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Archives of Animal Nutrition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gaan20
Adsorption of modified halloysite nanotubes in vitro and the protective effect in rats exposed to zearalenone a
a
a
a
Yuanyuan Zhang , Rui Gao , Min Liu , Changjiang Yan & Anshan a
Shan a
Institute of Animal Nutrition, Northeast Agricultural University, Harbin, P.R. China Published online: 30 Jun 2014.
To cite this article: Yuanyuan Zhang, Rui Gao, Min Liu, Changjiang Yan & Anshan Shan (2014) Adsorption of modified halloysite nanotubes in vitro and the protective effect in rats exposed to zearalenone, Archives of Animal Nutrition, 68:4, 320-335, DOI: 10.1080/1745039X.2014.927710 To link to this article: http://dx.doi.org/10.1080/1745039X.2014.927710
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Archives of Animal Nutrition, 2014 Vol. 68, No. 4, 320–335, http://dx.doi.org/10.1080/1745039X.2014.927710
Adsorption of modified halloysite nanotubes in vitro and the protective effect in rats exposed to zearalenone
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Yuanyuan Zhang, Rui Gao, Min Liu, Changjiang Yan and Anshan Shan* Institute of Animal Nutrition, Northeast Agricultural University, Harbin, P.R. China (Received 12 February 2014; accepted 8 May 2014) The aim of the present study was to investigate the sorption properties in vitro and the application feasibility of modified halloysite nanotubes (HNT) in reducing the toxic effect of ZEN in rats in vivo. HNT were modified using the surfactant stearyldimethylbenzylammonium chloride. Modified HNT (MHNT) was evaluated using electron microscopy, which revealed that the modification had successfully enlarged the nanotube inner diameter from 11.35 to 20.12 nm. In an in vitro study, the efficiency of MHNT to adsorb zearalenone (ZEN) from simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) was investigated in comparison with HNT or a montmorillonite mixture (MON). For all tested adsorbents, the adsorption efficiency for ZEN was increased when the adsorbent dose rose from 0.4 to 1.0 mg/ml and became stable beyond this dose. Moreover, the ZEN adsorption increased with the extension of adsorption times from 20 to 90 min (SGF) and from 30 to 120 min (SIF) without further increase afterwards. Already at the first measuring times (20 and 30 min for SGF and SIF, respectively) MHNT showed a higher adsorptive property then HNT. The ability of MHNT to prevent lesions caused by ZEN was evaluated in 60 female rats. The rats received five experimental diets for two weeks: Control (per kg diet 0.001 mg ZEN); ZEN (0.5 mg ZEN), HNT (0.5 mg ZEN + 1% HNT), MHNT (0.5 mg ZEN + 1% MHNT) and MON (0.5 mg ZEN + 1% MON). The results indicated that the tested adsorbents mitigated toxic and estrogenic effects of ZEN exposure including changes in oxidative stress biomarkers and organ weights. In some parameters (gain, oestradiol content in serum and ZEN concentration in reproductive organs), MHNT exceeded the effectiveness of HNT. Thus, it can be concluded that the modification enhances the adsorbent properties of HNT and that MHNT can bind to ZEN in animal feed or in the gastrointestinal tract. Keywords: adsorption; hormones; in vitro; nanotubes; rats; removal; zearalenone
1. Introduction It has been reported that worldwide 25% of food crops are contaminated with mycotoxins (Charmley et al. 1994). ZEN (zearalenone), a non-steroidal estrogenic mycotoxin that is produced as a secondary metabolite by a variety of Fusarium fungi, poses serious health problems for livestock (Zinedine et al. 2007). Several in vivo studies have reported that ZEN exposure results in an increased incidence of disease, alterations in the reproductive tract and lead to oxidative stress in laboratory animals (Dänicke et al. 2002). Recent studies have shown that ZEN enhances the formation of reactive oxygen species and causes oxidative damage in cells and organisms (Yu et al. 2011). Many attempts have been made to find ways to detoxify contaminated feedstuffs or diets in order to cope with the problem (Döll and Dänicke 2004). Montmorillonite (Jiang *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
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et al. 2010), activated carbon (Binder 2007), zeolite (Binder 2007), and kaolin (Figueroa et al. 2004) have been widely used for the adsorption of mycotoxins in feed. 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). It has been reported that organo-modified mineral adsorbents are more effective for ZEN adsorption compared with their raw analogues (Sprynskyy et al. 2012). Sanchez-Martin et al. (2006) reported the efficiency of a series of clay minerals (montmorillonite, illite, muscovite, sepiolite and palygorskite) that were modified using the cationic surfactant octadecyl trimethyl ammonium bromide in the adsorption of the pesticides penconazole, linuron, alachlor, atrazine and metalaxyl. Sunardi and Yateman (2011) reported that surface modification using a cationic surfactant showed that increasing surfactant content increased the adsorption capacity of kaolin. 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). Stearyl dimethyl benzyl ammonium chloride (SKC), which is non-toxic in humans, has been used as a cationic surfactant. Clays and soils (montmorillonite, illite, muscovite, sepiolite and palygorskite) that have been modified using 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). To the best of our knowledge, this is the first study to use SKC-modified HNT as an adsorbent of ZEN in feed. The aim of the present study was to investigate the sorption properties and application feasibility of modified halloysite nanotubes (MHNT) in reducing the toxic effect of ZEN.
