Food Additives & Contaminants: Part A, 2015 Vol. 32, No. 1, 87–99, http://dx.doi.org/10.1080/19440049.2014.987700

Alleviation of zearalenone toxicity by modified halloysite nanotubes in the immune response of swine Shutong Yin, Qingwei Meng, Boru Zhang, Baoming Shi, Anshan Shan* and Zhongyu Li Institute of Animal Nutrition, Northeast Agricultural University, Harbin, China (Received 23 July 2014; accepted 10 November 2014) Zearalenone (ZEN) has caused significant economic effects on swine production in China. There is growing concern that exposure to ZEN during pregnancy affects the health of the offspring due to changes in the development of the immune system. To assess the risks associated with maternal ZEN exposure, several immunological parameters were assessed in pregnant sows and their offspring. The main aim of the study was to determine if modified hallosite nanotubes (MHNTs) can be used to protect pigs against the adverse effects of ZEN. Eighteen pregnant sows (second parity Yorkshire sows) were randomly divided into three treatment groups: (1) basal diet (control group); (2) contaminated grain (instead of 50% mouldy corn); and (3) contaminated grain (instead of 50% mouldy corn) + 1% MHNTs. The pregnant sows were fed the different treated diets from days 35 to 70 of gestation. Dietary ZEN exposure decreased the organ coefficient and the mRNA expression levels of IFN-γ, TNF-α, and IL-10, and increased ZEN residues and IL-4 mRNA expression in the spleen of pregnant sows and neonatal piglets. Decreases in the serum IgA and IgG levels were observed in the pregnant sows. Maternal ZEN exposure decreased the organ coefficient and the mRNA expression levels of IFN-γ and IL-10, and increased IL-4 mRNA expression in the spleen of weaning piglets. Exposure to ZEN during pregnancy decreased the level of serum IgG in the weaning piglets. Maternal exposure to ZEN induced histopathological damage and oxidative stress in the spleens of pregnant sows and their piglets. The addition of MHNTs to ZEN-contaminated diets can mitigate the negative effects induced by ZEN in the swine. Keywords: zearalenone; immune response; pregnant sows; offspring; modified halloysite nanotubes

Introduction Zearalenone (ZEN) is a non-steroidal oestrogenic mycotoxin produced by Fusarium graminearum in cereal crops, such as corn, barley, wheat and sorghum, during the process of production and storage (Paganetto et al. 2000; Voss et al. 2007). F. graminearum becomes well established on grain at 28°C (Mylona et al. 2012). The rate of ZEN production varies depending on the incubation conditions, and the highest production is obtained with cultures transferred to 12°C (Sherwood & Peberdy 1972). ZEN is one of the most common contaminants in cereals. Yoshizawa (1997) observed high concentrations of ZEN in barley (Japan, 15 mg kg–1) and corn (New Zealand, 10.5 mg kg–1). Briones-Reyes et al. (2007) indicated that approximately 70% of 24 corn samples were contaminated with ZEN (3–83 μg kg–1) in the state of Tlaxcala, Mexico. ZEN has adverse effects on the spleen, liver, kidney and reproductive system. In farm animals, the consumption of a ZEN-contaminated diet leads to clinical signs, such as prolonged oestrus intervals, ovarian atrophy, decreased fertility, stillbirth and the persistent presence of corpus luteum (Fink-Gremmels & Malekinejad 2007). ZEN causes lymphoid infiltration in the liver and shrunken glomeruli in the kidney of mice (Abbès, Ouanes, et al. 2006). Other data show that ZEN leads to lymphoid depletion in the spleen of BALB/c mice (Salah-Abbès *Corresponding author. Email: [email protected] © 2014 Taylor & Francis

et al. 2010). Alterations in inflammatory cytokines, such as decreases in the TNF-α level in the spleen of mice and increases in the production of interleukin IL-2 and IL-5 in splenic lymphocytes of mice, have been found under conditions of high ZEN concentration (Eriksen 1998; SalahAbbès et al. 2008). ZEN is also able to affect the immunological system at nutritionally relevant concentrations (Whalen & Green 1998). Some information is available on the influence of indirect (maternal effect) exposure to stimulation on the health of offspring. The results reported by Urakubo et al. (2001) show that maternal exposure to infection alters the inflammatory cytokine levels in the foetal brain. Lipopolysaccharide (LPS) stimulation during pregnancy alters the immune response in developing offspring (Surriga et al. 2009). Reduced litter size, abnormal foetal development and decreased fertility have been major problems associated with livestock production in China. The detrimental effects of ZEN may be more obvious during pregnancy because the foetus is susceptible to toxins due to its fragile developmental state and lack of adequate defence mechanisms. Studies have indicated that maternal ZEN exposure induces a delay of foetal development in rats and reduced litter size in the sows (Diekman & Long et al. 1989; Zhang, Jia, et al. 2014). The early events of pregnancy are associated with rapid changes in the

