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Modified halloysite nanotubes and the alleviation of kidney damage induced by dietary zearalenone in swine a
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Zhiqiang Jia , Shutong Yin , Min Liu , Yuanyuan Zhang , Rui Gao , Baoming Shi , Anshan a
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Institute of Animal Nutrition, Northeast Agricultural University, Harbin, China Published online: 03 Jul 2015.
Click for updates To cite this article: Zhiqiang Jia, Shutong Yin, Min Liu, Yuanyuan Zhang, Rui Gao, Baoming Shi, Anshan Shan & Zhihui Chen (2015): Modified halloysite nanotubes and the alleviation of kidney damage induced by dietary zearalenone in swine, Food Additives & Contaminants: Part A, DOI: 10.1080/19440049.2015.1048748 To link to this article: http://dx.doi.org/10.1080/19440049.2015.1048748
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Food Additives & Contaminants: Part A, 2015 http://dx.doi.org/10.1080/19440049.2015.1048748
Modified halloysite nanotubes and the alleviation of kidney damage induced by dietary zearalenone in swine Zhiqiang Jia, Shutong Yin, Min Liu, Yuanyuan Zhang, Rui Gao, Baoming Shi, Anshan Shan* and Zhihui Chen Institute of Animal Nutrition, Northeast Agricultural University, Harbin, China
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(Received 12 January 2015; accepted 27 April 2015) The aims of this study were, first, to investigate the toxicity of zearalenone (ZEN) through the analysis of biochemical parameters, oxidative stress, pathological changes and inflammatory response in the kidney of gestation sows and offspring; and, second, to evaluate the efficacy of modified halloysite nanotubes (MHNTs) for the alleviation to the adverse effects induced by ZEN. This study focused on the period of organogenesis between days 35 and 70 of gestation, and treatments included (1) a control diet; (2) contaminated grain (50% control corn and 50% mouldy corn); and (3) contaminated grain (50% control corn and 50% mouldy corn) + 1% MHNTs. ZEN treatment significantly increased most of the biochemical parameters and inflammatory cytokines and degenerative changes in the kidney and induced oxidative damage in plasma, whereas the addition of MHNTs in combination with ZEN induced a re-establishment of the biochemical parameters, the plasma oxidative stress enzyme activities and the normal histology of the kidney. Thus, the data strongly suggest that the deleterious effects of ZEN can be significantly diminished by MHNTs. Keywords: zearalenone; oxidative stress; biochemical and pathological changes; kidney; sows; offspring
Introduction The use of natural and synthetic aluminosilicates in animal production has been the subject of numerous interdisciplinary research studies. These substances are characterised by their ability to prevent the growth of fungi, including toxigenic fungi (Kolacz et al. 2003), decrease the bioaccumulation of heavy metals in animal organisms (Dobrzanski et al. 2004), and enrich the diet with trace elements (Yablonska 2003). In addition, these compounds may influence the processes of digestion and bonding of metabolites, which decreases the emission of toxic gases from bedding (Tymczyna 1993). Zeolites, kaolins, bentonites, saponites and halloysites are the best-known aluminosilicates. These can be used as ‘inorganic sponges’ for the sequestration of mycotoxins, namely T-2 toxin, aflatoxin, ergotamine and zearalenone (ZEN) (Avantaggiato et al. 2005), from the gastrointestinal tract of farm animals (Ramos et al. 1996). Mycotoxins are produced by several fungi and comprise a group of different chemically toxic compounds (Sweeney & Dobson 1998). The most important Fusarium toxins that may potentially affect human and animal health are deoxynivalenol, moniliformin, fumonisin B1 and ZEN (Berek et al. 2001; Yen et al. 2014). ZEN is a non-steroidal estrogenic mycotoxin produced by fungi belonging to the Fusarium genus (Richard 2007). ZEN is a contaminant of cereals and plant products (Engelhardt et al. 2006; Tabuc et al. 2009), has a major effect on human and
*Corresponding author. Email: [email protected] © 2015 Taylor & Francis
animal health, and causes serious worldwide economic problems (FAO/WHO 2000). It has been shown that ZEN acts as a mammalian endocrine disrupter with effects on both males and females of different species (Collins et al. 2006; Zhang, Jia, et al. 2014). This effect results from the capacity of ZEN to bind estrogen receptors, leading to hyperestrogenicity in several animal species, particularly pigs (Takemura et al. 2007; Minervini & Dell’Aquila 2008). Previous investigations have reported that genital organs, such as the vulva, ovaries and testes, are important target organs (Farnworth & Trenholm 1981; NTP 1982). Once having entered the body, ZEN is mainly metabolised in the liver and spleen (Koraichi et al. 2012; Yin et al. 2015). ZEN has been shown to induce liver lesions with subsequent development of hepatocarcinoma (NTP 1982) and alterations in some enzymatic parameters associated with hepatic function in rats (Maaroufi et al. 1996) and gilts (Wang et al. 2012). Moreover, it has been reported that a single intraperitoneal dose of ZEN induces haematological and biochemical toxicity (Maaroufi et al. 1996). However, at present, limited information is available regarding the negative effects of ZEN on oxidative stress and the biochemical and pathological changes induced by this toxic compound in the kidney of pregnant sows. The positive effect of some aluminosilicates used in animal feeding has been proven by various researchers (Rudzik 1998; Zhang, Gao, et al. 2014; Yin et al. 2015).
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Phyllosilicate clay was found to be able to chemisorb aflatoxins from aqueous solutions (Harvey et al. 1994). Up to 85% of the toxic effects induced by aflatoxins were reversed by the addition of 0.5 g of clay kg–1 of contaminated diets, and this reversion prevented subsequent liver damage and chromosomal aberrations induced by this toxic compound (Abdel-Wahhab et al. 1998). The dietary inclusion of hydrated sodium calcium aluminosilicate has also been demonstrated to be an effective method to prevent the negative effects of ZEN on reproductive, haematological and biochemical processes (Abbès, Ouanes, et al. 2006). Kulok et al. (2005) indicated the high decontaminating efficiency of halloysite toward bacteria, fungi and aflatoxin B1, which proves that aluminosilicates may be used to reduce the contamination of feed mixtures. Moreover, Bueno et al. (2005) indicated that bentonite can bind to ZEN in vitro and proposed this sorbent as a good candidate for the detoxification of ZEN present in foods. Afriyie-Gyawu et al. (2005) suggested that the addition of activated carbon, hectorite or montmorillonite clay effectively decreases the effects of ZEN. The aims of the present study were to investigate the toxicity of ZEN through the analysis of biochemical parameters, oxidative stress, pathological changes and inflammatory response in the kidney of gestation sows and filial generation and to evaluate the efficacy of modified halloysite nanotubes (MHNTs) for the alleviation to the adverse effects induced by ZEN.
Materials and methods Sorbent material and modification The adsorbent used for the experiment was prepared as reported by Zhang, Gao, et al. (2014). Powder halloysite nanotubes (HNTs, Al2Si2O5(OH)4·nH2O) were refined from clay minerals from Henan province (China) with a purity of 95%. The powder was prepared according to the method described by Wang et al. (2010). HNTs were modified using stearyl dimethylbenzyl ammonium chloride (SKC) (Jingwei Chemical Co., Ltd, Shanghai, China) according to methods previously described by Lemke et al. (2001), with some modifications.
