Laboratory of Animal Biology, National Institute for Research and Development for Biology and Animal Nutrition, Calea Bucuresti No. 1, Balotesti, Ilfov 077015, Romania
National Institute of Pathology Victor Babes , Splaiul Independentei No. 99-101, 050096 Bucharest, Romania
a r t i c l e
i n f o
Article history:
Received 25 March 2013
Accepted 18 May 2013
Available online 28 May 2013
Keywords:
Zearalenone
Swine
Oxidative stress
Inammation
a b s t r a c t
Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by the fungi of Fusarium genera. Piglets were fed for 18 days with a control or a ZEN (316 ppb) contaminated diet. At the end of the experiment tissue samples were taken for assessment of: lymphocyte proliferation, monocytes and
granulocytes respiratory burst, inammatory cytokine synthesis in blood and liver, expression of genes
involved in oxidative stress or in inammation, plasma biochemical parameters, total antioxidant status
and nitric oxide synthesis. In blood, ZEN increases the respiratory burst of monocytes and the inammatory cytokine (TNF alpha, IL-1 beta, IFN gamma) synthesis, while in liver, ZEN decreases the synthesis of
all inammatory cytokines investigated. In liver and spleen, different effect on the expression of genes
involved in oxidative stress and inammation was observed. While in liver, ZEN decrease the expression
of cyclooxigenase gene, but increase the expression of glutathione peroxydase and catalase genes; in
spleen, ZEN induces a decrease of the superoxide dismutase gene expression together with an increase
of the cyclooxigenase. In conclusion, our results showed that liver, spleen and blood may also be target
tissues in weanling piglets fed ZEN contaminated diet, with different effects on oxidative stress and
inammation.
2013 Elsevier Ltd. All rights reserved.
1. Introduction
Mycotoxins are secondary metabolites produced by fungi
belonging mainly to the following genera: Aspergillus, Penicillium,
Fusarium, Alternaria and Claviceps (Blunden et al., 1991). Until
now, more than 400 secondary metabolites with toxigenic potential have been reported and Food and Agriculture Organization
(FAO) estimates that 25% of the worlds agricultural commodities
are contaminated with mycotoxins (WHO, 1991) with signicant
economic losses (Kabak et al., 2006; Hussein and Brasel, 2001).
Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by the fungi of Fusarium genera (Richard, 2007; Bennett and
Klich, 2003). ZEN is a contaminant of cereals and plant products
(MacDonald et al., 2005; Engelhardt et al., 2006; Tabuc et al.,
2009). ZEN has a major effect on human and animal health and
raises serious worldwide economic problems (FAO/WHO, 2000).
It was shown that ZEN has strong estrogenic and anabolic activities (Kuiper-Goodman et al., 1987; Pazaiti et al., 2012) causing
serious alterations in the reproductive system of laboratory and
domestic animals (Kuiper-Goodman et al., 1987; Diekman and
Green, 1992). This effect results from the capacity of ZEN to bind
Corresponding author. Tel.: +40 21 351 20 82; fax: +40 21 351 20 80.
E-mail address: daniela.marin@ibna.ro (D.E. Marin).
0278-6915/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.fct.2013.05.033
of the production of inammatory cytokines (Ruh et al., 1998; Salah-Abbs et al., 2008; Marin et al., 2010, 2011).
Inammation and the oxidative stress are tightly correlated, the
pathways that generate the mediators of inammation (e.g. adhesion molecules, interleukins, etc.) being all induced by oxidative
stress (Jenny, 2012; Kim et al., 2012). Despite this, only a few studies (Marin et al., 2010; Busk et al., 2011) investigating the effect of
ZEN on both inammation and oxidative stress have been reported
for swine, the most sensitive animal species to zearalenone and its
metabolites (EFSA, 2004).
In the present study we have investigated the effect of ZEN on
some parameters of oxidative stress and inammation in piglets
after an oral intoxication with zearalenone.
2. Materials and methods
2.1. Reagents
All chemicals, immunological reagents and media components were purchased
from Sigma (SigmaAldrich, Steinheim, Germany) unless otherwise noted.