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expression of genes required for nutrient transportation, cellular remodelling, angiogenesis and relaxation of vascular tissues, as well as cell proliferation and migration (Bazer et al. 2009). Sows are the most susceptible species to ZEN (Gajęcki et al. 2010). In pregnant sows, the placental transfer of ZEN may present a potential danger for direct effects on their neonatal and weaning piglets. It is important to minimise ZEN exposure to prevent its adverse effects. At present, the most potent dietary approach for the prevention of mycotoxicoses in livestock involves the use of mycotoxin adsorbents (Surai & Mezes 2005). Avantaggiato et al. (2003) found that cholestyramine is a useful compound for the prevention of ZEN-induced hyperestrogenism in pigs. However, the high cost of cholestyramine limits its applicability as a feed additive (Galvano et al. 2001). Phyllosilicate clay and hydrated sodium calcium aluminosilicate (HSCAS) are currently effective and inexpensive as absorbents for feedstuff and may prevent the detrimental effects associated with ZEN in livestock (Abbès, Ouanes, et al. 2006; Abbès, Salah-Abbès, et al. 2006). Halloysite, a natural nanomaterial, is a 1:1 clay mineral that is chemically similar to kaolinite, but its higher content of hydrated water results in a tubular morphology (Ismail et al. 2009). Halloysite is known as aluminosilicate, which is a porous, negatively charged substance, and has chemosorptive and ionexchanging capacities depending on its structure, the degree of polarisation of the particles, and the diameter of the pores. Clays and soils (montmorillonite, illite, muscovite, sepiolite and palygorskite) that have been modified using stearyl dimethyl benzyl ammonium chloride (SKC) could also be used as barriers to prevent the mobility of specific hydrophobic pesticides from a point source of pollution (Sanchez-Martin et al. 2006). With extensive raw material sources, low price, simple process and stable quality, there has been an increased interest in the application of halloysite nanotubes (HNT). After treatment with SKC, the dispersion of the halloysite was increased, the average internal diameter of the lumen was enlarged, the external surface of modified hallosite nanotubes (MHNTs) was cruder than HNT (Zhang, Gao, et al., 2014). Fatteners fed a diet containing HNTs exhibited a significantly better utilisation of protein, fat, fibre and mineral components, such as calcium, magnesium, zinc and copper, compared with the control group (Korniewicz et al. 2006). The results reported by Kulok et al. (2005) indicate the high decontaminating efficiency of HNTs toward bacteria, fungi and aflatoxin B1, which inferred that the addition of HNTs to feed mixtures may be used to reduce adverse effects. However, natural adsorbents do not exhibit notable binding effects against ZEN. In our study, HNTs were modified to increase the diameter of the pores and improve their ability to bind hydrophobically with ZEN. The trans-generational effects of ZEN have been studied in the reproductive system of rats, mice and pigs

(Nikaido et al. 2004; Schoevers et al. 2012; Zhang, Jia, et al. 2014). It is hypothesised that maternal ZEN exposure can damage the immune function of pregnant sows and the offspring, and increase the death rate of neonatal and weaning piglets. The addition of MHNTs may eliminate the ZEN-induced damage in the immune system. The immunological parameters were first measured in sows fed different treatment diets from days 35 to 70 of gestation. Several relevant immune parameters and immunotoxicological tests were subsequently performed in neonatal and weaning piglets.

Materials and methods Preparation of ZEN ZEN was mainly produced by a fungus (Fusarium graminearum) purchased from the Agricultural Culture Collection of China (No. ACCC36249). The fungus was mainly cultivated on potato dextrose agar medium (PDA, 0.4% potato extract, 2% glucose, and 1.5% agar, pH 5.6 ± 0.2), which was purchased from Fluka (Bornem, Belgium) (Demyttenaere et al. 2004). The corn used for the experiment was obtained from the Xiang Fang Experimental Establishment of Northeast Agricultural University and treated in a hammer mill with a 40-mesh screen (Trapp-TRF model 90). ZEN was produced in vitro according to the procedures outlined by Lígia and Marina (2002). The production of mycotoxin was conducted in duplicate on a sterile tray that contained 1000 g of sterilised cracked corn, 400 ml of distilled water and F. graminearum, and the activity of the water was adjusted to 0.97. The corn was sterilised by autoclaving. The autoclaved substrate was inoculated with the spore suspension according to the following procedure: 100 ml of sterile distilled water were added to each slant of 5-day-old solid culture medium (including fungus and spore), and the agar surface was then gently scraped to obtain a turbid suspension, which corresponded to 1 × 1014 spores ml–1. A total of 100 ml of this suspension were added to the cracked corn. The inoculated tray was stirred daily during the first 5 days. The culture was maintained at 28°C for the first 15 days and then maintained at 12°C. The production of ZEN reached its peak on the 35th day of incubation. These findings were reported by Martins and Marina Martins (2002).

Modification of the sorbent The adsorbent used for this study was reported by Zhang, Gao, et al. (2014). Powder HNTs (Al2Si2O5(OH)4•nH2O) were refined from clay minerals and obtained from Henan province (China) with a purity of 95%. The powder was prepared using the method described by Jinhua et al. (2010). HNTs were modified using SKC (Jingwei

Food Additives & Contaminants: Part A Chemical Co., Ltd, Shanghai, China) according to methods previously described by Tomašević-Čanović et al. (2003) with modifications. HNTs (100 g) were treated with 1000 ml distilled water containing SKC (0.5%) and mixed at a speed of 2300g with a reaction time of 10 min at 50°C. When the reaction was complete, the suspensions were filtered, washed three times with deionised water, dried at 80°C and crushed with 100 g of HNT to obtain particles that were less than 45 μm in a beater mill at 7400 g for 3 min (Lemke et al. 2001). Animals All the animal experimental procedures were approved by the Ethical and Animal Welfare Committee of Heilongjiang Province, China. All sows (second parity Yorkshire sows) were purchased from Dawan pigs farm (Mudanjiang, China). The sows were bred with semen from a pool of Landrace boars and housed in individual stalls after 35 gestation days. Each sow was fed restrictively from days 35 to 70 of gestation at a daily amount of 2 kg. The foetuses showed teratogenic effects or mortality from gestation day 35 to 70, which is the key period of organogenesis (Goyarts et al. 2007). One week before farrowing, the sows were moved to farrowing pens (4 m2), where the sows were unrestrained. After farrowing, the piglets stayed with their mothers during the suckling period until the age of 21 days. The sows and their litters were supplied water ad libitum. Experimental design and diets The experiment was performed in a completely randomised block designed with a total of 18 pregnant sows. The pregnant sows were divided into three treatment groups, which had mean initial body weights of 185.93 ± 3.57, 191.20 ± 2.61 and 187.37 ± 2.49 kg. The three treatments were the following (Table 1): (1) basal diet (control group), (2) ZEN-contaminated grains (instead of 50% mouldy corn), and (3) contaminated grains (instead of 50% mouldy corn) + 1% MHNTs (Jiang et al. 2010, 2012). The pregnant sows (six per treatment) were fed different treatment diets from Table 1.