Strain and moulding Fusarium graminearum, a ZEN-producing fungus (Eva 2007), was obtained from the Agricultural Culture Collection of China (No. ACCC36249). The fungus was cultivated on potato dextrose agar (PDA; 0.4% potato extract, 2% glucose and 1.5% agar, pH 5.6 ± 0.2). All culture media were obtained from Fluka (Bornem, Belgium) (Demyttenaere et al. 2004). The corn used for the experiments was obtained by Xiang Fang Experimental Bases of Northeast Agricultural
University and milled in a hammer mill with a 40-mesh screen (Trapp-TRF model 90). ZEN was produced in vitro according to the procedures outlined by Lı́gia and Marina (2002). The studies of mycotoxin production by F. graminearum were conducted in duplicate on trays containing 1000 g of sterilised cracked corn and 400 ml of distilled water with a water activity of 0.97. The corn contained no fungal infection or ZEN contamination. The autoclaved substrate was inoculated with 40 ml of the spore suspension according to the following procedure: 100 ml of sterile distilled water were added to each slant of 5-dayold culture, and the agar surface was gently scraped to obtain a turbid suspension corresponding to 1 × 1014 spores ml–1. Suspension (100 ml) was added to the cracked corn. The inoculated flasks were stirred daily during the first 5 days of culture. The following culture conditions were used in this experiment: the temperature was maintained at 28°C for the first 15 days and then at 12°C. ZEN production reached its peak after 35 days of incubation, as reported by Lı́gia and Marina (2002). To equalise the moisture of the samples, each sample was dried at 60°C for 96 h and stored in a freezer at −20°C until analysis (Queiroz et al. 2012). Animals and experimental design Pregnant sows were used in a completely randomised block design and divided into three groups with mean initial live weights of 185.93 ± 3.57, 191.20 ± 2.61 and 187.37 ± 2.49 kg. Eighteen second-parity Yorkshire sows (six per treatment) were bred with semen from a pool of Landrace boars. The pregnant sows were housed in individual stalls after 35 gestation days (GDs). During GDs 35–70, the feed was restricted to 2 kg per pig day–1. The period of organogenesis between days 35 and 70 of gestation was chosen for this study because the foetuses may show possible teratogenic effects or mortality (Goyarts et al. 2007) during this key period of organogenesis. The following treatments were included in this study: (1) control, (2) contaminated grains (50% control corn and 50% mouldy corn), and (3) contaminated grains (50% control corn and 50% mouldy corn) + 1% MHNTs. The doses of MHNTs were selected based on the work conducted by Jiang et al. (2010, 2012). All 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 19540–2004). ZEN was the major contaminant and was present at a concentration of 2.77 mg kg–1 in the ZEN-contaminated diets. Analysis of the corn and diets by GC-MS was performed to obtain a detailed characterisation of the trichothecene mycotoxin pattern, and the results revealed that levels of other B-trichothecene mycotoxins, such as 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol and nivalenol, and the A-
Food Additives & Contaminants: Part A trichothecene mycotoxins, such as HT-2 toxin, were lower than the detection limits (Tiemann et al. 2008). Water was provided ad libitum. Experimental diets (Table 1) were formulated to meet or exceed the National Research Council nutrient requirements (NRC 1998).
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Samples collection Three sows from each group were killed through an intraarterial injection of pentobarbital (200 mg kg–1) after general anaesthesia on day 70 of gestation after exposure to the experimental diets for 35 days. Blood of the sows was collected by puncture of the vena jugularis externa. Microcentrifuge tubes were coated with 10 ml of heparin sodium salt (20 U ml–1 in PBS). Whole blood was collected from the submandibular site as described above and maintained on ice for 30 min. The samples were centrifuged at 3000g and 4°C for 10 min. The plasma layers were removed, placed into sterile microcentrifuge tubes and stored at −40°C until analysis. Fragments of the maternal kidneys were quickly dissected, frozen in liquid nitrogen, stored at −80°C, and subjected to RNA extraction and quantitative reverse-transcription PCR. Other fragments were stored at −20°C for biochemical analysis and ZEN residue analysis. The remaining sows were allowed to farrow naturally and fed a mycotoxin-free
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diet until weaning on day 21 of lactation. Six neonatal or weanling piglets from each group were killed by an intra-arterial injection of pentobarbital. The methods of blood and kidney samples collection from the piglets were same as those for the sows on GD 70. All the animal experimental procedures were approved by the Ethical and Animal Welfare Committee of Heilongjiang Province, China.
Biochemical parameter determination The biochemical parameters were determined with an automated biochemical analyser (Synchron CX®4 Pro Beckman Coulter, Brea, CA, USA) using commercial diagnostics kits (Biosino Biotechnology and Science Inc., Beijing, China). The following biochemical characteristics in the plasma were determined: blood urea nitrogen (BUN), creatinine (CRE) and uric acid (UA).
Oxidative parameter determination The parameters of oxidative stress were determined with an ultraviolet spectrophotometer (UV-2410PC model, Shimadzu, Japan) using commercial diagnostics kits (Nanjing Jiancheng Biotechnology Co., Ltd., Jiangsu, China). The following plasma parameters were evaluated:
Table 1. Percentage composition of the diet. Controlc
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 in this study were according to the Tables of Feed Composition and Nutritive Values in China (Yin et al. 2015). c Control = control diet; contaminated grains = instead of 50% mouldy corn; contaminated grains = instead of 50% mouldy corn + 1% MHNTs; ZEN, zearalenone; MHNTs, modified halloysite nanotubes.
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malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSHPx).
were routinely stained with haematoxylin and eosin (H&E) and assessed using a light microscope (Nikon Eclipse E400). All alterations from the normal structure were registered.
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Total RNA and quantitative real-time polymerase chain reaction (PCR) The total RNA from the kidney tissue was isolated using a reagent box (E.Z.N.A.®Total RNA Kit, Omega Bio-tek, Inc., Doraville, GA, USA) according to the manufacturer’s recommended protocol. The concentration of RNA was estimated based on its absorbance at 260 nm, which was determined using a spectrophotometer. The RNA quality was determined by checking its integrity through agarose gel electrophoresis and by confirming that the A260/ A280 nm absorbance ratio was between 1.8 and 2.0. The first-strand cDNA was synthesised from 5 μg of the total RNA using oligo-dT primers and superscript II reverse transcriptase according to the manufacturer’s instructions (Tiangen Biotech Co., Ltd, Beijing, China). The expression levels of tumour necrosis factor (TNF)-a, interleukin (IL)-1β, IL-6 and β-actin were determined through quantitative real-time PCR using an ABI PRISM 7500 SDS thermal cycler apparatus (Applied Biosystems, Foster City, CA, USA) using the following temperature programme: one cycle of 95°C for 30 s and 40 cycles of 95°C for 5 s and 61°C for 34 s. The dissociation curves for each PCR reaction were analysed using the Dissociation Curve 1.0 software (Applied Biosystems) to detect and eliminate any possible primer–dimers and nonspecific amplifications. The relative expression ratios of the target genes were calculated based on the efficiencies and quantification cycle (Cq) deviations of the unknown samples compared with the controls and are expressed in comparison with the reference gene, as described by Pfaffl (2001). The primer sequences and product sizes are shown in Table 2.
Histopathological studies Slices of the left kidney were fixed in 10% formalin for 24 h and embedded in paraffin. Then, 3–5-μm sections
Statistical analysis The comparison of the treatment groups with the control group was conducted through analysis of variance (ANOVA) using Statistical Packages for Social Science 18.0 (SPSS, Chicago, IL, USA) software. The differences between the means were determined using Duncan’s multiple-range test. Differences with a probability (p) value of less than 0.05 were considered to be statistically significant. The results of the statistical analyses are shown as the mean ± standard error of the means (SEM).
Results Study of the plasma biochemical parameters The effects of ZEN with or without modified halloysite on some plasma biochemical parameters, including BUN, CRE and UA, are presented in Table 3.
Plasma CRE Compared with the control animals, the plasma CRE level was significantly decreased (p < 0.05) in the sows in the ZEN-treated group and their offspring (neonatal piglets and weanling pigs). The plasma CRE level in the animals treated with MHNTs in combination with ZEN was significantly increased compared with the ZEN-treated sows their offspring (neonatal and weanling piglets).