2. Materials and methods 2.1. Sorbent material and modification Powder HNT (Al2Si2O5(OH)4 · nH2O) were refined from clay minerals from the Henan Province (China) with a purity of 95%. The powder was prepared according to the method described by Jinhua et al. (2010). All solutions were prepared using distilled water. The powder was prepared as follows: A water suspension solution (5% in mass) was prepared by adding water to dry halloysite mineral. The suspension solution was intensively stirred for 2 h and sprayed to dry at 200°C to obtain fine powder. Before use, dry halloysite powder was sieved to eliminate aggregates. HNT were modified using SKC (Jingwei Chemical Co., Ltd., Shanghai, China) according to methods previously described by Tomašević-Čanović et al. (2003) with modifications. HNT (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 crush 100 g of HNT to obtain particles that were less than 45 μm in a beater mill at 7400g for 3 min (Lemke et al. 2001).
2.2. Microscopic imaging Transmission electron microscopy (EM) images were recorded using HITACHI H-7650 TEM. Analytical samples were prepared by dropping diluted nanotube dispersion in water onto carbon-coated copper grids and allowing the water to evaporate (Vergaro et al. 2010). For characterisation of HNT and MHNT, three samples were prepared. Six single pictures were made per sample, and in a single picture three inner diameters of different nanotubes
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were chosen for evaluating the effectiveness of modification of HNT. The width of inner diameter was measured by the transmission EM. The external surface morphology of nanotubes was characterised using a scanning EM type HITACHI S-3400N (Vergaro et al. 2010). Six samples were chosen before or after modification. Six single pictures were made for each sample to evaluate the effectiveness of modification.
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2.3. Efficiency of zearalenone removal ZEN was purchased from Fermentek Ltd. (Jerusalem, Israel), purity >98.5%. The substance was reported to be stable for at least 8 months at room temperature. The removal efficiency (R) of adsorbed ZEN was calculated using the following equation: R ½% ¼ 100% ðC0 CeÞ=C0; where C0 and Ce represent the initial and equilibrium concentrations of ZEN [mg/l], respectively (Jinhua et al. 2010). Simulated gastric fluids (SGF) and simulated intestinal fluids (SIF) were prepared freshly on the day of the experiment according to the United States Pharmacopoeia specifications (The United States Pharmacopeia 1995) for evaluating adsorption capacity of MHNT. SGF consists of porcine pepsin A (272 U/mg) in 35 mM NaCl at pH 1.2. Porcine pepsin was prepared in our laboratory as previously described (Zhou et al. 2007). The volume of the reaction solution was 10 ml. SIF was prepared as described in the United States Pharmacopoeia (1995) and consists of trypsin (1500 U/mg; 10 mg/ml SIF) in 0.05 M KH2PO4 at pH 7.5. In the in vitro experiments HNT, MHNT and a montmorillonite mixture (MON) were used as adsorbents. MON was obtained from Shangqiu IGM Animal Pharmaceutical Co., Ltd. (main ingredient: 95% improved nanometre montmorillonite, yeast cell wall, modified polyvinylpolypyrrolidon (PVPP) and calcium propionate and other ingredients). In the first adsorption experiment, the SGF and SIF solutions contained ZEN at 50 mg/l and the tested adsorbents (HNT, MHNT and MON) were added at 0.4, 0.7, 1.0, 1.3 and 1.6 mg/ml, respectively. The mixtures were shaken at 37°C for 2 h and 4 h in Erlenmeyer flasks (50 ml) to determine the removal efficiency in vitro and the process was immediately terminated by addition of 30 μl of 200 mM Na2CO3 (Huang et al. 2010). In the second adsorption experiment, the adsorbents (HNT, MHNT and MON) were added to the SGF and SIF solutions (10 ml, ZEN concentration 50 mg/l) at 1.0 mg/ml. The mixtures were shaken at 37°C for 20, 40, 60, 90 and 120 min (SGF) and 30, 60, 90, 120 and 240 min (SIF) to determine the time of degradation equilibrium in vitro. The reaction was stopped by addition of serine proteinase inhibitor pefabloc SC to a final concentration of 5 mM (Huang et al. 2010). The adsorption experiments were repeated three times. ZEN concentrations were compared using validated analytical methods according to the National Standards of the People’s Republic of China (GB/T 19,540–2004). The method uses immunoaffinity chromatography for purification and high-performance liquid chromatography (HPLC) for detection and quantification of the toxin. 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
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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 firstly 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) and fluorescence detection (λex = 274 nm, λem = 440 nm).