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days 35 to 70 of gestation and a basal diet on the remaining days of gestation. The ingredients and composition of the different treatment diets (Table 2) were formulated to meet or exceed the National Research Council nutrient requirements (1998). All the feedstuffs were subjected to post-processing analytical control. The concentrations of the feed composition were compared using validated analytical methods (National Standards of the People’s Republic of China, GB/T 19,540–2004). The method uses immunoaffinity chromatography for purification and HPLC for the detection and quantification of ZEN. Before extraction, samples were ground to ensure that 95% of weight passed through a 0.85-mm sieve. Subsamples (40 g) were extracted in 250 ml Erlenmeyer flasks by adding 2.0 g NaCl and 50 ml acetonitrile–water (90 + 10, v/v) and blended for 2 min. Extracts were filtered through a fluted filter paper and aliquots of 10 ml were diluted with 40 ml distilled water. This process was followed by a filtration through a 1.0 µm glass microfiber filter, and 10 ml (0.8 g sample equivalent) was applied to the ZearalaTest column. The columns were then washed with 10 ml distilled water and eluted with 1.5 ml HPLC grade methanol into a glass text tube. The extract was first concentrated to dry under nitrogen at 55°C and then dissolved in mobile phase (acetonitrile–water–methanol, 46 + 46 + 8, v/v). Samples were analysed by HPLC on an Agilent extent C18 column as mobile phase (1.0 ml min–1) and fluorescence detection (λ ex = 274 nm, λ em = 440 nm). Based on the analysis, the major contaminant in the contaminated diets was ZEN, which was present at a concentration of 2.77 mg kg–1. The analysis of the corn and diets by GC-MS, which was used to obtain a detailed characterisation of the trichothecene mycotoxin pattern, revealed that the concentrations of other B-trichothecene mycotoxins, such as 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol, nivalenol and A-trichothecene, were lower than the detection limits (Tiemann, Brüssow, Dänicke, et al. 2008).

Collection and storage of organ and blood samples Three pregnant sows from each treatment group were sacrificed through an intra-arterial injection of pentobarbital (200 mg kg–1) after general anaesthesia on day 70 of

Different treatment group and number of swine in the study. Concentration

Experimental group 1 2 3

Feeding (gestation 35–70 days) a Basal diet (control group) ZEN-contaminated grains (instead of 50% mouldy corn) Contaminated grains (instead of 50% mouldy corn) + 1% MHNTs b

Number of sows in each treatment

Number of neonatal piglets

Number of weaning piglet

6 6

6 6

6 6

6

6

6

Notes: a Pregnant sows were exposed to different treatments group on days 35–70 of gestation (n = 6). b MHNTs, modified halloysite nanotubes.

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Ingredients and composition of the diet. Gestation 35–70 days Controlc

ZEN-contaminated grainsc

Contaminated grains + 1% MHNTsc

Lactation

Ingredient (%) Control corn Contaminated corn Soybean meal Wheat bran MHNTs Full-fat soybean Limestone Dicalcium phosphate Salt Vitamin and mineral premixa

62.40 – 16.00 18.00 – – 1.00 1.10 0.50 1.00

31.20 31.20 16.00 18.00 – – 1.00 1.10 0.50 1.00

31.20 31.20 16.00 17.00 1.00 – 1.00 1.10 0.50 1.00

63.80 – 20.00 – – 12 0.93 1.77 0.50 1.00

Analysed composition Metabolisable energy (MJ kg–1)b Crude protein Calcium Total phosphorus Lysine Tryptophan Threonine Methionine + cystine Concentration of ZEN (mg kg–1)c

11.90 14.51 0.69 0.61 0.65 0.16 0.52 0.39 0.03

11.84 14.45 0.68 0.61 0.67 0.16 0.52 0.45 2.77

11.75 14.31 0.67 0.60 0.66 0.16 0.52 0.45 2.76

12.90 18.48 0.79 0.64 0.98 0.23 0.69 0.52 0.01

Parameters

Notes: aProvided the following per kilogram of diet: Cu, 18.2 mg; Zn, 126.0 mg; Se, 0.3 mg; Mn, 50.5 mg; Fe, 150.3 mg; I, 0.4 mg; vitamin A, 11, 050 IU; vitamin D, 2,310 IU; vitamin E, 62.8 IU; vitamin K, 2.6 mg; riboflavin, 5.8 mg; pantothenic acid, 20 mg; niacin, 25 mg; vitamin B12, 326 µg; folate, 6.5 mg; pyridoxine, 1.8 mg; biotin, 350 µg; and thiamin, 1.9 mg. b Calculated values according to the Tables of Feed Composition and Nutritive Values in China in this study. c Basal diet: control group; ZEN-contaminated grains: instead of 50% mouldy corn; contaminated grains: instead of 50% mouldy corn + 1% MHNTs; ZEN: zearalenone; and MHNTs: modified halloysite nanotubes.