Plasma UA The plasma UA level in the ZEN-treated sows and their offspring (neonatal piglets and weanling piglets) was significantly increased (p < 0.05) compared with the control group. The UA level in the group treated with MHNTs in combination with ZEN was not significantly decreased
Table 2. Primers used for quantitative real-time PCR. Gene
GenBank Accession Number
β-Actin
AY550069
IL-1β
NM_214055.1
IL-6
M 214399
TNF-α
NM_031329.2
Sequence
Fragment length (bp)
Forward primer 5ʹ-atgcttctaggcggactgt-3ʹ Reverse primer 5ʹ-ccatccaaccgactgct-3ʹ Forward primer 5ʹ-ggccgccaagatataactga-3ʹ Reverse primer 5ʹ-ggacctctgggtatggctttc-3ʹ Forward primer 5ʹ-tgatgccacctcagacaa-3ʹ Reverse primer 5ʹ-tcacacttctcatacttctcac-3ʹ Forward primer 5ʹ-cagcctcttctccttcct-3ʹ Reverse primer 5ʹ-cgatgatctgagtccttgg-3ʹ
211 70 123 142
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Table 3. Studying of the biochemical parameters in plasma. Animal Sow Neonatal piglets Weanling pigs
Item CRE (μmol l–1) BUN (mg l–1) UA (μmol l–1) CRE (μmol l–1) BUN (mg l–1) UA (μmol l–1) CRE (μmol l–1) BUN (mg l–1) UA (μmol l–1)
Control 166.58 119.06 23.18 117.26 105.98 17.42 123.77 107.02 13.69
Contaminated grains (ZEN)
6.51a 2.75c 1.71b 2.04a 9.64b 0.00b 0.93a 0.34c 1.24b
± ± ± ± ± ± ± ± ±
138.66 221.60 31.15 88.66 199.92 33.60 81.90 361.31 27.13
± ± ± ± ± ± ± ± ±
2.79c 1.38a 1.24a 0.99 c 12.73a 1.24a 1.86c 11.01a 0.24a
Contaminated grains +1% MHNTs 151.76 189.05 26.13 96.79 113.21 18.67 107.09 186.85 23.64
± ± ± ± ± ± ± ± ±
1.12b 1.86b 1.24ab 1.86b 9.29b 1.24b 2.86b 0.34b 1.24a
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Notes: Data are means ± SEM. a,b,c within a row with no common superscripts differ significantly (p < 0.05). ZEN, zearalenone; MHNTs, modified halloysite nanotubes; BUN, blood urea nitrogen; CRE, creatinine, UA, uric acid.
compared with the ZEN-treated sows and weanling piglets. However, the UA level was significantly decreased (p < 0.05) in the MHNTs + ZEN-treated neonatal piglets compared with ZEN-alone-treated neonatal piglets.
Plasma BUN The plasma BUN level was significantly increased (p < 0.05) in the ZEN-treated group compared with the control group. The use of MHNTs in combination with ZEN significantly decreased (p < 0.05) the BUN level compared with that obtained for the ZEN-treated group.
group. The use of MHNTs in combination with ZEN significantly increased (p < 0.05) the SOD level in the plasma compared with that obtained in the ZEN-treated group.
Plasma GSHPx Compared with the control group, the level of GSHPx was significantly decreased (p < 0.05) in the plasma in the ZEN-treated group. Treatment with MHNTs in combination with ZEN significantly increased (p < 0.05) the GSHPx level compared with the ZEN-treated group.
Analysis of oxidative parameters in the plasma The effects of ZEN with or without modified halloysite on some oxidative parameters in the plasma, including MDA, SOD and GSHPx, are presented in Table 4.
Plasma SOD The ZEN-treated group exhibited significantly decreased activities of SOD in the plasma compared with the control
Plasma MDA Compared with the control group, the level of MDA in the plasma was significantly increased (p < 0.05) in the ZENtreated group. The sows and neonatal piglets treated with MHNTs in combination with a ZEN exhibited significantly decreased (p < 0.05) MDA level compared with the ZEN-treated group. However, the MDA level was not significantly decreased in weanling piglets.
Table 4. Oxidative parameters determination in plasma. Animal Sow Neonatal piglets Weanling pigs
Item SOD (U ml–1) MDA (nmol ml–1) GSHPx (U ml–1) SOD (U ml–1) MDA (nmol ml–1) GSHPx (U ml–1) SOD (U ml–1) MDA (nmol ml–1) GSHPx (U ml–1)
Control 180.99 3.36 1348.48 68.34 3.01 649.24 78.98 3.35 1001.44
± ± ± ± ± ± ± ± ±
4.62a 0.59b 14.03a 1.03a 0.33b 10.77a 0.56a 0.61a 11.07a
Contaminated grains (ZEN) 97.99 11.41 858.61 20.84 8.73 281.25 33.97 6.04 678.87
± ± ± ± ± ± ± ± ±
2.13c 0.78a 8.61c 2.48c 0.67a 6.25c 0.70c 0.67a 8.64c
Contaminated grains +1% MHNTs 131.38 6.04 1123.64 52.60 3.36 571.94 69.05 4.70 867.56
± ± ± ± ± ± ± ± ±
2.89b 0.67b 11.14b 1.62b 0.67b 3.06b 0.29b 0.66a 7.45b
Notes: Data are means ± SEM. a,b,c within a row with no common superscripts differ significantly (p < 0.05). ZEN, zearalenone; MHNTs, modified halloysite nanotubes; MDA, malondialdehyde; SOD, superoxide dismutase; GSHPx, glutathione peroxidase.
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Analysis of the mRNA expression levels of cytokines The mRNA expression levels of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) were measured by quantitative RT-PCR, as shown in Table 5. With the exception of IL-1β in neonatal piglets, there was a significant increase in the mRNA levels of pro-inflammatory cytokines (TNFa, IL-1β and IL-6) in the ZEN-treated sow group and their offspring compared to controls.
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Histopathological study of the kidney As shown in Figure 1(a), the kidney sections from the control animals show normal renal tubules, normal glomeruli and normal epithelial cells lining the renal tubules. In contrast, the treatment of animals with ZEN alone revealed histopathological changes in the kidney sections, as shown in Figure 1(b). These changes included the presence of abnormal epithelial cells lining the renal tubules, protein deposition, renal interstitial fibrosis and shrunken glomeruli. The treatment with MHNTs in combination with ZEN induced a re-establishment of normal histopathology in the kidney sections, as shown in Figure 1(c). The kidney sections from neonatal and weaned piglets were similar. Discussion Many epidemiological studies have demonstrated that ZEN can induce several deleterious effects in many visceral organs of both humans and animals (Berek et al. 2001; Abbès, Salah-Abbès, et al. 2006). Based on its role in excretion, re-absorption and general homeostasis, the kidney has an extensive blood flow, receiving approximately 1.2 L of plasma min–1 and filtering an average of 125 ml of plasma min–1. To assess the changes in kidney function induced by ZEN, we monitored BUN, UA and CRE levels. Increases in BUN level have been described in most cases of nephrotoxicity, including those induced by mycotoxin exposure (An et al. 2011). In the present study, the BUN level was significantly increased
(p < 0.05) in the ZEN-treated group compared with the control group. The resulting elevation in the BUN level is not solely due to a decrease in the GFR and results in acute renal failure (Aronson et al. 2008). CRE is a byproduct of muscle energy metabolism that is filtered from the blood by the kidneys and excreted into the urine. Because creatinine is only slightly affected by the liver function, it is a sensitive indicator of kidney function (Vander et al. 1998). The low level of creatinine found in gestation sows and offspring treated with ZEN alone, as found in the current study, indicates degenerative changes in the kidney function, as described by Salah-Abbès, Abbès, Ouanes, et al. (2008), and the same results were obtained by Abbès, Salah-Abbès, et al. (2006). Moreover, UA is a byproduct of protein metabolism; this waste product is formed in the liver and is then filtered from the blood and excreted in the urine by the kidneys (Vander et al. 1998). In this study, an increase in the plasma UA level was observed in swine treated with ZEN alone, indicating kidney damage and in agreement with the results reported by Abbès, Ouanes, et al. (2006). Oxidative stress results in damage to cellular structures and has been linked to many diseases, including cancer (Marin & Taranu 2012). Recent studies have shown that ZEN enhances reactive oxygen species formation and causes oxidative damage (Yuk et al. 2011). The oxidative stress caused by ZEN may be one of the underlining mechanisms through which ZEN induces cell injury and DNA damage, which eventually lead to tumorigenesis (Abid-Essefi et al. 2009). In the current study, we showed that ZEN treatment induced a decrease in the expression of two enzymes involved in oxidative stress, namely SOD and GSHPx. SOD refers to a class of enzymes that catalyse the dismutation of superoxide into oxygen and hydrogen peroxide, which represents an important antioxidant defence in nearly all cells exposed to oxygen (Liska 1998). Higher concentrations (1.1–3.2 ppm) of ZEN in the diet have been found to be able to reduce SOD activity in the spleen of post-weaning gilts (Jiang et al. 2011) and in the liver and serum of weanling piglets (Jiang et al. 2010). The down-regulation of SOD gene expression and
Table 5. mRNA expression of cytokines in kidney. Animal Sow Neonatal piglets Weanling pigs
Item
Control
Contaminated grains (ZEN)
TNF-α IL-1β IL-6 TNF-α IL-1β IL-6 TNF-α IL-1β IL-6
0.0092 ± 0.0004c 0.0009 ± 0.0001c 0.0014 ± 0.0001c 0.0027 ± 0.0003b 0.0011 ± 0.0002a 0.0002 ± 0.0001b 0.0013 ± 0.001b 0.0023 ± 0.0003b 0.0001 ± 0.000b
0.0201 ± 0.0022a 0.0277 ± 0.0010a 0.0211 ± 0.0007a 0.0184 ± 0.0013a 0.0196 ± 0.0082a 0.0043 ± 0.0002a 0.0059 ± 0.0003a 0.0218 ± 0.0036a 0.0007 ± 0.0001a
Contaminated grains +1% MHNTs 0.0130 0.0029 0.0053 0.0043 0.0059 0.0003 0.0017 0.0073 0.0004
± ± ± ± ± ± ± ± ±
0.0006 0.0004b 0.0003b 0.0008b 0.0045a 0.0001b 0.0003b 0.0007b 0.000b
Notes: Data are means ± SEM. a,b,c within a row with no common superscripts differ significantly (p < 0.05). ZEN, zearalenone; MHNTs, modified halloysite nanotubes.