2.2. Animals and treatments
Ten, 4-week-old, TOPIGS-40 crossbred weanling piglets with an initial average
body weight of 9.5 0.6 kg were studied for 18 days. Animals were cared for in
accordance with the Romanian law 206/2004 for handling and protection of the
animals used for experimental purposes and of EU (Council Directive 98/58/EC)
concerning the protection of farmed animals. The study protocol was approved
by the Ethical Committee of the National Research-Development Institute for Animal Nutrition and Biology, Balotesti, Romania.
The animals were housed in pens, ve animals/group/pen, two groups, one control fed control diet and one experimental fed diet intoxicated with ZEN. Animals
were individually identied by ear tag. The body weight was measured two times
at the beginning (day 0) and at the end of the experiment (day 18) for each animal.
Feed and water were provided ad libitum every day of the experiment. Administered feed was recorded daily for each group and feed intake was calculated by difference between the total administered feed and the feed residues measured at the
end of the experiment. They were fed a maizesoybean-meal-based diet (Taranu
et al., 2011; Table 1) contaminated with 250 ppb pure ZEN. The content of ZEN
was analyzed by high performance liquid chromatography (HPLC) with uorescence detection after clean-up with an immune-afnity column (Inertsil ODS-3V)
and a detection limit of 6 ng/g. Other mycotoxins concentration (DON, FB, T-2/
HT-2, OTA, AF) were analyzed by ELISA using ELISA kit Veratox (Neogen, MI,
48912, USA/Canada) with a detection limit of 100, 50, 25, 1 ppb respectively and
were not detected. ZEN content was 40.92 0.15 ppb in the control diet, respectively 316 ppb 30.9 ppb in the zearalenone contaminated diet. Body weights
and food consumption were recorded twice throughout the experiment (day 0
Wheat
Corn
Contaminated corn
Soybean meal
Sunower meal
Powder milk
Gluten
Full fat Soybean
Salt
Monocalcium phosphate
Feed grade limestone
Methionine premix
Lysine premix
Choline premix
Vitamin mineral premixa
Analyzed composition
Crude protein (g/kg)
ZEN (lg/kg)
a
and day 18). Blood samples were aseptically collected in the day 18, by jugular vein
puncture, for oxidative stress, biochemical analysis, cell proliferation and cytokines
production. At the end of the experiment, animals were slaughtered. No alteration
was observed in the morfology of the internal organs. Organs were weighed and
samples of liver, spleen and mesenteric lymph nodes were collected on ice and
stored at 80 C until analyzed.
2.3. Cell proliferation
Peripheral blood mononuclear cells (PBMC) from blood, splenocytes from
spleen and lymphocytes from lymph nodes were isolated as already described by
Taranu et al. (2010). The ability of isolated and mitogen-activated lymphocytes to
proliferate ex vivo was measured by the [methyl-3H]-thymidine proliferation assay.
Cells (1 106 cells/mL), stimulated or not with 10 lg/mL concanavalin A (ConA)
(Type IV, Sigma, Steinheim, Germany), were cultured for 72 h at 37 C and 5% CO2
in 96-well at-bottom tissue culture plates (NUNC, Langelnselbold, Germany). For
the last 18 h of cultivation, cells were labeled with 1 lCi/well of [metyl-3H]-thymidine and than harvested through glass-ber lters (Skatron, Sterling, UK). Incorporation of [methyl-3H]-thymidine was measured with a Canberra-Packard Beta
Counter (PerkinElmer Life and Analytical Science, Downers Grove, IL, USA) and
the results were expressed in counts per minute (cpm).
2.4. Respiratory burst of granulocytes and monocytes
The intracellular production of hydrogen peroxide by peripheral granulocytes
and monocytes was performed by ow-cytometry in whole blood using the uorogenic substrate dihydrorhodamine (DHRA) 123 (BurstTest kit, ORPEGEN Pharma,
Heidelberg, Germany). Briey, ice-cold heparinized blood (100 lL) was activated
with unlabeled opsonised Escherichia coli for 10 min at 37.0 C. DHRA 123 was then
added and incubation continued for another 10 min at 37.0 C. Erythrocytes were
lysed and xed for 20 min at room temperature. Samples were washed twice with
washing solution by centrifugation (5 min, 1200 rpm, 4 C), and supernatant was
discarded. Finally, 200 lL of propidium iodide-PI (DNA staining solution) were
added whilst samples were kept on ice, in a dark place. Cell analysis was done by
ow cytometry using a FACSCanto ow-cytometer (Becton Dickinson, New Jersey,
USA) and the CellQuest software (Becton Dickinson). At least 10,000 events were
analyzed. By manual gating, we rst selected from forward scatter versus side
scatter plot the population of granulocytes. Then we selected single cells and we excluded aggregation artefacts, as dened by PI incorporation. Finally, ow-cytometry
data were expressed as percentage of responsive cells under basal conditions or to a
particular ex vivo stimulus, meaning the percentage of cells with uorescence intensity above a dened threshold (M2). The mean intensity of cellular response was
calculated as geometrical mean of uorescence channel.