gestation after exposure to the experimental diets for 35 days. The remaining pregnant sows delivered their foetuses on gestation day 114. Six foetuses of each treatment group were sacrificed at birth and weaning day, respectively. The breeding and sample collection from the neonatal and weaning piglets were performed according to Wang et al. (2013). The sows were weighed at insemination, gestation day 110, within 24 h of farrowing, and on weaning day 21 of lactation. The piglets were individually weighed at birth and at weaning of lactation. Blood samples from pregnant sows and six piglets from each treatment group were collected from the vena jugularis externa into microcentrifuge tubes with heparin sodium (20 U ml–1 in PBS). The blood samples were incubated on ice for 30 min and centrifuged at 3000g and 4°C for 10 min. The serum was separated and stored in 1.5-ml sterile Eppendorf tubes at −80°C for the analysis of immunoglobulins. The spleens were removed from the foetuses, neonatal piglets and weaning piglets. Thereafter, the spleens were weighed on an electronic analytical balance. A section of the spleen was dissected and fixed in neutral buffered 10% formalin solution at 4°C for

histopathological study. A second section was snap-frozen in liquid nitrogen and stored at −80°C for quantitative expression of inflammatory cytokine mRNA. Last sections were stored at −20°C for the measurements of antioxidant enzymes and ZEN residues.

Coefficients of the spleen of pregnant sows and their offspring The body and spleen weights were obtained as described above. The coefficient of the spleen was calculated as the ratio of the spleen weight to the body weight and is expressed as mg g–1 (Jiang et al. 2011). Measurement of ZEN residues in the spleen of pregnant sows and their offspring The ZEN residues were measured in the spleen of swine by enzyme-linked immunosorbent assay (ELISA) according to the procedure described by the manufacturer (Agraquant ZEN Test kit; Romer Labs, Union, MO, USA) (Kolosova et al. 2006).

Food Additives & Contaminants: Part A Measurement of immunoglobulins in pregnant sows and their offspring Total concentrations of the different immunoglobulin subsets were measured by the ELISA method. The serum total IgA, IgG and IgM levels were determined with an ultraviolet spectrophotometer (UV-2410PC model, Shimadzu, Kyoto, Japan) using commercial diagnostics kits (Nanjing Jiancheng Biotechnology Co., Ltd, Jiangsu, China) (Accensi et al. 2006).

Determination of antioxidative parameters for pregnant sows and their offspring The antioxidative parameters were determined with an ultraviolet spectrophotometer (UV-2410PC model, Shimadzu) using commercial diagnostics kits (Nanjing Jiancheng Biotechnology Co.). The parameters evaluated in the spleen were the following: malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GHS-Px) and total anti-oxidative capacity (T-AOC) (Liu et al. 2007).

Histopathological observation of pregnant sows and their offspring After fixation in formalin solution, the spleen tissue was embedded in paraffin. The samples were cut into 5-μm-thick sections and placed onto glass slides. The sections were stained with haematoxylin and eosin (H&E) for histopathological analysis and mounted under a glass coverslip (Li et al. 2010). The percentage of the stained areas was measured using the AnalySIStrame grabben CSIS system (AnalySIS 3.4, SIS), as previously described (Tiemann et al. 2006). Each slide was divided into 15 single segments, and a single picture was used for the evaluation. The slides were observed under 400× magnification using an optical microscope (Nikon, Japan). Table 3. Gene

SYBR green real-time RT-PCR for the determination of inflammatory cytokine mRNA expression in pregnant sows and their offspring The total RNA from the spleens of sows was extracted using the Trizol reagent (E.Z.N.A. ® Total RNA Kit, Omega Bio-tek, Inc., Doraville, GA, USA). The RNA concentration was measured by a spectrophotometer at 260/280 nm. The quality of the RNA was estimated by detecting two bands for 18 S and 28 S rRNA, and the ratio of the intensity of these bands was found to be 1:2 in an agarose gel. SYBR green I real-time polymerase chain reaction (RT-PCR) was used to measure the mRNA expression levels of IL-4, IFN-γ, TNF-α, IL-10, TGF-β and β-actin. First-strand cDNA was synthesised from 5 μg of total RNA (processed using DNase) using oligo (dT) primers and Superscript II reverse transcriptase according to the manufacturer’s instructions (Tiangen Biotech Co., Ltd, Beijing, China). Real-time PCR was performed in an ABI PRISM 7500 SDS thermal cycler (Applied Biosystems, Foster City, CA, USA). Each sample was analysed in triplicate. The primers used in the analysis are listed in Table 3. The reactions were performed with 2.0 μl of firststrand cDNA and 0.8 μl of sense and anti-sense primers in a final volume of 20 μl as recommended by the SYBR realtime PCR kit (TaKaRa ® Bio Catalog, Da Lian, China). The RT-PCR conditions were as follows: one cycle at 95°C for 30 s and 40 cycles at 95°C for 5 s and 60°C for 34 s. The relative expression level of mRNA of inflammatory cytokines was determined through the 2 –ΔCt method (Lee & Schmittgen 2006; Bousquet et al. 2009).