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Figure 1. (colour online) (a) Control, (b) contaminated grains (instead of 50% mouldy corn), and (c) contaminated grains (instead of 50% mouldy corn) + 1% modified HNTs. Normal glomeruli (NG), normal renal tubules (NRT), protein deposition (PD), renal interstitial fibrosis (RIF), tubular degeneration (TD), shrunken glomeruli (SG). (H&E, 400×).
the reduction of SOD activity can lead to the accumulation of superoxide anions within the mitochondria, leading to oxidative stress and thereby impairing some vital cellular functions (Marczuk-Krynicka et al. 2003). In agreement with our results, Salah-Abbès, Abbès, Ouanes, et al. (2008) reported that the activities of SOD and GSHPx decrease with progressive liver and kidney injury in ZEN-treated mice. Salah-Abbès et al. (2009) also observed that ZEN supplementation at a concentration of 40 mg kg–1 significantly reduces the activities of SOD and GSHPx in the testes of mice. The activities of SOD and GSHPx are known to serve as protective responses for the elimination of reactive free radicals (Cheung et al. 2001). Therefore, the decreased activities of SOD and GSHPx in the plasma found in the present study suggest that ZEN supplementation induces oxidative stress. In fact, the most common group of indices used to assess oxidative stress involves the peroxidation products of lipids. The most widely used index is MDA, which is the end product of lipid peroxidation and is considered a late biomarker of oxidative stress and cellular damage (Dotan et al. 2004). In the present study, although exposure to ZEN induced a marked increase in MDA formation in the plasma, the administration of MHNTs (even at the lowest dose tested) significantly reduced this induction to the basal level. The administration of MHNTs prevented the lipid peroxidation induced by ZEN treatment in sow and foetal piglets. It has been demonstrated that ZEN and its metabolites induce lipid oxidation and increase the production of malondialdehyde in several cell lines (Hassen et al. 2007; Othmen et al. 2008). ZEN has been described as both a suppressor and an inducer of inflammation (Marin et al. 2011). The results showed that ZEN induced pro-inflammatory effects in the kidney. With the exception of IL-1β in neonatal piglets, an inflammatory response was observed in the kidneys of the animals in the ZEN-treated sow group and their offspring, as suggested by the increase in the mRNA expression
levels of inflammatory cytokines (IL-1β, TNF-α and IL-6). These results are in agreement with those obtained in mice after the administration of DON through a single oral dose of 5 or 25 mg kg–1 bw, which resulted in significantly up-regulated mRNA levels of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α; the lower doses had no effect (Zhou et al. 1997). Chiba et al. (2007) reported that aflatoxin B1 can increase the production of TNF-α and IL-6 by inhibiting the synthesis of ceramide. In addition, the in vivo daily ingestion of 2.8 mmol of fumonisin B1 kg–1 bw decreased the expression levels of IL-6, IL-10, IL-18 and IFN-γ in the liver of piglets, whereas the expression levels of IL-1β and IL-8 were increased (Royaee et al. 2004). DON induces modest increases in the expression levels of TNF-α, IL-6 and IL-1β in the liver relative to those found in the spleen (Salah-Abbès, Abbès, Houas, et al. 2008). In other studies, ZEN and its derivatives have been described as suppressors (Salah-Abbès, Abbès, Houas, et al. 2008; Marin et al. 2011) of the expression of pro-inflammatory cytokines and as inducers of DNA fragmentation (Gazzah et al. 2010). The study conducted by Wang et al. (2012) revealed that the alteration in the expression levels of cytokines (IL-2, IL-6 and IFN-γ) in chicken splenocytes is associated in vitro with increasing concentrations of ZEN. DNA fragmentation was one of the results of the chronological succession of some events that characterise the toxicity of ZEN in human hepatocarcinoma cells (Gazzah et al. 2010). This divergent effect may be due to differences in the toxicities of these compounds and the animal models used. The results found from the analyses of the inflammatory cytokines and biological and oxidative stress parameters were confirmed through the histological findings in the kidneys. The histopathological examination of the kidneys of the animals in the ZEN-treated group revealed shrunken glomeruli, renal interstitial fibrosis, tubular degeneration and protein deposition. The acute cellular degeneration in the epithelial lining of the renal tubules
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is due to the toxic effect of ZEN and may be caused by cell membrane injury or an effect on the mitochondria that leads to the depletion ATP or a defect in the sodium– potassium pump and thus fluid disturbance in and outside of cells. These results are consistent with the results in mice treated with a single dose of 40 mg kg–1 bw of ZEN (Abbès, Salah-Abbès, et al. 2006). The pathology of the kidneys was described by Grosman et al. (1983), and similar results were obtained by Abdel-Wahhab et al. (2002), who demonstrated that rats are acutely sensitive to the same nephrotoxic effects caused by aflatoxin B1. Our study showed a clear protective effect of the sorbent MHNTs material against oxidative stress and the biochemical and pathological (renal tissues) changes induced by ZEN. These results were supported by Kulok et al. (2005), who demonstrated the high decontaminating efficiency of halloysite toward bacteria, fungi and aflatoxin B1, which proves that aluminosilicates may be used to reduce the contamination of feed mixtures. Montmorillonite, another sorbent material, was found to be able to bind efficiently to ZEN (Lemke et al. 2001). Recently, Bueno et al. (2005) indicated that bentonite can bind to ZEN in vitro. AfriyieGyawu et al. (2005) suggested that the addition of either activated carbon or hectorite effectively decreases the toxic effects of ZEN on the reproductive system of Hydra attenuata. As a result, this type of sorbent material can be considered a good candidate for the prevention of the toxic effects of ZEN or for the detoxification of this toxin in contaminated foods.
Conclusions The results of the current study indicated that ZEN not only caused damage to both oxidative stress and biochemical parameters but also induced the degeneration of renal tissues in pregnant sows and their offspring. We also found that MHNTs were able to prevent most of the alterations induced by ZEN, which suggested that this material efficiently reduced the bioavailability of ZEN.
Disclosure statement No potential conflict of interest was reported by the authors.