2.4. Animals and experimental design Sixty female Sprague–Dawley (SD) rats were obtained from the Jilin University Laboratory Animal Centre (Changchun, China) and were acclimated for 2 weeks prior to experiment. The weights of rats ranged from 190 to 210 g (9 weeks of age). The experiments began on the tenth week of age and lasted for 2 weeks. The rats received diets containing different concentrations of ZEN, HNT, MHNT and MON. The rats were divided into five groups. The Control group received the basal diet (Table 1) without any further addition. The other experimental diets contained the basal diet and a supplementation of ZEN at 0.5 mg/kg diet (Table 2). Furthermore, to the diets of Groups HNT, MHNT and MON the respective adsorbents were added at 1% of the diet at the expense of cellulose (Table 2). Rats were kept individually in polycarbonate metabolic cages (Tecniplast, Hohenpeißenberg, Germany) and had free access to food and water. They were maintained on a 12/12 h light/dark cycle at 24 ± 2°C in the study room. The relative humidity was maintained between 47% and 55% (Collins et al. 2006). Throughout the study, the rats were fed experimental diets according to AIN-93G (Reeves et al. 1993). The feed was prepared in an experimental feed mill using high-precision mixers and all feedstuffs were prepared for the detection of mycotoxin contents. There were no other mycotoxin residuals in feedstock and adsorbents.
Table 1. Composition and nutrient levels of the basal diet (air-dry basis) fed the in vivo trial on rats. Ingredients Corn starch Dextrin starch Casein Soybean oil Cellulose Cane sugar L-cysteine CaHPO4 Limestone Choline Premix* Total
Contents [%] 41.65 13.20 20.00 6.00 5.00 10.00 0.30 0.80 1.30 0.25 1.50 100.00
Nutrient levels (analysed) Gross energy [MJ/kg] Crude protein [%] Crude fibre [%] Calcium [%] Phosphorus [%]
Contents 16.80 17.84 10.86 0.87 0.52
Notes: *Provided per kg of the diet: Fe (as ferrous sulphate) 35 mg, Cu (as copper sulphate) 6 mg, Zn (as zinc sulphate) 30 mg, Mn 10 mg, vitamin A 14,000 IU, vitamin D3 1500 IU, vitamin E 80.04 mg, riboflavin 12 mg, nicotinic acid 60 mg, D-pantothenic acid 24 mg, vitamin B12 1.2 mg, biotin 0.2 m.
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Supplementation of the basal diet in the in vivo trial on rats. Experimental groups
ZEN [mg/kg diet] Adsorbent [%]
Control
ZEN†
HNT*
MHNT◊
MON♦
– –
0.5 –
0.5 1
0.5 1
0.5 1
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Notes: †ZEN, Zearalenone; *HNT, Natural halloysite nanotubes (adsorbent); ◊MHNT, Modified natural halloysite nanotubes (adsorbent); ♦MON, Montmorillonite mixture (adsorbent).
2.5. Ethics statement This study was performed in strict accordance with the recommendations of the National Research Council (1996), and all animal experimental procedures were approved by the Ethical and Animal Welfare Committee of Heilongjiang Province, China. All surgeries were performed under anaesthesia using an intraperitoneal injection of sodium pentobarbital and every effort was made to minimise animal suffering.
2.6. Collection of physiological samples The following data were monitored during the animal trial: body weight (BW), average daily feed intake (ADFI), average daily gain (ADG) and feed conversion ratio (FCR). The animals were sacrificed under anaesthesia using an intraperitoneal injection of sodium pentobarbital (0.5 ml/kg BW) at the end of the experiments. Whole blood was collected from the submandibular site and placed into microcentrifuge tubes coated with 10 μl of heparin sodium salt (20 U/ml in PBS). Samples were kept on ice for 30 min, then centrifuged at 3000g for 10 min at 4°C. The serum layers were removed and placed into sterile microcentrifuge tubes and stored at −80° C until further analyses. The organs (liver, kidney, spleen, ovary and uterus) were quickly removed and weighed. The reproductive organs were prepared for HPLC analysis.
2.7. Quantification of zearalenone Preliminary treatments were performed according to the procedures described by Koraichi et al. (2012) with modifications. The samples (2 g of tissue) were extracted with 40 ml of methanol (Sigma-Aldrich, St. Louis, MO, USA) for 1 min. The extract was centrifuged at 825g for 5 min. The supernatant was transferred to a flask and evaporated at 50°C with a rotary evaporator under reduced pressure until 5 ml of residual water remained. pH was adjusted to 6.8 with 1 N NaOH. The sample was adjusted to 8 ml with 50 mM phosphate buffer, pH 6.8. Tube was incubated with βglucuronidase (200 U) at 37°C overnight to cleave the conjugates. Each sample was extracted twice with chloroform (Sigma-Aldrich, St. Louis, MO, USA). The chloroform layer was evaporated with a rotary evaporator at 50°C under reduced pressure. The flask was rinsed three times with 1 ml of methanol which was evaporated to dryness under a gentle nitrogen flow. The final purified sample was dissolved in 200 μl methanol and 50 μl was injected for analysis by HPLC. ZEN and metabolite concentrations were determined by HPLC according to Videmann et al. (2008) and Koraichi et al. (2012) with modifications. The HPLC
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system (Hitachi-MercK Product) consisted of a L7100 pump, an AS2000 sampler, a L4000 UV detector (wavelength of 235 nm) and D7000 HSM software (VWR, Nogent-sur-Marne, France). Samples (50 μl) or standard solution were injected in a LIChrospher ®100RP18 en-capped column (5 μM granule size, 250 × 4 mm) (VWR International, Fontenay-sous-Bois, France) was used. A gradient elution system was used: from 75% water (acidified with 0.2% H3PO4 and adjusted to pH 3 with 5 N NaOH), 25% acetonitrile at time zero, isocratic for 0.2 min, to 35% water, 65% acetonitrile at 13 min. The flow rate was set at 1.4 ml/min. The retention time of ZEN was 8 min.