Statistical analysis All the data were analysed using SPSS software (SPSS Inc., Chicago, IL, USA) and are expressed as the means ± standard error of the mean (SEM). The data were first analysed as a randomised design with individual swine as the random factor to examine the overall effect of the

Primers used for quantitative real-time PCR. GenBank accession numbera

IL-4

X68330

IL-10

JQ687536.1

TGF-β

X14150.1

IFN-γ

NM_213948.1

TNF-α

JF831365.1

β-actin

AY550069

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Primerb

Fragment length (bp)a

5ʹ-gcgacatcaccttacaagagat-3´ 5ʹ-cagcttcaacactttgagtatttc-3´ 5ʹ-aggaggagaaggggtaggtaag-3´ 5ʹ-cgaaggataaggaggaagtagaaa-3´ 5ʹ-acacaaaagcaacagacctcac-3´ 5ʹ-agccagcaagaaagagaaatgt-3´ 5ʹ-aagataaccaggccattcaaag-3´ 5ʹ-tcccagagctaccatttaggaa-3´ 5ʹ-accacgctcttctgcctact-3´ 5ʹ-gggcttatctgaggtttgagac-3´ 5ʹ-atgcttctaggcggactgt-3´ 5ʹ-ccatccaaccgactgct-3´

61 105 110 81 129 211

Notes: a Serial number and fragment length were obtained from GenBank. b Primers were designed by Sangon Biotech (Shanghai) Co. Ltd. Reactions were performed with the primer and reagents in a final volume of 20 μl as recommended by the SYBR real-time PCR kit.

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treatments. If differences between the treatments were detected by analysis of variance (ANOVA), the significance of the differences was determined by Duncan’s multiple range tests. Significance was considered at the probability of p < 0.05.

ZEN, there were significant decreases in the serum IgA and IgG levels observed in pregnant sows (p < 0.05; Table 5). Maternal ZEN exposure significantly decreased the level of serum IgG in the weaning piglets (p < 0.05; Table 5). There was no significant change in serum IgA level of weaning piglets (Table 5). However, the level of IgM exhibited no obvious changes in both pregnant sows and offspring. The addition of MHNTs to ZEN-contaminated diets ameliorated the changes in the levels of serum IgA and IgG in pregnant sows and offspring (p < 0.05, Table 5).

Results Splenic coefficient and ZEN residues in the spleen The results of the determination of the splenic coefficients and ZEN residue are shown in Table 4. ZEN exposure significantly decreased the splenic coefficient, and increased ZEN residues in the spleen of pregnant sows compared with the control group (p < 0.05; Table 4). The feeding of ZEN-containing diets to pregnant sows induced obvious changes in the splenic coefficient and ZEN residues in the spleen of neonatal and weaning piglets (p < 0.05). Maternal treatment with MHNTs alleviated the adverse effect of ZEN on the splenic coefficients and ZEN residues in the spleen of pregnant sows and their offspring (p < 0.05, Table 4). The addition of MHNTs to ZEN-contaminated diets reduced ZEN residues in the spleen of swine.

Histopathological examination Microscopically, maternal treatment with ZEN-induced atrophied white pulp (black arrow) and expanded red pulp (red arrow) in the spleen of pregnant sows and their piglets (Figures 1A, 1B, 1C, 2A, 2B, 2C, 3A, 3B and 3C). MHNTs induced an improvement in the splenic damage observed in the pregnant sows and their offspring. The control groups produced normal histological images.

Antioxidative parameters To evaluate the oxidative stress induced by ZEN and MHNTs, several enzymatic indicators, such as MDA, SOD, GSH-Px and T-AOC, were measured in the spleen of pregnant sows and their spring. As shown in Table 6,

Changes in immunoglobulin subsets There were some changes in haematological parameters (Table 5). Thirty-five days after maternal administration of Table 4.

Splenic coefficient and residue of ZEN in the spleen of swine (means ± standard error). Treatment

Stage

Item

Pregnant sows Neonatal piglets Weaning piglets Note:

1

Splenic coefficient (g kg–1 BW) Residue of ZEN (ppb) Splenic coefficient (g kg–1 BW) Residue of ZEN (ppb) Splenic coefficient (g kg–1 BW) Residue of ZEN (ppb)

2.76 0.82 1.28 0.31 2.17 0.29

± ± ± ± ± ±

2 0.05a 0.18c 0.02a 0.01c 0.04a 0.05c

2.13 32.47 1.03 13.15 1.98 8.98

± ± ± ± ± ±

3 0.08b 1.28a 0.01b 0.69a 0.05b 0.63a

2.69 12.27 1.29 5.33 2.15 3.64

± ± ± ± ± ±

0.02a 2.94b 0.05a 3.02b 0.02a 0.32b

a–c

Means within a line with no similar superscript letters are significantly different (p < 0.05).

Table 5. Effects of maternal exposure to ZEN-treated diets with or without MHNTs on immunoglobulin (IgM, IgA, IgG) in pregnant sows and of piglets (means ± standard error). Treatment Stage

Item

Pregnant sows Weaning piglets

Note:

a–b

IgM (mg ml–1) IgA (mg ml–1) IgG (mg ml–1) IgM (mg ml–1) IgA (mg ml–1) IgG (mg ml–1)

1 1.91 0.26 14.95 1.05 0.20 5.51

± ± ± ± ± ±

2 0.03 0.02a 0.48a 0.03 0.04 0.21a

Means within a line with no similar superscript letters are significantly different (p < 0.05).