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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Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
Modified halloysite nanotubes and the alleviation of kidney damage induced by dietary zearalenone in swine a
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Zhiqiang Jia , Shutong Yin , Min Liu , Yuanyuan Zhang , Rui Gao , Baoming Shi , Anshan a
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Institute of Animal Nutrition, Northeast Agricultural University, Harbin, China Published online: 03 Jul 2015.
Click for updates To cite this article: Zhiqiang Jia, Shutong Yin, Min Liu, Yuanyuan Zhang, Rui Gao, Baoming Shi, Anshan Shan & Zhihui Chen (2015): Modified halloysite nanotubes and the alleviation of kidney damage induced by dietary zearalenone in swine, Food Additives & Contaminants: Part A, DOI: 10.1080/19440049.2015.1048748 To link to this article: http://dx.doi.org/10.1080/19440049.2015.1048748
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Food Additives & Contaminants: Part A, 2015 http://dx.doi.org/10.1080/19440049.2015.1048748
Modified halloysite nanotubes and the alleviation of kidney damage induced by dietary zearalenone in swine Zhiqiang Jia, Shutong Yin, Min Liu, Yuanyuan Zhang, Rui Gao, Baoming Shi, Anshan Shan* and Zhihui Chen Institute of Animal Nutrition, Northeast Agricultural University, Harbin, China
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(Received 12 January 2015; accepted 27 April 2015) The aims of this study were, first, to investigate the toxicity of zearalenone (ZEN) through the analysis of biochemical parameters, oxidative stress, pathological changes and inflammatory response in the kidney of gestation sows and offspring; and, second, to evaluate the efficacy of modified halloysite nanotubes (MHNTs) for the alleviation to the adverse effects induced by ZEN. This study focused on the period of organogenesis between days 35 and 70 of gestation, and treatments included (1) a control diet; (2) contaminated grain (50% control corn and 50% mouldy corn); and (3) contaminated grain (50% control corn and 50% mouldy corn) + 1% MHNTs. ZEN treatment significantly increased most of the biochemical parameters and inflammatory cytokines and degenerative changes in the kidney and induced oxidative damage in plasma, whereas the addition of MHNTs in combination with ZEN induced a re-establishment of the biochemical parameters, the plasma oxidative stress enzyme activities and the normal histology of the kidney. Thus, the data strongly suggest that the deleterious effects of ZEN can be significantly diminished by MHNTs. Keywords: zearalenone; oxidative stress; biochemical and pathological changes; kidney; sows; offspring
Introduction The use of natural and synthetic aluminosilicates in animal production has been the subject of numerous interdisciplinary research studies. These substances are characterised by their ability to prevent the growth of fungi, including toxigenic fungi (Kolacz et al. 2003), decrease the bioaccumulation of heavy metals in animal organisms (Dobrzanski et al. 2004), and enrich the diet with trace elements (Yablonska 2003). In addition, these compounds may influence the processes of digestion and bonding of metabolites, which decreases the emission of toxic gases from bedding (Tymczyna 1993). Zeolites, kaolins, bentonites, saponites and halloysites are the best-known aluminosilicates. These can be used as ‘inorganic sponges’ for the sequestration of mycotoxins, namely T-2 toxin, aflatoxin, ergotamine and zearalenone (ZEN) (Avantaggiato et al. 2005), from the gastrointestinal tract of farm animals (Ramos et al. 1996). Mycotoxins are produced by several fungi and comprise a group of different chemically toxic compounds (Sweeney & Dobson 1998). The most important Fusarium toxins that may potentially affect human and animal health are deoxynivalenol, moniliformin, fumonisin B1 and ZEN (Berek et al. 2001; Yen et al. 2014). ZEN is a non-steroidal estrogenic mycotoxin produced by fungi belonging to the Fusarium genus (Richard 2007). ZEN is a contaminant of cereals and plant products (Engelhardt et al. 2006; Tabuc et al. 2009), has a major effect on human and
*Corresponding author. Email: [email protected] © 2015 Taylor & Francis
animal health, and causes serious worldwide economic problems (FAO/WHO 2000). It has been shown that ZEN acts as a mammalian endocrine disrupter with effects on both males and females of different species (Collins et al. 2006; Zhang, Jia, et al. 2014). This effect results from the capacity of ZEN to bind estrogen receptors, leading to hyperestrogenicity in several animal species, particularly pigs (Takemura et al. 2007; Minervini & Dell’Aquila 2008). Previous investigations have reported that genital organs, such as the vulva, ovaries and testes, are important target organs (Farnworth & Trenholm 1981; NTP 1982). Once having entered the body, ZEN is mainly metabolised in the liver and spleen (Koraichi et al. 2012; Yin et al. 2015). ZEN has been shown to induce liver lesions with subsequent development of hepatocarcinoma (NTP 1982) and alterations in some enzymatic parameters associated with hepatic function in rats (Maaroufi et al. 1996) and gilts (Wang et al. 2012). Moreover, it has been reported that a single intraperitoneal dose of ZEN induces haematological and biochemical toxicity (Maaroufi et al. 1996). However, at present, limited information is available regarding the negative effects of ZEN on oxidative stress and the biochemical and pathological changes induced by this toxic compound in the kidney of pregnant sows. The positive effect of some aluminosilicates used in animal feeding has been proven by various researchers (Rudzik 1998; Zhang, Gao, et al. 2014; Yin et al. 2015).
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Phyllosilicate clay was found to be able to chemisorb aflatoxins from aqueous solutions (Harvey et al. 1994). Up to 85% of the toxic effects induced by aflatoxins were reversed by the addition of 0.5 g of clay kg–1 of contaminated diets, and this reversion prevented subsequent liver damage and chromosomal aberrations induced by this toxic compound (Abdel-Wahhab et al. 1998). The dietary inclusion of hydrated sodium calcium aluminosilicate has also been demonstrated to be an effective method to prevent the negative effects of ZEN on reproductive, haematological and biochemical processes (Abbès, Ouanes, et al. 2006). Kulok et al. (2005) indicated the high decontaminating efficiency of halloysite toward bacteria, fungi and aflatoxin B1, which proves that aluminosilicates may be used to reduce the contamination of feed mixtures. Moreover, Bueno et al. (2005) indicated that bentonite can bind to ZEN in vitro and proposed this sorbent as a good candidate for the detoxification of ZEN present in foods. Afriyie-Gyawu et al. (2005) suggested that the addition of activated carbon, hectorite or montmorillonite clay effectively decreases the effects of ZEN. The aims of the present study were to investigate the toxicity of ZEN through the analysis of biochemical parameters, oxidative stress, pathological changes and inflammatory response in the kidney of gestation sows and filial generation and to evaluate the efficacy of modified halloysite nanotubes (MHNTs) for the alleviation to the adverse effects induced by ZEN.
Materials and methods Sorbent material and modification The adsorbent used for the experiment was prepared as reported by Zhang, Gao, et al. (2014). Powder halloysite nanotubes (HNTs, Al2Si2O5(OH)4·nH2O) were refined from clay minerals from Henan province (China) with a purity of 95%. The powder was prepared according to the method described by Wang et al. (2010). HNTs were modified using stearyl dimethylbenzyl ammonium chloride (SKC) (Jingwei Chemical Co., Ltd, Shanghai, China) according to methods previously described by Lemke et al. (2001), with some modifications.