2.8. Hormone levels Hormone levels were determined using an ELISA Kit according to the manufacturer’s recommended protocol (R&D Systems, Inc., USA). The parameters evaluated in the serum included prolactin, follicle-stimulating hormone (FSH), luteinising hormone (LH), oestradiol and progesterone.
2.9. Determination of antioxidative parameters The antioxidative parameters were determined using an ultraviolet spectrophotometer (UV-2410PC model, SHIMADZU, Japan) with commercial diagnostic kits (Nanjing Jiancheng Biotechnology Co., Ltd, Jiangsu, China). The parameters evaluated in serum and liver were malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx).
2.10. Statistical analysis Statistical analysis was performed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) for a randomised complete design. The data of inner diameter before or after modification was analysed using the unpaired t-test. The linear and quadratic effects of ZEN the removal efficiency in SGF and SIF in vivo were assessed. Because the data were normally distributed, indices in vitro were analysed using ANOVA and the Duncan multiple range test. Data were expressed as the mean ± standard errors (SEM) of the indicated number of experiments; p-values less than 0.05 were considered to be statistically significant.
3. Results 3.1. Modification and appraisal of halloysite nanotubes Figure 1 shows the images after EM. It can be seen that the samples consisted of cylindrical tubes. After treatment with SKC, the dispersion of the halloysite (Figure 1B) was increased compared to the untreated HNT powder (Figure 1A). Moreover, the lumen of modified nanotubes shown in Figure 1D was enlarged compared with untreated nanotubes (Figure 1C). By the surface modification, the average internal diameter of the lumen was significantly enlarged from 11.4 ± 0.31 nm (n = 54) to 20.1 ± 0.04 nm (n = 54) (p < 0.05). As shown in Figure 1C and D, the external surface of the treated HNT was cruder than the natural halloysite.
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(A)
(B)
(C)
(D)
Figure 1. (A) Scanning electron microscopy (EM) image of the halloysite nanotubes (HNT) powder. (B) Scanning EM image of modified HNT powder with increased dispersity. (C) Transmission EM images of the HNT. (D) Transmission EM image of modified HNT (MHNT) with enlarged internal diameters.
3.2. Efficiency of zearalenone removal in simulated gastric and intestinal fluid For the determination of the adsorption capacity, simulated gastric and intestinal fluids were used in an in vitro study. The removal efficiency of ZEN by HNT, MHNT and MON in SGF and SIF is shown in Tables 3–5. For all tested adsorbents, the adsorption efficiency for ZEN was increased linearly and quadratically (p < 0.01) when the adsorbent dose rose from 0.4 to 1.0 mg/ml (Table 3). Furthermore, for all testes adsorbents the adsorption efficiency became stable at 1.0 mg/ml. Moreover, the adsorption efficiency for ZEN increased with the extension of adsorption times from 20 to 90 min (SGF) and from 30 to 120 min (SIF) (Tables 4 and 5). For example, the removal efficiency by the MON mixture increased during this period from 7.6% to 27.4% and from 19.9% to 55.3% for SGF and SIF, respectively. The equilibrium times of adsorption were 90 min and 120 min for SGF and SIF, respectively. In case of treatment MHNT, the surfactant SKC increased the adsorptive property of HNT already at the first measuring times of 20 and 30 min for SGF and SIF, respectively (Tables 4 and 5). Consequently, the results proofed that the removal efficiency of HNT was enhanced after modification by SKC at all conditions. The removal efficiency of the MHNT approached the MON mixture.
Archives of Animal Nutrition Table 3.
Removal efficiency [%] of zearalenone (ZEN) adsorbents at different doses in vitro. p-Value#
Adsorbents dose [mg/ml] 0.4
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0.7
In simulated gastric fluid (SGF) 9.80 13.51 HNT†§ 11.39 15.82 MHNT†◊ 10.47 15.50 MON†♦ In simulated intestinal fluid (SIF) HNT 12.55 23.64 MHNT 20.27 38.86 MON 16.66 38.92
1.0
1.3
1.6
SEM*
Linear
Quadratic
18.81 23.74 24.06
18.13 23.90 24.25
18.26 23.63 24.52
0.993 1.407 1.565
Archives of Animal Nutrition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gaan20
Adsorption of modified halloysite nanotubes in vitro and the protective effect in rats exposed to zearalenone a
a
a
a
Yuanyuan Zhang , Rui Gao , Min Liu , Changjiang Yan & Anshan a
Shan a
Institute of Animal Nutrition, Northeast Agricultural University, Harbin, P.R. China Published online: 30 Jun 2014.