2.06 0.13 10.79 1.09 0.05 3.07

± ± ± ± ± ±

3 0.10 0.04b 0.57b 0.12 0.02 0.31b

1.90 0.24 13.77 1.06 0.18 5.47

± ± ± ± ± ±

0.45 0.01a 0.54a 0.03 0.08 0.59a

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Figure 1. (colour online) (A) Normal morphological appearances in spleen tissue dyed with haematoxylin and eosin in pregnant sows at 400× original magnifications; (B) representative photomicrographs of pathological changes in spleen tissue dyed with haematoxylin and eosin in pregnant sows exposed to ZEN at 400× original magnifications; and (C) representative photomicrographs of pathological changes in spleen tissue dyed with haematoxylin and eosin in pregnant sows treated with MHNTs at 400× original magnifications.

Figure 2. (colour online) (A) Normal morphological appearances in spleen tissue dyed with haematoxylin and eosin in neonatal piglets at 400× original magnifications; (B) representative photomicrographs of pathological changes in spleen tissue dyed with haematoxylin and eosin in neonatal piglets exposed to ZEN at 400× original magnifications; and (C) representative photomicrographs of pathological changes in spleen tissue dyed with haematoxylin and eosin in neonatal piglets treated with MHNTs at 400× original magnifications.

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Figure 3. (colour online) (A) Normal morphological appearances in spleen tissue dyed with haematoxylin and eosin in weaning piglets at 400× original magnifications; (B) representative photomicrographs of pathological changes in spleen tissue dyed with haematoxylin and eosin in weaning piglets exposed to ZEN at 400× original magnifications; and (C) representative photomicrographs of pathological changes in spleen tissue dyed with haematoxylin and eosin in weaning piglets treated with MHNTs at 400× original magnifications.

Table 6. Effects of maternal exposure to ZEN-treated diets with or without MHNTs on antioxidative parameters in pregnant sows and piglets (means ± standard error). Treatment Stage

Item

Pregnant sows

Neonatal piglets

Weaning piglets

Note:

a–b

MDA (nmol mg–1 protein) SOD (u mg–1 protein) GPx (u mg–1 protein) T-AOC (U mg–1 protein) MDA (nmol mg–1 protein) SOD (u mg–1 protein) GPx (u mg–1 protein) T-AOC (U mg–1 protein) MDA (nmol mg–1 protein) SOD (u mg–1 protein) GPx (u mg–1 protein) T-AOC (U mg–1 protein)

1 62.77 105.27 124.31 27.68 17.76 44.97 50.80 10.82 30.89 65.32 77.46 19.26

± ± ± ± ± ± ± ± ± ± ± ±

2 1.06b 6.65a 15.14a 3.29a 0.61b 0.91a 2.39a 0.67a 1.46b 1.20a 1.42a 1.41a

76.92 66.97 79.25 18.17 27.26 34.36 39.21 8.14 41.37 49.15 61.66 10.50

± ± ± ± ± ± ± ± ± ± ± ±

3 6.36a 2.19b 2.06b 1.38b 0.96a 2.16b 2.72b 0.46b 2.36a 2.07b 1.76b 0.56b

63.76 111.03 124.50 24.21 19.63 41.57 51.01 10.27 31.44 61.04 74.38 17.68

± ± ± ± ± ± ± ± ± ± ± ±

0.55b 6.20a 8.06a 1.11ab 5.00b 0.94a 2.52a 0.42a 2.67b 4.16a 1.12a 0.75a

Means within a line with no similar superscript letters are significantly different (p < 0.05).

ZEN induced a significant increase in the level of MDA and obvious decreases in the levels of SOD, GSH-Px and T-AOC in pregnant sows, neonatal piglets and weaning piglets (p < 0.05). The addition of MHNTs decreased the risk of ZEN in pregnant sows, neonatal, and weaning piglets (p < 0.05, Tables 6).

Inflammatory cytokine mRNA expression ZEN exposure during pregnancy decreased the mRNA expression levels of IFN-γ, TNF-α and IL-10, and increased the mRNA expression level of IL-4 in the spleen of pregnant sows and neonatal piglets, indicating that ZEN can have a direct trans-generational impact on the expression of

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Table 7. Effects of maternal ZEN exposure with or without MHNTs on the mRNA expression of inflammatory cytokines in the spleen of offspring (means ± standard error). Treatment Stage Pregnant sows

Neonatal piglets

Weaning piglets

Item IL-4 IFN-γ TNF-α IL-10 TGF-β IL-4 IFN-γ TNF-α IL-10 TGF-β IL-4 IFN-γ TNF-α IL-10 TGF-β

1 0.89 1.37 1.30 1.26 1.24 0.73 1.23 1.21 1.20 0.99 0.77 1.29 1.26 1.23 1.05

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2 0.01b 0.08a 0.08a 0.05a 0.10 0.03b 0.07a 0.06a 0.04a 0.06 0.02b 0.06a 0.05 0.04a 0.07

1.07 0.98 0.97 0.94 1.14 0.88 0.93 0.87 0.83 0.96 0.86 0.95 1.14 0.91 0.98

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3 0.04a 0.16b 0.07b 0.02b 0.08 0.01a 0.16b 0.03b 0.05b 0.08 0.03a 0.05b 0.09 0.05b 0.12

0.90 ± 0.04b 1.38 ± 0.01a 1.20 ± 0.04a 1.14 ± 0.06a 1.19 ± 0.05 0.75 ± 0.02b 1.19 ± 0.06a 1.16 ± 0.03a 1.08 ± 0.06a 0.97 ± 0.08 0.78 ± 0.02b 1.21 ± 0.09a 1.20 ± 0.11 1.11 ± 0.03a 1.08 ± 0.12