Strain and moulding Fusarium graminearum, a ZEN-producing fungus (Eva 2007), was obtained from the Agricultural Culture Collection of China (No. ACCC36249). The fungus was cultivated on potato dextrose agar (PDA; 0.4% potato extract, 2% glucose and 1.5% agar, pH 5.6 ± 0.2). All culture media were obtained from Fluka (Bornem, Belgium) (Demyttenaere et al. 2004). The corn used for the experiments was obtained by Xiang Fang Experimental Bases of Northeast Agricultural
University and milled in a hammer mill with a 40-mesh screen (Trapp-TRF model 90). ZEN was produced in vitro according to the procedures outlined by Lı́gia and Marina (2002). The studies of mycotoxin production by F. graminearum were conducted in duplicate on trays containing 1000 g of sterilised cracked corn and 400 ml of distilled water with a water activity of 0.97. The corn contained no fungal infection or ZEN contamination. The autoclaved substrate was inoculated with 40 ml of the spore suspension according to the following procedure: 100 ml of sterile distilled water were added to each slant of 5-dayold culture, and the agar surface was gently scraped to obtain a turbid suspension corresponding to 1 × 1014 spores ml–1. Suspension (100 ml) was added to the cracked corn. The inoculated flasks were stirred daily during the first 5 days of culture. The following culture conditions were used in this experiment: the temperature was maintained at 28°C for the first 15 days and then at 12°C. ZEN production reached its peak after 35 days of incubation, as reported by Lı́gia and Marina (2002). To equalise the moisture of the samples, each sample was dried at 60°C for 96 h and stored in a freezer at −20°C until analysis (Queiroz et al. 2012). Animals and experimental design Pregnant sows were used in a completely randomised block design and divided into three groups with mean initial live weights of 185.93 ± 3.57, 191.20 ± 2.61 and 187.37 ± 2.49 kg. Eighteen second-parity Yorkshire sows (six per treatment) were bred with semen from a pool of Landrace boars. The pregnant sows were housed in individual stalls after 35 gestation days (GDs). During GDs 35–70, the feed was restricted to 2 kg per pig day–1. The period of organogenesis between days 35 and 70 of gestation was chosen for this study because the foetuses may show possible teratogenic effects or mortality (Goyarts et al. 2007) during this key period of organogenesis. The following treatments were included in this study: (1) control, (2) contaminated grains (50% control corn and 50% mouldy corn), and (3) contaminated grains (50% control corn and 50% mouldy corn) + 1% MHNTs. The doses of MHNTs were selected based on the work conducted by Jiang et al. (2010, 2012). All 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 19540–2004). ZEN was the major contaminant and was present at a concentration of 2.77 mg kg–1 in the ZEN-contaminated diets. Analysis of the corn and diets by GC-MS was performed to obtain a detailed characterisation of the trichothecene mycotoxin pattern, and the results revealed that levels of other B-trichothecene mycotoxins, such as 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol and nivalenol, and the A-
Food Additives & Contaminants: Part A trichothecene mycotoxins, such as HT-2 toxin, were lower than the detection limits (Tiemann et al. 2008). Water was provided ad libitum. Experimental diets (Table 1) were formulated to meet or exceed the National Research Council nutrient requirements (NRC 1998).
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Samples collection Three sows from each group were killed through an intraarterial injection of pentobarbital (200 mg kg–1) after general anaesthesia on day 70 of gestation after exposure to the experimental diets for 35 days. Blood of the sows was collected by puncture of the vena jugularis externa. Microcentrifuge tubes were coated with 10 ml of heparin sodium salt (20 U ml–1 in PBS). Whole blood was collected from the submandibular site as described above and maintained on ice for 30 min. The samples were centrifuged at 3000g and 4°C for 10 min. The plasma layers were removed, placed into sterile microcentrifuge tubes and stored at −40°C until analysis. Fragments of the maternal kidneys were quickly dissected, frozen in liquid nitrogen, stored at −80°C, and subjected to RNA extraction and quantitative reverse-transcription PCR. Other fragments were stored at −20°C for biochemical analysis and ZEN residue analysis. The remaining sows were allowed to farrow naturally and fed a mycotoxin-free
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diet until weaning on day 21 of lactation. Six neonatal or weanling piglets from each group were killed by an intra-arterial injection of pentobarbital. The methods of blood and kidney samples collection from the piglets were same as those for the sows on GD 70. All the animal experimental procedures were approved by the Ethical and Animal Welfare Committee of Heilongjiang Province, China.
Biochemical parameter determination The biochemical parameters were determined with an automated biochemical analyser (Synchron CX®4 Pro Beckman Coulter, Brea, CA, USA) using commercial diagnostics kits (Biosino Biotechnology and Science Inc., Beijing, China). The following biochemical characteristics in the plasma were determined: blood urea nitrogen (BUN), creatinine (CRE) and uric acid (UA).
Oxidative parameter determination The parameters of oxidative stress were determined with an ultraviolet spectrophotometer (UV-2410PC model, Shimadzu, Japan) using commercial diagnostics kits (Nanjing Jiancheng Biotechnology Co., Ltd., Jiangsu, China). The following plasma parameters were evaluated:
Table 1. Percentage composition of the diet. Controlc
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 in this study were according to the Tables of Feed Composition and Nutritive Values in China (Yin et al. 2015). c Control = control diet; contaminated grains = instead of 50% mouldy corn; contaminated grains = instead of 50% mouldy corn + 1% MHNTs; ZEN, zearalenone; MHNTs, modified halloysite nanotubes.
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malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSHPx).
were routinely stained with haematoxylin and eosin (H&E) and assessed using a light microscope (Nikon Eclipse E400). All alterations from the normal structure were registered.
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Total RNA and quantitative real-time polymerase chain reaction (PCR) The total RNA from the kidney tissue was isolated using a reagent box (E.Z.N.A.®Total RNA Kit, Omega Bio-tek, Inc., Doraville, GA, USA) according to the manufacturer’s recommended protocol. The concentration of RNA was estimated based on its absorbance at 260 nm, which was determined using a spectrophotometer. The RNA quality was determined by checking its integrity through agarose gel electrophoresis and by confirming that the A260/ A280 nm absorbance ratio was between 1.8 and 2.0. The first-strand cDNA was synthesised from 5 μg of the total RNA using oligo-dT primers and superscript II reverse transcriptase according to the manufacturer’s instructions (Tiangen Biotech Co., Ltd, Beijing, China). The expression levels of tumour necrosis factor (TNF)-a, interleukin (IL)-1β, IL-6 and β-actin were determined through quantitative real-time PCR using an ABI PRISM 7500 SDS thermal cycler apparatus (Applied Biosystems, Foster City, CA, USA) using the following temperature programme: one cycle of 95°C for 30 s and 40 cycles of 95°C for 5 s and 61°C for 34 s. The dissociation curves for each PCR reaction were analysed using the Dissociation Curve 1.0 software (Applied Biosystems) to detect and eliminate any possible primer–dimers and nonspecific amplifications. The relative expression ratios of the target genes were calculated based on the efficiencies and quantification cycle (Cq) deviations of the unknown samples compared with the controls and are expressed in comparison with the reference gene, as described by Pfaffl (2001). The primer sequences and product sizes are shown in Table 2.
Histopathological studies Slices of the left kidney were fixed in 10% formalin for 24 h and embedded in paraffin. Then, 3–5-μm sections
Statistical analysis The comparison of the treatment groups with the control group was conducted through analysis of variance (ANOVA) using Statistical Packages for Social Science 18.0 (SPSS, Chicago, IL, USA) software. The differences between the means were determined using Duncan’s multiple-range test. Differences with a probability (p) value of less than 0.05 were considered to be statistically significant. The results of the statistical analyses are shown as the mean ± standard error of the means (SEM).
Results Study of the plasma biochemical parameters The effects of ZEN with or without modified halloysite on some plasma biochemical parameters, including BUN, CRE and UA, are presented in Table 3.
Plasma CRE Compared with the control animals, the plasma CRE level was significantly decreased (p < 0.05) in the sows in the ZEN-treated group and their offspring (neonatal piglets and weanling pigs). The plasma CRE level in the animals treated with MHNTs in combination with ZEN was significantly increased compared with the ZEN-treated sows their offspring (neonatal and weanling piglets).