To cite this article: Yuanyuan Zhang, Rui Gao, Min Liu, Changjiang Yan & Anshan Shan (2014) Adsorption of modified halloysite nanotubes in vitro and the protective effect in rats exposed to zearalenone, Archives of Animal Nutrition, 68:4, 320-335, DOI: 10.1080/1745039X.2014.927710 To link to this article: http://dx.doi.org/10.1080/1745039X.2014.927710
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Archives of Animal Nutrition, 2014 Vol. 68, No. 4, 320–335, http://dx.doi.org/10.1080/1745039X.2014.927710
Adsorption of modified halloysite nanotubes in vitro and the protective effect in rats exposed to zearalenone
Downloaded by [Memorial University of Newfoundland] at 02:59 01 August 2014
Yuanyuan Zhang, Rui Gao, Min Liu, Changjiang Yan and Anshan Shan* Institute of Animal Nutrition, Northeast Agricultural University, Harbin, P.R. China (Received 12 February 2014; accepted 8 May 2014) The aim of the present study was to investigate the sorption properties in vitro and the application feasibility of modified halloysite nanotubes (HNT) in reducing the toxic effect of ZEN in rats in vivo. HNT were modified using the surfactant stearyldimethylbenzylammonium chloride. Modified HNT (MHNT) was evaluated using electron microscopy, which revealed that the modification had successfully enlarged the nanotube inner diameter from 11.35 to 20.12 nm. In an in vitro study, the efficiency of MHNT to adsorb zearalenone (ZEN) from simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) was investigated in comparison with HNT or a montmorillonite mixture (MON). For all tested adsorbents, the adsorption efficiency for ZEN was increased when the adsorbent dose rose from 0.4 to 1.0 mg/ml and became stable beyond this dose. Moreover, the ZEN adsorption increased with the extension of adsorption times from 20 to 90 min (SGF) and from 30 to 120 min (SIF) without further increase afterwards. Already at the first measuring times (20 and 30 min for SGF and SIF, respectively) MHNT showed a higher adsorptive property then HNT. The ability of MHNT to prevent lesions caused by ZEN was evaluated in 60 female rats. The rats received five experimental diets for two weeks: Control (per kg diet 0.001 mg ZEN); ZEN (0.5 mg ZEN), HNT (0.5 mg ZEN + 1% HNT), MHNT (0.5 mg ZEN + 1% MHNT) and MON (0.5 mg ZEN + 1% MON). The results indicated that the tested adsorbents mitigated toxic and estrogenic effects of ZEN exposure including changes in oxidative stress biomarkers and organ weights. In some parameters (gain, oestradiol content in serum and ZEN concentration in reproductive organs), MHNT exceeded the effectiveness of HNT. Thus, it can be concluded that the modification enhances the adsorbent properties of HNT and that MHNT can bind to ZEN in animal feed or in the gastrointestinal tract. Keywords: adsorption; hormones; in vitro; nanotubes; rats; removal; zearalenone
1. Introduction It has been reported that worldwide 25% of food crops are contaminated with mycotoxins (Charmley et al. 1994). ZEN (zearalenone), a non-steroidal estrogenic mycotoxin that is produced as a secondary metabolite by a variety of Fusarium fungi, poses serious health problems for livestock (Zinedine et al. 2007). Several in vivo studies have reported that ZEN exposure results in an increased incidence of disease, alterations in the reproductive tract and lead to oxidative stress in laboratory animals (Dänicke et al. 2002). Recent studies have shown that ZEN enhances the formation of reactive oxygen species and causes oxidative damage in cells and organisms (Yu et al. 2011). Many attempts have been made to find ways to detoxify contaminated feedstuffs or diets in order to cope with the problem (Döll and Dänicke 2004). Montmorillonite (Jiang *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
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et al. 2010), activated carbon (Binder 2007), zeolite (Binder 2007), and kaolin (Figueroa et al. 2004) have been widely used for the adsorption of mycotoxins in feed. 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). It has been reported that organo-modified mineral adsorbents are more effective for ZEN adsorption compared with their raw analogues (Sprynskyy et al. 2012). Sanchez-Martin et al. (2006) reported the efficiency of a series of clay minerals (montmorillonite, illite, muscovite, sepiolite and palygorskite) that were modified using the cationic surfactant octadecyl trimethyl ammonium bromide in the adsorption of the pesticides penconazole, linuron, alachlor, atrazine and metalaxyl. Sunardi and Yateman (2011) reported that surface modification using a cationic surfactant showed that increasing surfactant content increased the adsorption capacity of kaolin. 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). Stearyl dimethyl benzyl ammonium chloride (SKC), which is non-toxic in humans, has been used as a cationic surfactant. Clays and soils (montmorillonite, illite, muscovite, sepiolite and palygorskite) that have been modified using 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). To the best of our knowledge, this is the first study to use SKC-modified HNT as an adsorbent of ZEN in feed. The aim of the present study was to investigate the sorption properties and application feasibility of modified halloysite nanotubes (MHNT) in reducing the toxic effect of ZEN.