Notes: a mRNA expression of inflammatory cytokines was expressed in the spleen of pregnant sows by SYBR green I real-time PCR. a–b Means within a line with no similar superscript letters are significantly different (p < 0.05).

inflammatory cytokines (p < 0.05; Table 7). Significant decreases in the mRNA expression levels of IFN-γ and IL-10, and an increase in the mRNA expression level of IL-4, were found in the spleen of weaning piglets (p < 0.05, Table 7). No obvious changes were found in the mRNA expression levels of TNF-α and TGF-β in the spleen of weaning piglets. The TGF-β mRNA expression level during pregnancy was not markedly altered in the spleen of pregnant sows and their offspring compared with the control group (Table 7). The combination of ZEN and MHNTs in the diets ameliorated the adverse effects of ZEN in pregnant sows and their offspring (p < 0.05; Table 7).

Discussion Decreased fertility and abnormal foetal development are the primary problems found in livestock production in China. These phenomena may be due to mycotoxin ingestion during pregnancy. In fact, it has been reported that in utero exposure to cigarette smoke can have negative effects on the health of the child (Tsai et al. 2010). Sharkhuu et al. (2011) demonstrated that maternal exposure to O3 produces decreases in immune function and indicators of allergic lung disease in the offspring. Maternal exposure to ZEN can induce adverse effects on the reproductive system of the offspring (Diekman & Long 1989; Schoevers et al. 2012). Immunity to infections is the most important physiological function due to its relationship with health. The purpose of this experimental study in sows was to determine whether maternal exposure to ZEN can affect the immune function of the offspring (neonatal and weaning piglets). In addition, the present investigation was conducted to detect the protective effect and the possible

ameliorative role of MHNTs on ZEN-induced toxicity in pregnant sows and their offspring. The results of this study indicate that dietary ZEN exposure during pregnancy decrease the splenic coefficient of early pregnant sows and their piglets. The results were similar to those reported by Jiang et al. (2011), who found that diets containing 2.0 or 3.2 mg kg–1 ZEN decrease the relative spleen weight of piglets. Maternal exposure to deoxynivalenol (DON) and ZEN significantly decreases the relative spleen weight of the piglets (Tiemann, Brüssow, Dannenberger, et al. 2008). However, Thuvander et al. (1996) found that prenatal exposure to the ochratoxin-A results in no obvious changes in relative spleen weight of mice offspring, and this apparent conflict may be due to the species, feeding time, features and mycotoxin were different from ours. Smith (1982) found that residues of ZEN were measured in the liver and kidney of male weanling rats exposed to a single oral dose of ZEN for 18 h. The heifers were fed for 84 days with 2 kg of ZENcontaminated oats (1370 μg kg–1). The content of ZEN was measured in urine and tissue samples (back, femoral region and liver) (Kleinova et al. 2002). These results were accorded with our study and suggest that ZEN induces damage in lymphoid organ and immune system. The decrease in both IgA and IgG concentrations observed in the ZEN-treated sows corresponds to the development of active humoral immunity, which indicates that ZEN affects the humoral immune system (Abbès, SalahAbbès, et al. 2006). In our study, maternal exposure to ZEN decreased the levels of serum IgA and IgG in pregnant sows and piglets during the first day of life compared with the control group. Prenatal ZEN exposure induced a lower

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serum IgG concentration in weaning piglets compared with those from control sows. Previous studies have demonstrated that treatment with mycotoxin induces significant decreases in the serum IgA and IgG levels in mice (Abbès, SalahAbbès, et al. 2006; Kubosaki et al. 2008). The porcine epitheliochorial placenta is a barrier to the transport of maternal immunoglobulins to the foetus. The decrease in the serum IgG level in weaning piglets may be due to ZENinduced decreases in the permeability of immunoglobulins to the offspring via the maternal placenta and breast milk. These findings were supported by the histological changes observed in spleen tissues, which is considered the target organ of the infectious agent and product. Oxidative stress refers to a disturbance in the balance between pro-oxidants and antioxidants that causes potential cell damage. MDA is the product of lipid peroxidation products. SOD and GSH-Px are enzymes that form part of the enzymatic antioxidant defence system of the cell and play crucial roles in the conversion of free radicals into harmless compounds. T-AOC represents the total anti-oxidative capacity. In our study, maternal exposure to ZEN decreased the levels of SOD, GSH-Px and T-AOC and increased the level of MDA in pregnant sows and their offspring. The results suggest that ZEN induces lipid peroxidation and damage in the spleen of pregnant sows and their offspring. Lipid peroxidation is observed as the peroxidation of unsaturated fatty acids of the cell membrane and organelles by free radicals. Peroxidation is confirmed as damage to protein and lipid distribution, decreased resistance and changes in or disruption of the selective permeability of the cell membrane (Eraslan et al. 2013). All these processes alter the extracellular and intracellular ion concentrations and disrupt the function of transporter proteins found in the cell membrane (Evans & Halliwell 2001; Halliwell 2007; Ogino & Wang 2007; Niki 2009). The results are in agreement with the findings reported by Ben Salah-Abbès et al. (2009), who found that male BALB/c mice orally treated for 28 days with ZEN exhibited significantly decreased GSH-Px, and SOD activities in their testes. Eraslan et al. (2013) also reported that oral exposure to aflatoxin induces oxidative stress in the spleen of mice. Histopathological examination is regarded as the standard method through which the degree of tissue injury is measured. Numerous spherical collections of blue-staining cells have been identified as white pulp in pathological sections stained with H&E. The remaining red tissues between these focal aggregates of lymphoid tissue are considered red pulp. Mice treated with 40/80 mg of ZEN kg–1 BW exhibited atrophy of the white pulp and expansion of the red pulp in the spleen (Salah-Abbès et al. 2008). Tiemann, Brüssow, Dannenberger, et al. (2008) found that exposure to a diet with a high concentration of Fusarium toxin (9.57 mg DON and 0.35 mg ZEN kg–1 diet) causes significant histological alterations in the spleen cells of pregnant sows compared with control