2.5. Plasma biochemical parameters
Plasma concentrations of glucose, calcium, phosphorus, iron, magnesium, urea,
glucose, cholesterol, triglycerides, total protein, albumin, total bilirubin, creatinin
and concentrations of alanine aminotransferase (ALAT), aspartate aminotransferase
(ASAT), alkaline phosphatise (AP), gamma glutamyl transferase (GGT) were determined on a BS-130 Chemistry analyzer (Bio-Medical Electronics Co., LTD, China).
2.6. Determination of total antioxidant status
Table 1
Composition of experimental diet (%).
Ingredients
409
Control
15.00
53.31
0.00
3.00
8.00
5.00
2.00
9.00
0.20
1.30
1.60
0.10
0.40
0.09
1.00
164.0
40.92
Contaminated diet
15.00
0.00
53.31
3.00
8.00
5.00
2.00
9.00
0.20
1.30
1.60
0.10
0.40
0.09
1.00
164.0
316
Total antioxidant status (TAS) assay was based on the absorption of 2, 20-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid cation (ABTS+) as described by Scislowski et al. (2005). Briey, the production of ABTS+ was initiated by adding
450 lM H2O2 in 1 mm quartz microcells containing 12 lL of plasma samples,
0.5 mM ABTS and 48.8 lM metmyoglobin diluted in 5 mM, pH 7.4 phosphate buffer
solution (PBS). Absorbance at 732 nm was measured immediately (A0) and 3 min
after the addition of hydrogen peroxide at 37 C (A3), using a Uvikon 923 doublebeam spectrophotometer (Kontron Instruments, Zurich, Switzerland). Absorbance
of the blank (i.e., containing PBS) was also determined to calculate the inhibition
percentage of the reaction as follows:
%inhibition
A0 A3 blank A0 A3
100
A0 A3 blank
410
Recombinant swine IL-1b, TNF-a, IFN-c IL-10, IL-4 were used as standards and results were expressed as picograms of cytokine/mL, after normalization to the total
protein content of the samples.
2.10. Statistical analysis
2.8. Quantitative real-time polymerase chain reaction (qRT-PCR) and data analysis
Tissue samples were taken from the liver and spleen and stored at 80 C until
RNA extraction. Tissue samples were homogenized in liquid nitrogen using an Ultra-Turrax homogenizer (IKA-Werke GmbH & Co. KG, Germany) and total RNAs
were extracted using Qiagen RNeasy midi kit (QIAGEN GmbH, Germany), according
to the manufacturers recommendations. After extraction, RNA was treated with a
ribonuclease inhibitor (RNasin Plus RNase Inhibitor; Promega Corp., USA). Quantity of extracted total RNA were measured on a Nanodrop ND-1000 spectrophotometer (Thermo Fischer Scientic, USA) and the quality was veried by agarose gel
electrophoresis. In order to quantify the expression levels of selected genes, equal
amounts of cDNA were synthesized using 1 lg of puried RNA and M-MuLV reverse
transcriptase (Fermentas, Thermo Fischer Scientic, USA), as well as oligo(dT) primers (Fermentas, Thermo Fischer Scientic, USA). Fluorescent real-time PCR was used
to evaluate the expression of: catalase (CAT), cyclooxygenase (COX2), glutathione
peroxydase (GPx) 1, superoxide dismutase 1 (SOD), inducible nitric oxide synthase
2 (iNOS). Synthesized cDNA was diluted 1:50 with nuclease-free water and used for
the qRT-PCR together with Maxima SYBR Green/Fluorescein qPCR Master Mix 2X
(Fermentas, Thermo Fischer Scientic, USA) and 0.3 lM of both forward and reverse
primers. Gene-specic primer pairs obtained from Eurogentec (San Diego, USA)
were found in the literature or designed using Primer3 and BLAST, as shown in Table 2. Thermal cycling was carried out with a Rotor Gene-Q Pure Detection (QIAGEN, Hilden, Germany) using the following conditions: 50 C, 2 min; 95 C,
10 min; and 40 cycles at 95 C, 15 s; 60 C, 1 min; 72 C, 30 s and nal elongation
step at 72 C for 10 min). Each gene was measured in triplicate and the formation
of single PCR products was conrmed using melting curves. Negative controls,
which consisted of all of the components of the qPCR mix except cDNA, were used
for all primers. The relative quantication of gene expression changes was recorded
after normalizing for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene
expression computed by using the 2(DD CT) method (Livak and Schmittgen, 2001)
and data were expressed as relative fold increase or decrease from control weanling
piglets.