2. Materials and methods 2.1. Sorbent material and modification Powder HNT (Al2Si2O5(OH)4 · nH2O) were refined from clay minerals from the Henan Province (China) with a purity of 95%. The powder was prepared according to the method described by Jinhua et al. (2010). All solutions were prepared using distilled water. The powder was prepared as follows: A water suspension solution (5% in mass) was prepared by adding water to dry halloysite mineral. The suspension solution was intensively stirred for 2 h and sprayed to dry at 200°C to obtain fine powder. Before use, dry halloysite powder was sieved to eliminate aggregates. HNT were modified using SKC (Jingwei Chemical Co., Ltd., Shanghai, China) according to methods previously described by Tomašević-Čanović et al. (2003) with modifications. HNT (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 crush 100 g of HNT to obtain particles that were less than 45 μm in a beater mill at 7400g for 3 min (Lemke et al. 2001).
2.2. Microscopic imaging Transmission electron microscopy (EM) images were recorded using HITACHI H-7650 TEM. Analytical samples were prepared by dropping diluted nanotube dispersion in water onto carbon-coated copper grids and allowing the water to evaporate (Vergaro et al. 2010). For characterisation of HNT and MHNT, three samples were prepared. Six single pictures were made per sample, and in a single picture three inner diameters of different nanotubes
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were chosen for evaluating the effectiveness of modification of HNT. The width of inner diameter was measured by the transmission EM. The external surface morphology of nanotubes was characterised using a scanning EM type HITACHI S-3400N (Vergaro et al. 2010). Six samples were chosen before or after modification. Six single pictures were made for each sample to evaluate the effectiveness of modification.
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2.3. Efficiency of zearalenone removal ZEN was purchased from Fermentek Ltd. (Jerusalem, Israel), purity >98.5%. The substance was reported to be stable for at least 8 months at room temperature. The removal efficiency (R) of adsorbed ZEN was calculated using the following equation: R ½% ¼ 100% ðC0 CeÞ=C0; where C0 and Ce represent the initial and equilibrium concentrations of ZEN [mg/l], respectively (Jinhua et al. 2010). Simulated gastric fluids (SGF) and simulated intestinal fluids (SIF) were prepared freshly on the day of the experiment according to the United States Pharmacopoeia specifications (The United States Pharmacopeia 1995) for evaluating adsorption capacity of MHNT. SGF consists of porcine pepsin A (272 U/mg) in 35 mM NaCl at pH 1.2. Porcine pepsin was prepared in our laboratory as previously described (Zhou et al. 2007). The volume of the reaction solution was 10 ml. SIF was prepared as described in the United States Pharmacopoeia (1995) and consists of trypsin (1500 U/mg; 10 mg/ml SIF) in 0.05 M KH2PO4 at pH 7.5. In the in vitro experiments HNT, MHNT and a montmorillonite mixture (MON) were used as adsorbents. MON was obtained from Shangqiu IGM Animal Pharmaceutical Co., Ltd. (main ingredient: 95% improved nanometre montmorillonite, yeast cell wall, modified polyvinylpolypyrrolidon (PVPP) and calcium propionate and other ingredients). In the first adsorption experiment, the SGF and SIF solutions contained ZEN at 50 mg/l and the tested adsorbents (HNT, MHNT and MON) were added at 0.4, 0.7, 1.0, 1.3 and 1.6 mg/ml, respectively. The mixtures were shaken at 37°C for 2 h and 4 h in Erlenmeyer flasks (50 ml) to determine the removal efficiency in vitro and the process was immediately terminated by addition of 30 μl of 200 mM Na2CO3 (Huang et al. 2010). In the second adsorption experiment, the adsorbents (HNT, MHNT and MON) were added to the SGF and SIF solutions (10 ml, ZEN concentration 50 mg/l) at 1.0 mg/ml. The mixtures were shaken at 37°C for 20, 40, 60, 90 and 120 min (SGF) and 30, 60, 90, 120 and 240 min (SIF) to determine the time of degradation equilibrium in vitro. The reaction was stopped by addition of serine proteinase inhibitor pefabloc SC to a final concentration of 5 mM (Huang et al. 2010). The adsorption experiments were repeated three times. ZEN concentrations were compared using validated analytical methods according to the National Standards of the People’s Republic of China (GB/T 19,540–2004). The method uses immunoaffinity chromatography for purification and high-performance liquid chromatography (HPLC) for detection and quantification of the toxin. 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
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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 firstly 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) and fluorescence detection (λex = 274 nm, λem = 440 nm).
