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Evaluation of efficacy and toxicity of exfoliated silicate nanoclays as a feed additive for fumonisin detoxification Chiao-Wei Yuan, Jie-Ting Huang, Ching-Chin Chen, Pin-Chi Tang, JennWen Huang, Jiang-Jen Lin, San-Yuan Huang, and Shuen-Ei Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02224 • Publication Date (Web): 17 Jul 2017 Downloaded from http://pubs.acs.org on July 17, 2017

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Journal of Agricultural and Food Chemistry

Evaluation of efficacy and toxicity of exfoliated silicate nanoclays as a feed additive for fumonisin detoxification Chiao-Wei Yuan1+, Jie-Ting Huang1+, Ching-Chin Chen1, Pin-Chi Tang1, 2, 3, Jenn-Wen Huang4, Jiang-Jen Lin6, San-Yuan Huang1, 2, 3 ,5 *, Shuen-Ei Chen1, 2, 3 * 1

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Department of Animal Science, National Chung Hsing University, Taichung, Taiwan

Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan 3

Center for the Integrative and Evolutionary Galliformes Genomics, iEGG Center, National Chung Hsing University, Taichung, Taiwan 4

Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan

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Research Center for Sustainable Energy and Nanotechnology, National Chung Hsing University, Taichung, Taiwan 6

Institute of Polymer Science and Engineering, National Taiwan University, Taipei, Taiwan

RUNNING TITLE: Nanosilicate platelets detoxifies mycotoxins All authors listed have contributed to the work and have agreed to submit the manuscript to J. Agri. Food Chem. in exclusive manner. All human and animal studies have been reviewed by the appropriate ethics committees. 1

ACS Paragon Plus Environment


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These authors contributed equally to this work

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To whom correspondence should be addressed; e-mail:

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[email protected], [email protected]

ABSTRACT The efficacy of nano-silicate clay platelets (NSCP), exfoliated silicates from natural montmorillonites, as a feed additive for ameliorating fumonisin B1 (FB1) toxicosis was evaluated. Toxicological mechanisms by NSCP were examined through proteomic and biochemical analyses. Dietary supplementation with NSCP at a low level of 40 mg/kg feed improved growth performances in chickens with respect to FB1 toxicosis. Other issues of ameliorated symptoms including serum and/or hepatic AST activity, oxidative stress indicators, and sphinganine/sphingosine ratio, a hallmark of FB1 toxicosis were considered. Chickens with NSCP inclusion alone at 1000 mg/kg feed exhibited no changes in hepatic histology, oxidative status, and serum parameters, and even had higher feed intake. Proteomic analysis with liver tissues identified 45 distinct proteins differentially affected by FB1 and/or NSCP, in which proteins involved in thiol metabolism and redox regulation, glycolysis, carcinogenesis, and detoxification by glutathione S-transferase (GST) were promoted by FB1, whereas NSCP caused differential changes of protein abundances related to methionine/cysteine and choline/glycine interconversion for glutathione synthesis, redox regulation by

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peroxiredoxin, toxin/metabolite delivery by albumin, glycolysis, TCA cycle, ATP synthesis, and chaperon escort for ER stress relief. Functional analyses confirmed the enhancement of hepatic metabolic processes for ATP and NAD(P)H production to meet the need for detoxification, anti-oxidative defense, and toxin/metabolite clearance by FB1 or NSCP ingestion. Based on the amelioration of FB1 toxicosis, global profile of hepatic protein expressions, and validated toxicological mechanisms, NSCP were concluded as a safe and effective agent for FB1 detoxification. Key words; nanosilicate platelets, fumonisin, detoxification, liver, proteomics ■

INTRODUCTION Mycotoxins are toxic secondary metabolites produced by various fungi. It was estimated that globally 25% of crops are contaminated by

mycotoxins1. In the case of fumonisins, as high as 59% raw corns and related products in America and Europe were contaminated with detectable fumonisn B1 (FB1)2. Prevalent contamination of mycotoxins in animal and human food therefore is a worldwide problem and potential hazard risk factor to human and animal health. In most practical cases of farm animals, moderate contamination by mycotoxins in diets tends to suppress growth, reproductive performances, and immune responses3. In order to counteract mycotoxins in feedstuffs, a variety of approaches have been tried, but only few of them are practically appliacble, in which dietary inclusion with mycotoxin binders is commonly used to decrease harmfulness to animal health in livestock management. For years, montmorillonite (MMT) clays have been used as a raw material to produce remedy drugs for diarrhea in humans due to their antibacterial activity4. In feedstuff industry, hydrated sodium calcium aluminosilicates (HSCAS) and clays such as bentonites, montmorillonites,