ANOVA tests followed by a Fisher PSLD test were used to analyze the differences
in terms of animal performances, plasma biochemistry, antibodies, cytokines synthesis, oxidative burst, expression of genes involved in the oxidative stress, total
antioxidant status, nitric oxide synthesis. The P values lower than 0.05 were considered signicant.
3. Results
3.1. Effect of dietary zearalenone on piglets performances and organ
weight
We rst investigated the effect of dietary treatment on clinical
signs and animal performance. Piglets were fed with control or
ZEN-contaminated diets for a period of 18 days. Control animals
as well as piglets fed ZEN appeared clinically normal during the
whole experiment and no deaths resulted from the ZEN exposure.
Animal performances are reported in Table 3. ZEN does not affect
the body weight, average daily gain, feed intake or feed: gain ratio
of the intoxicated animals. Also, the concentration of 316 ppb of
ZEN has no effect on weight (kg0.75) of liver, kidney, spleen or heart
of the piglets.
3.2. Effect of dietary zearalenone on cell proliferation
The [methyl-3H]-thymidine proliferation assay was used to
determine the proliferation of the PBMC isolated from the blood
of the control or intoxicated animals, cultivated for 72 h at 37 C
and 5% CO2. The results presented in Fig. 1 showed that ZEN induced a decrease of unstimulated PBMC proliferation from
586 144 cpm in cells from control animals to 198 28 cpm in
cells from ZEN animals, which represents a 66.15% decrease from
the control (P = 0.0298). No effects were observed on lymphocytes
from spleen or lymph nodes of the treated group.
3.3. Effect of dietary zearalenone on respiratory burst of granulocytes
and monocytes
To assess the effect of ZEN on the respiratory burst, the
intracellular production of H2O2, induced ex vivo by opsonised
E. coli, was evaluated by ow cytometry in peripheral granulocytes
and monocytes derived from the two control and ZEN contaminated groups (Fig. 2). As expected, E. coli stimulation induced a signicant increase of both monocytes and granulocytes response.
Table 2
Nucleotide sequences of primers for real-time PCR.
No
Gene
Genbank no.
References
GAPDH
100
NM_001206359.1
CAT
241
NM_214301.2
COX2
119
NM_214321.1
GPx
62
NM_214201.1
iNOS
73
NM_001143690.1
SOD
Fw: ACTCACTCTTCTACCTTTGATGCT
R: TGTTGCTGTAGCCAAATTCA
Fw: CTTGGAACATTGTACCCGCT
R: GTCCAGAAGAGCCTGAATGC
Fw: CCCAATTTGTTGAATCATTT
R: TCTCATCTCTCTGCTCTGGT
Fw: GGAGATCCTGAATTGCCTCAAG
R: GCATGAAGTTGGGCTCGAA
Fw: GGAGCCATCATGAACCCCAA
R: GTAGAAGCTCGTCTGGTGGG
Fw: GAGACCTGGGCAATGTGACT
R: CTGCCCAAGTCATCTGGTTT
139
NM_001190422.1
GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; CAT: catalase; COX2: cyclooxygenase; GPx: glutathione peroxydase; iNOS: inducible nitric oxyde synthese; SOD:
superoxyde dismutase.
