SEM. n ¼ 6 in each group).

Controlgroup

Group 1 (lowermycotoxin levels)*

Group 2 (highermycotoxin levels)#

Duodenal mucosaMDA [nmol � g71 protein] 41.4 + 8.9 51.9 + 8.4 35.5 + 3.8GPx [U � g71 protein] 5.4 + 1.5 8.4 + 1.3 5.6 + 1.5

LiverMDA [nmol � g71 protein] 137.3 + 23.6a 397.3 + 54.7b 319.7 + 32.4b

GPx [U � g71 protein] 13.8 + 1.7a 7.9 + 1.3b 8.3 + 1.0b

TrxR [U � g71 protein ] 26.5 + 5.6 24.9 + 8.7 30.3 + 6.3

KidneyMDA [nmol � g71 protein] 56.1 + 7.0a 93.2 + 6.8b 65.8 + 8.0a

GPx [U � g71 protein] 15.4 + 3.5 13.9 + 1.5 13.0 + 3.8

Notes: *Both, DON and ZEA, 3.4 mg � kg71 complete feed; #DON and ZEA, 8.2 and 8.3 mg � kg71 completefeed, respectively. Different superscripts within a row indicate significant differences (p 5 0.05).

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portal blood system (Awad et al. 2006). Significant species differences in the liverbiotransformation of ZEA have recently been described. In poultry, after absorption ZEAwas found to be metabolised mainly into b–zearalenone in the liver (b-ZOL) (Malekinejadet al. 2006). Metabolic derivates of ZEA have been shown to be several times more activethan ZEA itself (Gaumy et al. 2001).

Our results demonstrate highly-increased production of MDA with concomitantsignificant reduction of GPx activity in liver tissue of broilers fed on diets contaminatedwith DON and ZEA. It has been reported that lower GPx activity in tissues is generallyaccompanied by an increase in MDA concentration (Balogh et al. 2004). DONcontaminated feed has been reported as a factor increasing TBARS formation in rat(Rizzo et al. 1994) and mice liver (Karppanen et al. 1989). It was found that dietaryinclusion of another trichothecene mycotoxin T-2 toxin also increased lipid peroxidationin the livers of quails (Dvorska and Surai 2001), chickens, ducks and geese (Mezes et al.1999).

Studies on the K562 human erythroleukemia cell line have shown that DNA damageand apoptosis rather than plasma membrane damage and necrosis may be responsible fortoxicity of DON (Minervini et al. 2004). In the liver, the gluthathione and thioredoxinsystems are major players in the regulation of cell redox status (Gromer et al. 2004). Thedecreased activity of GPx in liver tissue together with almost 3-fold greater MDAproduction indicate that combinations of DON and ZEA in concentrations from 3 to8 mg � kg71 feed are capable of inducing significant oxidative stress in chickens.

On the other hand, the liver tissue activity of another specific selenoenzyme TrxR wasnot affected by dietary mycotoxins; we have no rational explanation for this finding,however.

The adverse effects of mycotoxin contaminated diet on the livers of our chickens aredemonstrated also by findings of significant increased plasma activities of g-glutamyl-transferase (GGT). This enzyme is known to be involved in the transfer of amino acidsacross the cellular membrane and in glutathione metabolism. Serum GGT activity iswidely used as an index of liver tissue integrity or a marker of liver dysfunction. Thecurrent as well as previous studies show that serum GGT activity may be an early markerof oxidative stress (Lim et al. 2004).

Table 5. Activity of glutathione peroxidase (GPx) in blood, superoxid dismutase (SOD) in redblood cells (RBC), activity of g-glutamyltransferase (GGT) and concentration of a-tocopherol inplasma, as well as blood phagocytic activity and its index in broilers fed diets contaminated withdifferent concentrations of mycotoxins (Means + SEM. n ¼ 6 in each group).

Controlgroup

Group 1(lower mycotoxin

levels)*

Group 2(higher mycotoxin

levels)#

GPx in blood [U � g71 Hb] 38.1 + 3.3a 37.7 + 4.1a 70.1 + 10.3b

SOD in RBC [U � g71 Hb] 1566 + 113.0 1447 + 40.7 1572 + 52.6GGT in plasma [U � l71] 34.1 + 2.7a 42.5 + 6.8a 64.9 + 7.8b

a-tocopherol [mmol � l71] 16.5 + 1.7 15.8 + 1.2 22.1 + 2.4Phagocytic activity [%]{ 33.0 + 1.4a 25.2 + 1.8b 18.6 + 1.9c

Index of phagocytic activity{ 1.6 + 0.2a 1.0 + 0.2ab 0.8 + 0.1b

Notes: *Both, DON and ZEA, 3.4 mg � kg71 complete feed; #DON and ZEA, 8.2 and 8.3 mg � kg71 completefeed, respectively. {Number of white cells containing at least three engulfed particles/100 white cells (neutrophilsand monocytes); {Number of engulfed particles per total number of neutrophils and monocytes observed.Different superscripts within a row indicate significant differences (p 5 0.05).