animals fed a diet with a low concentration of Fusarium toxin (0.210 mg DON and 0.004 mg ZEN kg–1 diet). In our study, ZEN exposure induced the atrophy of white pulp and the expansion of red pulp in pregnant sows and offspring (neonatal and weaning piglets). The results are consistent with those reported by Tiemann, Brüssow, Dannenberger, et al. (2008), who showed that exposure to ZEN and DON induces changes in the red pulp of spleen. A histopathology examination revealed that prenatal exposure to air pollution causes prominent damage in the spleen of mice offspring (Hong et al. 2013). Thus, variations in histopathology indicate splenic dysfunction in the absence of clinical signs. As mentioned previously, cytokines are small peptide molecules that are important mediators of immune and inflammatory responses (Bouhet & Oswald 2005). T-helper cells are the effector and regulatory cells in immune reaction. Interferon (IFN)-γ is produced by Th1 cells and it promotes inflammatory and cytotoxic T-lymphocyte response. IL-4 is produced by Th2 cells and it promotes B-cell responses. In our study, maternal ZEN exposure induced fluctuations of inflammatory cytokines mRNA levels in pregnant sows and their piglets. A previous study found that ZEN significantly decreases the mRNA expression of TNF-α in the spleen of mice (Abbès et al. 2012). Our findings suggest that the splenic responses to ZEN exposure, including changes in the levels of inflammatory cytokines during gestation, lead to secondary effects in the neonatal and weaning piglets. Maternal exposure to infection alters inflammation cytokine levels in the foetal environment, and has a significant impact on the developing brain (Urakubo et al. 2001). Yessoufou and Moutairou (2011) found that gestational diabetes mellitus and maternal obesity are closely associated with the expression of inflammation cytokines in the offspring. Th1 and Th2 are functionally balanced under normal conditions. The imbalance in Th1/Th2 is closely associated with immunofunctional disorders (Hong et al. 2013). Maternal exposure to ZEN exaggerated Th2 immune response in the offspring postnatally, as indicated by elevated the expression of IL-4 mRNA. Decreased IFN-γ production in spleen implied a suppressed Th1 function. The results found maternal exposure to ZEN potentially modulated both Th1 and Th2 responses in pregnant sows and their piglets. Inflammatory cytokines may have crossed placenta and breast gland to the foetus, and have detrimental effect on the spleen of piglets. ZEN exposure during gestation can damage the newborn immune system, resulting in postnatal immune dysfunction by exacerbation of Thl/Th2 deviation. Further investigation is needed to study the specific mechanism through which the maternal and placental immune systems interact with the newborn and its immune system. ZEN causes a threat to animal and human health (Hassen et al. 2007; Bouaziz et al. 2008; Wang et al. 2012). Effective and practical methods to detoxify ZEN-

Food Additives & Contaminants: Part A contaminated feed and foodstuffs are in great demand. However, to date, no method has successfully removed ZEN from contaminated feeds. HNTs is one of best known aluminosilicates and results in a better utilisation of the protein, fat, fibre and mineral components in fatteners (Korniewicz et al. 2006). Kulok et al. (2005) found that HNTs has the ability to prevent the growth of toxigenic organisms, decrease the bioaccumulation of heavy metals in animal organisms and enrich the diet with trace elements. The HNTs used in this study were modified to increase its porosity and surface area such that it binds effectively to ZEN. The results of the present study revealed that the addition of MHNTs to a ZEN-contaminated diet resulted in a significant improvement in the tested parameters, including the splenic coefficient, ZEN residues, immunoglobulin, antioxidative parameters, histopathology and inflammatory cytokine mRNA expression, in the pregnant sows and their offspring. The protective effects of MHNTs may be explained by the ability of this absorbent to bind ZEN in the gastrointestinal tract and thereby reduce the toxin’s bioavailability. Jiang et al. (2012) mentioned that ZEN induces deleterious effects on nutrient availability, genital organs and serum hormones in piglets and that these effects are ameliorated by the dietary supplementation of modified montmorillonite. Therefore, MHNTs may be the better absorbent for the reduction of the toxic effects of ZEN and the alleviation of the contamination of feed mixtures.

Conclusions Maternal exposure to ZEN through contaminated diets resulted in damaged immune function in the pregnant sows and their offspring (neonatal and weaning piglets), as demonstrated by the analysis of their splenic coefficients, ZEN residues, immunoglobulin levels, antioxidative parameters, histopathological examination and inflammatory cytokine mRNA expression levels. In addition, the dietary addition of MHNTs can improve the adverse effects induced by ZEN in the swine. Further studies are warranted to investigate the specific mechanism through which the maternal immune systems interact with the new born immune system, and determine the efficacy, safety and specificity of MHNTs.

Acknowledgements The authors declare that there are no conflicts of interest.

Funding This work was supported by the National Basic Research Program [grant number 2012CB124703]; the China Agriculture Research System [grant number CARS-36]; and the Program for Innovative Research Team of Universities in Heilongjiang Province [grant number 2012TD003].

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