2.4. Animals and experimental design Sixty female Sprague–Dawley (SD) rats were obtained from the Jilin University Laboratory Animal Centre (Changchun, China) and were acclimated for 2 weeks prior to experiment. The weights of rats ranged from 190 to 210 g (9 weeks of age). The experiments began on the tenth week of age and lasted for 2 weeks. The rats received diets containing different concentrations of ZEN, HNT, MHNT and MON. The rats were divided into five groups. The Control group received the basal diet (Table 1) without any further addition. The other experimental diets contained the basal diet and a supplementation of ZEN at 0.5 mg/kg diet (Table 2). Furthermore, to the diets of Groups HNT, MHNT and MON the respective adsorbents were added at 1% of the diet at the expense of cellulose (Table 2). Rats were kept individually in polycarbonate metabolic cages (Tecniplast, Hohenpeißenberg, Germany) and had free access to food and water. They were maintained on a 12/12 h light/dark cycle at 24 ± 2°C in the study room. The relative humidity was maintained between 47% and 55% (Collins et al. 2006). Throughout the study, the rats were fed experimental diets according to AIN-93G (Reeves et al. 1993). The feed was prepared in an experimental feed mill using high-precision mixers and all feedstuffs were prepared for the detection of mycotoxin contents. There were no other mycotoxin residuals in feedstock and adsorbents.
Table 1. Composition and nutrient levels of the basal diet (air-dry basis) fed the in vivo trial on rats. Ingredients Corn starch Dextrin starch Casein Soybean oil Cellulose Cane sugar L-cysteine CaHPO4 Limestone Choline Premix* Total
Contents [%] 41.65 13.20 20.00 6.00 5.00 10.00 0.30 0.80 1.30 0.25 1.50 100.00
Nutrient levels (analysed) Gross energy [MJ/kg] Crude protein [%] Crude fibre [%] Calcium [%] Phosphorus [%]
Contents 16.80 17.84 10.86 0.87 0.52
Notes: *Provided per kg of the diet: Fe (as ferrous sulphate) 35 mg, Cu (as copper sulphate) 6 mg, Zn (as zinc sulphate) 30 mg, Mn 10 mg, vitamin A 14,000 IU, vitamin D3 1500 IU, vitamin E 80.04 mg, riboflavin 12 mg, nicotinic acid 60 mg, D-pantothenic acid 24 mg, vitamin B12 1.2 mg, biotin 0.2 m.
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Supplementation of the basal diet in the in vivo trial on rats. Experimental groups
ZEN [mg/kg diet] Adsorbent [%]
Control
ZEN†
HNT*
MHNT◊
MON♦
– –
0.5 –
0.5 1
0.5 1
0.5 1
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Notes: †ZEN, Zearalenone; *HNT, Natural halloysite nanotubes (adsorbent); ◊MHNT, Modified natural halloysite nanotubes (adsorbent); ♦MON, Montmorillonite mixture (adsorbent).
2.5. Ethics statement This study was performed in strict accordance with the recommendations of the National Research Council (1996), and all animal experimental procedures were approved by the Ethical and Animal Welfare Committee of Heilongjiang Province, China. All surgeries were performed under anaesthesia using an intraperitoneal injection of sodium pentobarbital and every effort was made to minimise animal suffering.
2.6. Collection of physiological samples The following data were monitored during the animal trial: body weight (BW), average daily feed intake (ADFI), average daily gain (ADG) and feed conversion ratio (FCR). The animals were sacrificed under anaesthesia using an intraperitoneal injection of sodium pentobarbital (0.5 ml/kg BW) at the end of the experiments. Whole blood was collected from the submandibular site and placed into microcentrifuge tubes coated with 10 μl of heparin sodium salt (20 U/ml in PBS). Samples were kept on ice for 30 min, then centrifuged at 3000g for 10 min at 4°C. The serum layers were removed and placed into sterile microcentrifuge tubes and stored at −80° C until further analyses. The organs (liver, kidney, spleen, ovary and uterus) were quickly removed and weighed. The reproductive organs were prepared for HPLC analysis.
2.7. Quantification of zearalenone Preliminary treatments were performed according to the procedures described by Koraichi et al. (2012) with modifications. The samples (2 g of tissue) were extracted with 40 ml of methanol (Sigma-Aldrich, St. Louis, MO, USA) for 1 min. The extract was centrifuged at 825g for 5 min. The supernatant was transferred to a flask and evaporated at 50°C with a rotary evaporator under reduced pressure until 5 ml of residual water remained. pH was adjusted to 6.8 with 1 N NaOH. The sample was adjusted to 8 ml with 50 mM phosphate buffer, pH 6.8. Tube was incubated with βglucuronidase (200 U) at 37°C overnight to cleave the conjugates. Each sample was extracted twice with chloroform (Sigma-Aldrich, St. Louis, MO, USA). The chloroform layer was evaporated with a rotary evaporator at 50°C under reduced pressure. The flask was rinsed three times with 1 ml of methanol which was evaporated to dryness under a gentle nitrogen flow. The final purified sample was dissolved in 200 μl methanol and 50 μl was injected for analysis by HPLC. ZEN and metabolite concentrations were determined by HPLC according to Videmann et al. (2008) and Koraichi et al. (2012) with modifications. The HPLC
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system (Hitachi-MercK Product) consisted of a L7100 pump, an AS2000 sampler, a L4000 UV detector (wavelength of 235 nm) and D7000 HSM software (VWR, Nogent-sur-Marne, France). Samples (50 μl) or standard solution were injected in a LIChrospher ®100RP18 en-capped column (5 μM granule size, 250 × 4 mm) (VWR International, Fontenay-sous-Bois, France) was used. A gradient elution system was used: from 75% water (acidified with 0.2% H3PO4 and adjusted to pH 3 with 5 N NaOH), 25% acetonitrile at time zero, isocratic for 0.2 min, to 35% water, 65% acetonitrile at 13 min. The flow rate was set at 1.4 ml/min. The retention time of ZEN was 8 min.