411
Performances
Initial weight (kg)
Final weight (kg)
Average daily gain (kg/animal/day)
Feed intake (kg/animal/day)
Feed: Gain (kg:kg)
P-value
Control
Zearalenone
9.4 0.20
16.0 0.50
0.367 0.02
0.681 0.03
1.87 0.08
9.5 0.20
15.7 0.70
0.344 0.04
0.716 0.12
2.17 0.23
0.80
0.75
0.58
0.41
0.25
39.2 1.1
7.69 0.3
2.77 0.1
6.15 0.3
41.0 1.9
7.50 0.3
2.49 0.1
5.70 0.3
0.44
0.75
0.22
0.37
Mycotoxin-contaminated feed signicantly increased the percentage of responsive monocytes cells in both unstimulated (27.25 in
ZEN versus 9.77% in control) and stimulated conditions (62.54 versus 46.08% in the control). As can be seen in Fig. 2, ZEN was not
able to increase the percentage of responsive granulocytes stimulated or not with E. coli.
3.4. Effect of dietary zearalenone on selected blood biochemical
parameters in piglets
Biochemistry analysis was also performed in the plasma of animals receiving the control or ZEN supplemented diets. As can be
seen in Table 4, ZEN treatment resulted in a slightly increase of
the concentration of triglycerides and iron, while no effect was observed for cholesterol, total protein, albumin, total bilirubin, creatinin, phosphorus, calcium, magnesium. Compared with the
control, the ingestion of the mycotoxin contaminated feed induced a signicant decrease of activity of ALAT (18.35% decrease;
P = 0.004), associated with a slight decrease of activity of GGT
(26.9% decrease; P = 0.182) and of the glucose concentration
(21.31% decrease; P = 0.056).
the results are presented in Table 5. ZEN does not affect the antioxidant status, no differences being observed between groups. The
nitric oxide synthesis slightly increased after the ZEN intoxication,
with a concentration of 91.4 8.87 in the treated group compared
with 81.5 11.9 in the control group, but the difference was not
signicant (P = 0.174).
Glucose (mg/dL)
Cholesterol (mg/dL)
Triglycerides (mg/dL)
Total protein (g/dL)
Albumin (g/dL)
Total bilirubin (mg/dL)
Creatinin (mg/dL)
Urea (mg/dL)
Phosphorus (mg/dL)
Calcium (mg/dL)
Magnesium (mg/dL)
Iron (lg/dL)
Alanine aminotransferase (IU/L)
Aspartate aminotransferase (IU/L)
Alkaline phosphatase (IU/L)
Gamma glutamyl transferase (IU/L)
P-value
Control
Zearalenone
122 12.2
56.3 12.1
40.5 10.6
5.30 0.30
3.20 0.10
0.05 0.01
1.19 0.09
1.00 0.00
9.86 1.04
13.2 0.69
2.54 0.76
1216 43
37.7 3.55
51.8 5.21
184 35.8
34.8 11.3
96.0 23.8
58.0 15.7
54.1 13.7
5.24 0.83
3.07 0.40
0.09 0.01
1.14 0.26
1.00 0.00
9.29 1.43
12.9 1.60
3.12 1.43
1264 55
30.8 1.59
49.2 3.97
162 28.0
25.4 8.77
0.056
0.853
0.118
0.746
0.513
0.385
0.690
0.489
0.671
0.445
0.191
0.004
0.410
0.329
0.182
412
Table 5
Effect of dietary zearalenone on different plasma parameters involved in the oxidative
stress.
Treatments
Pvalue
Control
Zearalenone
1.13 0.05
1.15 0.06
0.562
81.5 11.9
91.4 8.87
0.174
4. Discussion
spleen samples from control or treated animals using quantitative
Real-Time PCR and the results are showed in Fig. 3. In liver, ZEN induced an increase by 2.6 times for GPx (P = 0.028) and 3.25 times
for catalase expression (P = 0.080), while no effect was observed
for SOD expression. On the other hand, ZEN induced a decrease
of the expression of genes involved in inammation, with a fold
change decrease of 0.21 for COX2 (P = 0.0015) and of 0.59 for iNOS
(P = 0.224). In spleen, ZEN induced a signicant decrease of the
SOD expression (P = 0.049), while an increase was observed for
the genes involved in the inammation with a fold change of
3.29 for COX2 (P = 0.128) and of 2.38 for iNOS (P = 0.209).
3.7. Effect of dietary zearalenone on cytokine synthesis in blood and
liver
We next examined the ability of ZEN and its metabolites to
modulate the synthesis of cytokines involved in the inammatory
response in blood or in liver of the control or treated animals. As
shown in Fig. 4, ZEN intoxication induced a decrease of the
lower effects on these parameters, but when ZEN was administered together with other Fusarium mycotoxins they were able to
decrease the body weight in swine (Tiemann et al., 2006).