Plasma UA The plasma UA level in the ZEN-treated sows and their offspring (neonatal piglets and weanling piglets) was significantly increased (p < 0.05) compared with the control group. The UA level in the group treated with MHNTs in combination with ZEN was not significantly decreased
Table 2. Primers used for quantitative real-time PCR. Gene
GenBank Accession Number
β-Actin
AY550069
IL-1β
NM_214055.1
IL-6
M 214399
TNF-α
NM_031329.2
Sequence
Fragment length (bp)
Forward primer 5ʹ-atgcttctaggcggactgt-3ʹ Reverse primer 5ʹ-ccatccaaccgactgct-3ʹ Forward primer 5ʹ-ggccgccaagatataactga-3ʹ Reverse primer 5ʹ-ggacctctgggtatggctttc-3ʹ Forward primer 5ʹ-tgatgccacctcagacaa-3ʹ Reverse primer 5ʹ-tcacacttctcatacttctcac-3ʹ Forward primer 5ʹ-cagcctcttctccttcct-3ʹ Reverse primer 5ʹ-cgatgatctgagtccttgg-3ʹ
211 70 123 142
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Table 3. Studying of the biochemical parameters in plasma. Animal Sow Neonatal piglets Weanling pigs
Item CRE (μmol l–1) BUN (mg l–1) UA (μmol l–1) CRE (μmol l–1) BUN (mg l–1) UA (μmol l–1) CRE (μmol l–1) BUN (mg l–1) UA (μmol l–1)
Control 166.58 119.06 23.18 117.26 105.98 17.42 123.77 107.02 13.69
Contaminated grains (ZEN)
6.51a 2.75c 1.71b 2.04a 9.64b 0.00b 0.93a 0.34c 1.24b
± ± ± ± ± ± ± ± ±
138.66 221.60 31.15 88.66 199.92 33.60 81.90 361.31 27.13
± ± ± ± ± ± ± ± ±
2.79c 1.38a 1.24a 0.99 c 12.73a 1.24a 1.86c 11.01a 0.24a
Contaminated grains +1% MHNTs 151.76 189.05 26.13 96.79 113.21 18.67 107.09 186.85 23.64
± ± ± ± ± ± ± ± ±
1.12b 1.86b 1.24ab 1.86b 9.29b 1.24b 2.86b 0.34b 1.24a
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Notes: Data are means ± SEM. a,b,c within a row with no common superscripts differ significantly (p < 0.05). ZEN, zearalenone; MHNTs, modified halloysite nanotubes; BUN, blood urea nitrogen; CRE, creatinine, UA, uric acid.
compared with the ZEN-treated sows and weanling piglets. However, the UA level was significantly decreased (p < 0.05) in the MHNTs + ZEN-treated neonatal piglets compared with ZEN-alone-treated neonatal piglets.
Plasma BUN The plasma BUN level was significantly increased (p < 0.05) in the ZEN-treated group compared with the control group. The use of MHNTs in combination with ZEN significantly decreased (p < 0.05) the BUN level compared with that obtained for the ZEN-treated group.
group. The use of MHNTs in combination with ZEN significantly increased (p < 0.05) the SOD level in the plasma compared with that obtained in the ZEN-treated group.
Plasma GSHPx Compared with the control group, the level of GSHPx was significantly decreased (p < 0.05) in the plasma in the ZEN-treated group. Treatment with MHNTs in combination with ZEN significantly increased (p < 0.05) the GSHPx level compared with the ZEN-treated group.
Analysis of oxidative parameters in the plasma The effects of ZEN with or without modified halloysite on some oxidative parameters in the plasma, including MDA, SOD and GSHPx, are presented in Table 4.
Plasma SOD The ZEN-treated group exhibited significantly decreased activities of SOD in the plasma compared with the control
Plasma MDA Compared with the control group, the level of MDA in the plasma was significantly increased (p < 0.05) in the ZENtreated group. The sows and neonatal piglets treated with MHNTs in combination with a ZEN exhibited significantly decreased (p < 0.05) MDA level compared with the ZEN-treated group. However, the MDA level was not significantly decreased in weanling piglets.
Table 4. Oxidative parameters determination in plasma. Animal Sow Neonatal piglets Weanling pigs
Item SOD (U ml–1) MDA (nmol ml–1) GSHPx (U ml–1) SOD (U ml–1) MDA (nmol ml–1) GSHPx (U ml–1) SOD (U ml–1) MDA (nmol ml–1) GSHPx (U ml–1)
Control 180.99 3.36 1348.48 68.34 3.01 649.24 78.98 3.35 1001.44
± ± ± ± ± ± ± ± ±
4.62a 0.59b 14.03a 1.03a 0.33b 10.77a 0.56a 0.61a 11.07a
Contaminated grains (ZEN) 97.99 11.41 858.61 20.84 8.73 281.25 33.97 6.04 678.87
± ± ± ± ± ± ± ± ±
2.13c 0.78a 8.61c 2.48c 0.67a 6.25c 0.70c 0.67a 8.64c
Contaminated grains +1% MHNTs 131.38 6.04 1123.64 52.60 3.36 571.94 69.05 4.70 867.56
± ± ± ± ± ± ± ± ±
2.89b 0.67b 11.14b 1.62b 0.67b 3.06b 0.29b 0.66a 7.45b
Notes: Data are means ± SEM. a,b,c within a row with no common superscripts differ significantly (p < 0.05). ZEN, zearalenone; MHNTs, modified halloysite nanotubes; MDA, malondialdehyde; SOD, superoxide dismutase; GSHPx, glutathione peroxidase.
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Analysis of the mRNA expression levels of cytokines The mRNA expression levels of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) were measured by quantitative RT-PCR, as shown in Table 5. With the exception of IL-1β in neonatal piglets, there was a significant increase in the mRNA levels of pro-inflammatory cytokines (TNFa, IL-1β and IL-6) in the ZEN-treated sow group and their offspring compared to controls.
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Histopathological study of the kidney As shown in Figure 1(a), the kidney sections from the control animals show normal renal tubules, normal glomeruli and normal epithelial cells lining the renal tubules. In contrast, the treatment of animals with ZEN alone revealed histopathological changes in the kidney sections, as shown in Figure 1(b). These changes included the presence of abnormal epithelial cells lining the renal tubules, protein deposition, renal interstitial fibrosis and shrunken glomeruli. The treatment with MHNTs in combination with ZEN induced a re-establishment of normal histopathology in the kidney sections, as shown in Figure 1(c). The kidney sections from neonatal and weaned piglets were similar. Discussion Many epidemiological studies have demonstrated that ZEN can induce several deleterious effects in many visceral organs of both humans and animals (Berek et al. 2001; Abbès, Salah-Abbès, et al. 2006). Based on its role in excretion, re-absorption and general homeostasis, the kidney has an extensive blood flow, receiving approximately 1.2 L of plasma min–1 and filtering an average of 125 ml of plasma min–1. To assess the changes in kidney function induced by ZEN, we monitored BUN, UA and CRE levels. Increases in BUN level have been described in most cases of nephrotoxicity, including those induced by mycotoxin exposure (An et al. 2011). In the present study, the BUN level was significantly increased
(p < 0.05) in the ZEN-treated group compared with the control group. The resulting elevation in the BUN level is not solely due to a decrease in the GFR and results in acute renal failure (Aronson et al. 2008). CRE is a byproduct of muscle energy metabolism that is filtered from the blood by the kidneys and excreted into the urine. Because creatinine is only slightly affected by the liver function, it is a sensitive indicator of kidney function (Vander et al. 1998). The low level of creatinine found in gestation sows and offspring treated with ZEN alone, as found in the current study, indicates degenerative changes in the kidney function, as described by Salah-Abbès, Abbès, Ouanes, et al. (2008), and the same results were obtained by Abbès, Salah-Abbès, et al. (2006). Moreover, UA is a byproduct of protein metabolism; this waste product is formed in the liver and is then filtered from the blood and excreted in the urine by the kidneys (Vander et al. 1998). In this study, an increase in the plasma UA level was observed in swine treated with ZEN alone, indicating kidney damage and in agreement with the results reported by Abbès, Ouanes, et al. (2006). Oxidative stress results in damage to cellular structures and has been linked to many diseases, including cancer (Marin & Taranu 2012). Recent studies have shown that ZEN enhances reactive oxygen species formation and causes oxidative damage (Yuk et al. 2011). The oxidative stress caused by ZEN may be one of the underlining mechanisms through which ZEN induces cell injury and DNA damage, which eventually lead to tumorigenesis (Abid-Essefi et al. 2009). In the current study, we showed that ZEN treatment induced a decrease in the expression of two enzymes involved in oxidative stress, namely SOD and GSHPx. SOD refers to a class of enzymes that catalyse the dismutation of superoxide into oxygen and hydrogen peroxide, which represents an important antioxidant defence in nearly all cells exposed to oxygen (Liska 1998). Higher concentrations (1.1–3.2 ppm) of ZEN in the diet have been found to be able to reduce SOD activity in the spleen of post-weaning gilts (Jiang et al. 2011) and in the liver and serum of weanling piglets (Jiang et al. 2010). The down-regulation of SOD gene expression and
Table 5. mRNA expression of cytokines in kidney. Animal Sow Neonatal piglets Weanling pigs
Item
Control
Contaminated grains (ZEN)
TNF-α IL-1β IL-6 TNF-α IL-1β IL-6 TNF-α IL-1β IL-6
0.0092 ± 0.0004c 0.0009 ± 0.0001c 0.0014 ± 0.0001c 0.0027 ± 0.0003b 0.0011 ± 0.0002a 0.0002 ± 0.0001b 0.0013 ± 0.001b 0.0023 ± 0.0003b 0.0001 ± 0.000b
0.0201 ± 0.0022a 0.0277 ± 0.0010a 0.0211 ± 0.0007a 0.0184 ± 0.0013a 0.0196 ± 0.0082a 0.0043 ± 0.0002a 0.0059 ± 0.0003a 0.0218 ± 0.0036a 0.0007 ± 0.0001a
Contaminated grains +1% MHNTs 0.0130 0.0029 0.0053 0.0043 0.0059 0.0003 0.0017 0.0073 0.0004
± ± ± ± ± ± ± ± ±
0.0006 0.0004b 0.0003b 0.0008b 0.0045a 0.0001b 0.0003b 0.0007b 0.000b
Notes: Data are means ± SEM. a,b,c within a row with no common superscripts differ significantly (p < 0.05). ZEN, zearalenone; MHNTs, modified halloysite nanotubes.