2.8. Hormone levels Hormone levels were determined using an ELISA Kit according to the manufacturer’s recommended protocol (R&D Systems, Inc., USA). The parameters evaluated in the serum included prolactin, follicle-stimulating hormone (FSH), luteinising hormone (LH), oestradiol and progesterone.
2.9. Determination of antioxidative parameters The antioxidative parameters were determined using an ultraviolet spectrophotometer (UV-2410PC model, SHIMADZU, Japan) with commercial diagnostic kits (Nanjing Jiancheng Biotechnology Co., Ltd, Jiangsu, China). The parameters evaluated in serum and liver were malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx).
2.10. Statistical analysis Statistical analysis was performed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) for a randomised complete design. The data of inner diameter before or after modification was analysed using the unpaired t-test. The linear and quadratic effects of ZEN the removal efficiency in SGF and SIF in vivo were assessed. Because the data were normally distributed, indices in vitro were analysed using ANOVA and the Duncan multiple range test. Data were expressed as the mean ± standard errors (SEM) of the indicated number of experiments; p-values less than 0.05 were considered to be statistically significant.
3. Results 3.1. Modification and appraisal of halloysite nanotubes Figure 1 shows the images after EM. It can be seen that the samples consisted of cylindrical tubes. After treatment with SKC, the dispersion of the halloysite (Figure 1B) was increased compared to the untreated HNT powder (Figure 1A). Moreover, the lumen of modified nanotubes shown in Figure 1D was enlarged compared with untreated nanotubes (Figure 1C). By the surface modification, the average internal diameter of the lumen was significantly enlarged from 11.4 ± 0.31 nm (n = 54) to 20.1 ± 0.04 nm (n = 54) (p < 0.05). As shown in Figure 1C and D, the external surface of the treated HNT was cruder than the natural halloysite.
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(A)
(B)
(C)
(D)
Figure 1. (A) Scanning electron microscopy (EM) image of the halloysite nanotubes (HNT) powder. (B) Scanning EM image of modified HNT powder with increased dispersity. (C) Transmission EM images of the HNT. (D) Transmission EM image of modified HNT (MHNT) with enlarged internal diameters.
3.2. Efficiency of zearalenone removal in simulated gastric and intestinal fluid For the determination of the adsorption capacity, simulated gastric and intestinal fluids were used in an in vitro study. The removal efficiency of ZEN by HNT, MHNT and MON in SGF and SIF is shown in Tables 3–5. For all tested adsorbents, the adsorption efficiency for ZEN was increased linearly and quadratically (p < 0.01) when the adsorbent dose rose from 0.4 to 1.0 mg/ml (Table 3). Furthermore, for all testes adsorbents the adsorption efficiency became stable at 1.0 mg/ml. Moreover, the adsorption efficiency for ZEN increased with the extension of adsorption times from 20 to 90 min (SGF) and from 30 to 120 min (SIF) (Tables 4 and 5). For example, the removal efficiency by the MON mixture increased during this period from 7.6% to 27.4% and from 19.9% to 55.3% for SGF and SIF, respectively. The equilibrium times of adsorption were 90 min and 120 min for SGF and SIF, respectively. In case of treatment MHNT, the surfactant SKC increased the adsorptive property of HNT already at the first measuring times of 20 and 30 min for SGF and SIF, respectively (Tables 4 and 5). Consequently, the results proofed that the removal efficiency of HNT was enhanced after modification by SKC at all conditions. The removal efficiency of the MHNT approached the MON mixture.
Archives of Animal Nutrition Table 3.
Removal efficiency [%] of zearalenone (ZEN) adsorbents at different doses in vitro. p-Value#
Adsorbents dose [mg/ml] 0.4
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0.7
In simulated gastric fluid (SGF) 9.80 13.51 HNT†§ 11.39 15.82 MHNT†◊ 10.47 15.50 MON†♦ In simulated intestinal fluid (SIF) HNT 12.55 23.64 MHNT 20.27 38.86 MON 16.66 38.92
1.0
1.3
1.6
SEM*
Linear
Quadratic
18.81 23.74 24.06
18.13 23.90 24.25
18.26 23.63 24.52
0.993 1.407 1.565