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and zeolites are usually used as feed additives due to the effectiveness and lower cost4. The clays all contain aluminates, silicates and interchangeable ions, mainly alkali and alkaline earth ions and exhibit a strong interaction to attract polar functional groups of mycotoxins, thereby serving as an effective binder to expel mycotoxins from digestive absorption. The high affinity of aluminosilicates to aflatoxins depends on the interaction of the β-carbonyl group with aluminium ions6. However, aluminosilicate products have been shown unable to counteract toxic effects of Fusarium mycotoxins, such as trichothecenes and fumonisins, despite quite effective for aflatoxin detoxification5. Nano-silicate clay platelets (NSCP) were produced by the exfoliation process of natural montmorillonites in rendering the highly dispersed form of nanometer thin platelets, which were characterized by polygonal structure (ca. 80×80×1 nm), high surface areas (ca. 720 m2/g), cationic exchange capacity (1.20 meq/g), and ionic charges (ca. 20,000 ions/platelet)7-9. These unique characteristics promote NSCP with a high binding affinity to adhere onto the surface of bacteria leading to a strong bacteriostatic activity9-12. For applications in animals, the acute toxicity of NSCP was evaluated and results suggested a low lethal dose (LD50)(>5700 mg/kg bodyweight) in rats receiving daily oral administration of NSCP for 2 weeks12. No genotoxicity and very low cytotoxicity were observed12. Furthermore, oral administration of NSCP ameliorated embryo teratogenesis and improved fetus health in pregnant mice toxified by FB113, one of the most prevalent mycotoxins, suggesting NSCP as an effective mycotoxin detoxifying agent.

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In the study, we aimed to evaluate the efficacy of NSCP as a mycotoxin binder supplemented in feedstuffs to ameliorate FB1 toxicosis in chickens. Besides, proteomic approaches to profile hepatic protein expressions were used to annotate possible toxicological mechanisms by NSCP and functional analyses were used to confirm the proteomic analysis. ■

MATERIALS AND METHODS

Preparation of NSCP. Nano-silicate clay platelets were derived from the naturally occurring clay, sodium montmorillonite (Na+-MMT) by the process of exfoliating the layered silicates into individual NSCP using polyamine-salts as the exfoliating agent. Detailed preparation procedures and characteristics of the NSCP products were described previously7-9. Images of NSCP by transmission electron microscopy (TEM) were shown in Figure S1.  Fumonisin production. The Fusarium colony was isolated from corn kernel by Nash-PCNB selective medium and was identified as Fusarium proliferatum (Matsushima) Nirenberg based on conidial morphology, conidigenous cell, and ribosomal DNA (rDNA) LSU-D1/D2 domain sequences 14. Production and extraction procedure were performed as described previously and aliquots of extract powder were dissolved in 70% methanol and further diluted in water for FB1concentration determination using a commercial ELISA kit (R-Biopharm AG, Germany)14, 15. The yield of FB1 by the Fusarium proliferatum was ranged from 180-370 ppm based potato dextrose broth. An HPLC system with fluorescent derivatization method by o-phthaldialdehyde (OPA) was used to confirm the structure of FB1 product14. The average purity of FB1was calculated around 57%.

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Animal management and tissue collection. One-day old chicks (Arbor Acres, n=192) from a local commercial hatchery were randomly allocated into 8 groups; 2 FB1 levels (0 and 50 mg/kg feed) × 4 NSCP levels (0, 40, 200, 1000 mg/kg feed) in dietary supplementation with 3 repetitions (3 pans per group, 8 birds per pan). Chicks were provided with nutritionally adequate diets and maintained on a photoschedule 14L:10D with feed provided at 08:30AM and free access to feed and water throughout the experiment. At day 42, chickens were necropsied by cervical dislocation. All chicken husbandry and tissue collections were conducted in accordance with an approved animal care protocol by the Institutional Animal Care and Use Committee (IACUC, No:104-056) of National Chung Hsing University, Taiwan. Chickens with the highest dose of NSCP inclusion (1000 mg/kg feed) were used for NSCP toxicity studies, in which randomly selected 6 chickens from each group (2 birds per pan) with or without FB1 (50 mg/kg feed) and NSP (1000 mg/kg feed) inclusion were used for biochemical analyses. Liver slices from 3 males of each pan were pooled for proteomic study and Western blot validation. Proteomic analysis. Protein extraction with the liver tissues and 2-DE analysis using a 2-D Quant Kit (GE Healthcare Bio-Science AB) with bovine serum albumin as a standard were conducted as described previously16. The 2-DE procedure was performed according to the method from Gorg et al.17 with some modifications18. Gels were stained with colloidal Coomassie blue (Serva Electrophoresis GmbH, Germany) for at least 14 h and scanned (Image Scanner III, Lab Scan 6.0, GE Healthcar Bio-Science AB) for further analysis. Protein spots were detected and analyzed by the Melanie 7 software package (GeneBio, Geneva, Switzerland)18. The expression level of individual protein spot was obtained as the relative volume to the total volume of all spots on each gel (RVol)19. The expression ratio of each spot was calculated by dividing the Rvol of

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the highest group to that of the lowest group. Differentially expressed protein spots were excised from the gels and subjected to in-gel digestion as described previously16. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI TOF/TOF MS/MS analysis were performed through a modified procedure16. Digested samples were spotted directly onto an AnchorChip sample target (Bruker Daltonics, Bremen, Germany), and added with an equal volume of matrix solution (1 mg/mL alpha-cyano-4-hydroxycinnamic acid, 0.1% TFA, and 50% ACN). MALDI and MS/MS spectra were obtained from a Bruker UltraFlex III TOF/TOF MS (Bruker Daltonics). The MALDI spectra were searched against comprehensive non-redundant protein sequence databases (NCBInr 20141222 version with 54027943 sequences; 19470615453 residues) using the BioTools 3.0 software (Bruker Daltonics) in combination with the Mascot program20. The search conditions included taxonomy of Gallus gallus, fixed modification of carbamidomethylation, variable modification of oxidation, mass accuracy of 100 ppm, and significance threshold at P

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