Also, 316 ppb of ZEN was not able to affect the weight of liver,
kidney, spleen or heart of the piglets which was in accord with
other results on the relative weight of the heart, liver, lung, kidney
and spleen in female pigs (75-day-old) treated for 21 days with
2 ppm ZEN (Wang et al., 2012). By contrast, other studies showed
that the relative weight of liver and kidney increased linearly in
postweaning gilts (21-day-old) treated for 18 days with 1.1 to
3.2 ppm ZEN (Jiang et al., 2012). These apparent contradictory results could maybe be explained by the different age of the animals
and the different number of the animals used in the two experiments. However, both studies have shown increase of the relative
weight of the reproductive organs (including uterus and vagina)
after the different ZEN treatment (Jiang et al., 2011), showing once
more that the rst effect of ZEN is on the reproductive system.
In swine, ZEN was already described as a toxin capable to decrease the in vitro cell proliferation of blood cells subsets (Marin
et al., 2010; Marin et al., 2011) or of porcine granulosa cells (Zhu
et al., 2012). This effects was conrmed in the present study, where
ZEN decreased the cell proliferation in blood, but not in spleen or
lymph nodes. According to our results, it seems that blood lymphocytes are more exposed to the ZEN action, as compared with the
splenocytes or lymphocytes from lymph nodes. In other toxicological studies, parallel assessment of the lymphocyte proliferation in
blood and immune organs (spleen) indicate a compartmentalization of the response with different effects (Waclavicek et al.,
2009; Taranu et al., 2010). In addition, other studies showed that
T-lymphocyte proliferations varied between the different immune
organs or tissues used according to their source, suggesting that Tlymphocyte proliferation is tissue/matrix dependent (Frouin et al.,
2010). A dose-dependent reduction of the lymphocyte proliferation
induced by ZEN was also observed in rats (Atkinson and Miller,
1984), humans (Forsell and Pestka, 1985; Vlata et al., 2006) or in
a monkey cell line (Bouaziz et al., 2012). It was suggested that
the decrease of cell proliferation induced by ZEN could be the result of the cell arrest in G2/M phase and the formation of DNA-adducts (Abid-Esse et al., 2003). Indeed, some studies showed that
ZEN induces DNA fragmentation and blockade of cell cycle characterized by an increase in the number of cells in the G2/M phase
(Abid-Esse et al., 2003; Minervini et al., 2006).
413
414
suggesting that ZEN was responsible for the induction of an inammatory status at least at the level of spleen and blood. Indeed, Kostro et al. (2012) showed that during the ZEN intoxication,
metabolism of arachidonic acid increased, mainly due to enzymes
of the cyclooxygenase group, which are responsible for the increase of the concentration of prostaglandin F2a and thromboxane
B2. ZEN was already described as responsible for inammation of
the reproductive organs: vulva and vagina (Mitak et al., 2002),
prostate gland (Zinedine et al., 2007), ovaries (Jiang et al., 2010)
and our results showed that ZEN could be also an inductor of the
inammation in immune organs, as spleen.
In conclusion, our results showed that in liver, ZEN has mainly
hepatotoxic effects associated with a depreciation of the hepatic
function and secondary an immunotoxic effects through the decrease of the inammatory response. In spleen and blood, ZEN
was responsible for the induction of an inammatory status and
it was responsible for the impossibility to realize an adequate response to the oxidative stress triggered by ZEN. Taken together,
our results showed that besides genital organs, liver, spleen and
blood may also be target tissues in weanling piglets fed diets containing 316 ppb ZEN for 18 days, with different effects concerning
oxidative stress and inammation.
Conict of Interest
The authors declare that there are no conicts of interest.
Acknowledgements
This work was nanced through the Project PNII: 2008-2011,
Parteneriate 5122 and PNII: 2011-2014, IDEI 101 and Nucleu
Project: 09-38 0104 granted by the Romanian Ministry of Research
and Technology. The authors want to thanks to Dr Denys Durand
and Dr. Dominique Bauchart and to all the team for their kindness
and help in measuring the nitric oxide and total antioxidant status
concentrations. The authors thank Dr. Olga Starodub for her help
with the English corrections.
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