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Figure 1. (colour online) (a) Control, (b) contaminated grains (instead of 50% mouldy corn), and (c) contaminated grains (instead of 50% mouldy corn) + 1% modified HNTs. Normal glomeruli (NG), normal renal tubules (NRT), protein deposition (PD), renal interstitial fibrosis (RIF), tubular degeneration (TD), shrunken glomeruli (SG). (H&E, 400×).
the reduction of SOD activity can lead to the accumulation of superoxide anions within the mitochondria, leading to oxidative stress and thereby impairing some vital cellular functions (Marczuk-Krynicka et al. 2003). In agreement with our results, Salah-Abbès, Abbès, Ouanes, et al. (2008) reported that the activities of SOD and GSHPx decrease with progressive liver and kidney injury in ZEN-treated mice. Salah-Abbès et al. (2009) also observed that ZEN supplementation at a concentration of 40 mg kg–1 significantly reduces the activities of SOD and GSHPx in the testes of mice. The activities of SOD and GSHPx are known to serve as protective responses for the elimination of reactive free radicals (Cheung et al. 2001). Therefore, the decreased activities of SOD and GSHPx in the plasma found in the present study suggest that ZEN supplementation induces oxidative stress. In fact, the most common group of indices used to assess oxidative stress involves the peroxidation products of lipids. The most widely used index is MDA, which is the end product of lipid peroxidation and is considered a late biomarker of oxidative stress and cellular damage (Dotan et al. 2004). In the present study, although exposure to ZEN induced a marked increase in MDA formation in the plasma, the administration of MHNTs (even at the lowest dose tested) significantly reduced this induction to the basal level. The administration of MHNTs prevented the lipid peroxidation induced by ZEN treatment in sow and foetal piglets. It has been demonstrated that ZEN and its metabolites induce lipid oxidation and increase the production of malondialdehyde in several cell lines (Hassen et al. 2007; Othmen et al. 2008). ZEN has been described as both a suppressor and an inducer of inflammation (Marin et al. 2011). The results showed that ZEN induced pro-inflammatory effects in the kidney. With the exception of IL-1β in neonatal piglets, an inflammatory response was observed in the kidneys of the animals in the ZEN-treated sow group and their offspring, as suggested by the increase in the mRNA expression
levels of inflammatory cytokines (IL-1β, TNF-α and IL-6). These results are in agreement with those obtained in mice after the administration of DON through a single oral dose of 5 or 25 mg kg–1 bw, which resulted in significantly up-regulated mRNA levels of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α; the lower doses had no effect (Zhou et al. 1997). Chiba et al. (2007) reported that aflatoxin B1 can increase the production of TNF-α and IL-6 by inhibiting the synthesis of ceramide. In addition, the in vivo daily ingestion of 2.8 mmol of fumonisin B1 kg–1 bw decreased the expression levels of IL-6, IL-10, IL-18 and IFN-γ in the liver of piglets, whereas the expression levels of IL-1β and IL-8 were increased (Royaee et al. 2004). DON induces modest increases in the expression levels of TNF-α, IL-6 and IL-1β in the liver relative to those found in the spleen (Salah-Abbès, Abbès, Houas, et al. 2008). In other studies, ZEN and its derivatives have been described as suppressors (Salah-Abbès, Abbès, Houas, et al. 2008; Marin et al. 2011) of the expression of pro-inflammatory cytokines and as inducers of DNA fragmentation (Gazzah et al. 2010). The study conducted by Wang et al. (2012) revealed that the alteration in the expression levels of cytokines (IL-2, IL-6 and IFN-γ) in chicken splenocytes is associated in vitro with increasing concentrations of ZEN. DNA fragmentation was one of the results of the chronological succession of some events that characterise the toxicity of ZEN in human hepatocarcinoma cells (Gazzah et al. 2010). This divergent effect may be due to differences in the toxicities of these compounds and the animal models used. The results found from the analyses of the inflammatory cytokines and biological and oxidative stress parameters were confirmed through the histological findings in the kidneys. The histopathological examination of the kidneys of the animals in the ZEN-treated group revealed shrunken glomeruli, renal interstitial fibrosis, tubular degeneration and protein deposition. The acute cellular degeneration in the epithelial lining of the renal tubules
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is due to the toxic effect of ZEN and may be caused by cell membrane injury or an effect on the mitochondria that leads to the depletion ATP or a defect in the sodium– potassium pump and thus fluid disturbance in and outside of cells. These results are consistent with the results in mice treated with a single dose of 40 mg kg–1 bw of ZEN (Abbès, Salah-Abbès, et al. 2006). The pathology of the kidneys was described by Grosman et al. (1983), and similar results were obtained by Abdel-Wahhab et al. (2002), who demonstrated that rats are acutely sensitive to the same nephrotoxic effects caused by aflatoxin B1. Our study showed a clear protective effect of the sorbent MHNTs material against oxidative stress and the biochemical and pathological (renal tissues) changes induced by ZEN. These results were supported by Kulok et al. (2005), who demonstrated the high decontaminating efficiency of halloysite toward bacteria, fungi and aflatoxin B1, which proves that aluminosilicates may be used to reduce the contamination of feed mixtures. Montmorillonite, another sorbent material, was found to be able to bind efficiently to ZEN (Lemke et al. 2001). Recently, Bueno et al. (2005) indicated that bentonite can bind to ZEN in vitro. AfriyieGyawu et al. (2005) suggested that the addition of either activated carbon or hectorite effectively decreases the toxic effects of ZEN on the reproductive system of Hydra attenuata. As a result, this type of sorbent material can be considered a good candidate for the prevention of the toxic effects of ZEN or for the detoxification of this toxin in contaminated foods.
Conclusions The results of the current study indicated that ZEN not only caused damage to both oxidative stress and biochemical parameters but also induced the degeneration of renal tissues in pregnant sows and their offspring. We also found that MHNTs were able to prevent most of the alterations induced by ZEN, which suggested that this material efficiently reduced the bioavailability of ZEN.
Disclosure statement No potential conflict of interest was reported by the authors.
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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