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Unexpectedly, the MDA level in kidney tissue was increased only in the birds fed onthe diet with lower DON and ZEA contents (both 3.4 mg � kg71 feed), whereas it did notchange in chickens on the diet with DON and ZEA levels 8.2 and 8.3 mg � kg71 feed,respectively. We have no evidence producing a rational explanation for these findings. Onecould object that when DON undergoes more extensive absorption from the GI tract, ahighly efficient hepatic and/or renal first-pass effect effectively removes the toxin fromsystemic circulation (Rotter et al. 1996). We did not measure the circulating DON levels insystemic or portal blood plasma, so we can only speculate that this discrepancy could becaused by the presence of some other mycotoxins in diets which were not analysed, or byconjugated mycotoxins. Such conjugated DON-3-glucoside referred to as so-calledmasked mycotoxin has been shown to escape routine analysis (Berthiller et al. 2005), suchas was done on the maize used in our experiment. The maize levels of ergosterol, as afurther toxic substance associated with fungi contamination, followed the same pattern asthe DON and ZEA levels determined by our analysis. The possible higher presence offumonisins or other Fusarium toxins cannot be excluded in the diet with DON and ZEAlower levels. However, it should be stressed that the same strain of Fusarium graminearumwas used for inoculation of both sterilised identical maize batches, and their cultivationwas done at the same time and under the same conditions.

The kidney tissue activities of GPx did not show any differences between dietarytreatments. This could be in part ascribed to a considerably smaller MDA response inkidney tissue to contaminated diets than that in the liver in the case of the diet with lowerDON and ZEA contents, while the diet with DON and ZEA higher levels did not induceany response in kidney MDA production.

In contrast to the liver tissue showing reduced GPx activity, this parameter in wholeblood, which mostly represents the actual enzyme activity in red blood cells (RBC),increased to almost doubled values in the third group of birds with higher DON and ZEAintake when compared to control. Explanation of the higher GPx activity in RBC inbroilers fed on the diet with 8.3 mg ZEA per kg feed is based on the ability of estrogens toupregulate the activity of this selenoenzyme (Flohe and Brigelius-Flohe 2001). ZOL-derivates of ZEA are known to possess strong binding activity to estrogenic receptors.Although ZEA does not have estrogenic structure, it has estrogenic effects on animals.ZEA and its metabolites have been reported to influence the serum levels of progesteroneand estradiol too (Gajecki 2002).

No effects of dietary contamination with mycotoxins on plasma levels of a-tocopherolwere found in our experiment. Its concentration in birds with highest DON and ZEAintake showed some trend towards increased values, but the difference was not significant.It has been shown that another dietary trichocetene T-2 toxin is able to reduce the a-tocopherol levels in chicken plasma (Coffin and Combs 1981). The adverse effects of T-2toxin on chicken liver concentration of vitamin E has been recently described (Dvorskaet al. 2007). We measured the levels of a-tocopherol in plasma only. One of theexplanations why we did not find any response of plasma vitamin E to our DON and ZEAcontaminated diets could be that liver measurements better reflect the actual bodydeposits. The second reason might be the relatively short duration of bird exposure to themycotoxin-contaminated diets, and continual delivery of a-tocopherol by dietary intake.In general, when radicals are formed within the phospholipid bilayer, a-tocopheroldonates its own hydrogen atom and electron to the radical, stabilising the radical intononreactive form. It is oxidised into an unreactive tocopheroxyl radical which can beconverted into non-radical form by ascorbic acid. It is well established that deficiency ofone antioxidant can to some extent induce a higher production of another antioxidant.

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Moreover, currently it is not clear if mycotoxins stimulate lipid peroxidation directly byenhancing free radicals production, or if the increased tissue susceptibility to lipidperoxidation is a result of a compromised antioxidant system (Surai 2006).

Two-week intake of the diets contaminated with lower or higher concentrations ofDON and ZEA resulted in suppressed blood phagocytic activity in the chickens. DON hasbeen shown to reduce phagocytic and microbicidal activity and superoxide anionproduction using an in vitro experimental model (Ayral et al. 1992). In chickens, aflatoxinsand ochratoxin A have been shown to suppress blood phagocytosis (Chang and Hamilton1980; Neldon-Ortiz and Qureshi 1992). Reduced phagocytic activity of leukocytes due toDON consumption could be the consequence of breaking the important communicationsnetwork between cellular and humoral components, leading to dysbalance of the immunesystem and potential immunosuppression (Corrier 1991). Compromised immunity isusually associated with higher susceptibility of chickens to infection diseases. Leukocytesare an ideal target for free radicals due to the high levels of polyunsaturated fatty acids intheir membranes (Surai 2006).

In conclusion, the results of this experiment demonstrate the adverse effects ofFusarium mycotoxins on the antioxidative status and blood phagocytic activity in chickensfor fattening. It is shown that diets contaminated with DON and ZEA at medium levels of3.4 mg � kg71 feed are already able to induce oxidative stress in liver tissue andcompromise the blood phagocytic activity.

Acknowledgements

The study was supported by the Grant Agency for Science (VEGA) of the Slovak Republic, GrantNo. 2/6173/6, and by the Science and Technology Assistance Agency, Slovak Republic, Grants No.APVT-51-004804 and LPP-0213-06.

Our thanks belong to Dr Christine Lang from University for Veterinary Medicine, Vienna foranalysis of mycotoxins and Dr Jean-Denis Bailly from National Veterinary School in Toulousefor analysis of ergosterol levels in experimental diets. We are grateful to Mrs M. Stavrovska for theexcellent technical assistance.

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