Influence of deoxynivalenol and T-2 toxin on the intestinal barrier and liver function in broiler chickens Ann Osselaere Dissertation submitted in fulfillment of the requirements for the degree of Doctor of Philosophy (PhD) in Veterinary Science 2013 Promoters Prof. Dr. S. Croubels Prof. Dr. P. De Backer Faculty of Veterinary Medicine Department of Pharmacology, Toxicology and Biochemistry This PhD was funded by the Bijzonder Onderzoeksfonds (01J08309) of Ghent University. This work was printed by University Press, Zelzate | www.universitypress.be Osselaere, Ann Influence of deoxynivalenol and T-2 toxin on the intestinal barrier and liver function in broiler chickens Ghent University, Faculty of Veterinary Medicine This thesis is lovingly dedicated to my father Table of Contents ABBREVIATION KEY .................................................................................................................................................................... 1 GENERAL INTRODUCTION .......................................................................................................................................................... 7 1. The poultry industry with emphasis on broiler chickens ............................................................................................. 9 2. Mycotoxins as contaminants of animal feed ............................................................................................................. 10 2.1 Classification and occurrence of mycotoxins ................................................................................................... 10 2.2 Toxicity and metabolism of Fusarium mycotoxins ........................................................................................... 14 2.3 Implication for the health of broilers ............................................................................................................... 16 2.3.1 DON mycotoxicosis ...................................................................................................................................... 17 2.3.2 T-2 mycotoxicosis ........................................................................................................................................ 20 2.4 Implications for men’s health ........................................................................................................................... 22 2.5 Principles of mycotoxin management .............................................................................................................. 22 3 The gastro-intestinal tract and the liver as a target for trichothecene mycotoxins ................................................... 27 3.1 Effects at the gastro-intestinal level ..................................................................................................................... 29 3.1.1 Gut wall morphology ................................................................................................................................... 32 3.1.2 The intestinal functional barrier .................................................................................................................. 33 3.1.2.1 Trichothecenes interact with the paracellular pathway .............................................................................. 33 3.1.2.2. Trichothecenes interact with efflux transporters involved in the transcellular pathway………………. .......... 36 3.1.2.3 Trichothecenes interact with drug metabolism in the GI tract ................................................................... 38 3.2 Effects at the hepatic level .................................................................................................................................... 40 SCIENTIFIC AIMS ....................................................................................................................................................................... 59 EXPERIMENTAL STUDIES .......................................................................................................................................................... 63 Chapter 1. ............................................................................................................................................................................ 65 Toxicokinetic studies of three important Fusarium mycotoxins: deoxynivalenol, T-2 toxin and zearalenone ........................ Chapter 2. ............................................................................................................................................................................ 85 Evaluation of different biomarkers to assess deoxynivalenol exposure and efficacy and safety testing of mycotoxin detoxifiers ................................................................................................................................................................................ Chapter 3. .......................................................................................................................................................................... 111 The effects of deoxynivalenol and an adsorbing agent on the intestinal barrier and liver function ....................................... Chapter 4. .......................................................................................................................................................................... 135 CYP3A in the liver and the small intestine of healthy broiler chickens .................................................................................... Chapter 5. .......................................................................................................................................................................... 155 Effects of T-2 toxin on intestinal and hepatic biotransformation mechanisms and transporter systems ............................... GENERAL DISCUSSION ............................................................................................................................................................ 179 SUMMARY .............................................................................................................................................................................. 197 SAMENVATTING ..................................................................................................................................................................... 205 CURRICULUM VITAE ............................................................................................................................................................... 215 BIBLIOGRAPHY ........................................................................................................................................................................ 219 DANKWOORD ......................................................................................................................................................................... 227 Abbreviation key ABBREVIATION KEY α-ZAL α-zearalanol α-ZEL α-zearalenol ABC ATP-binding cassette ACN acetonitrile AcNIV mono-acetylnivalenol a-DON mono-acetyldeoxynivalenol ANOVA analysis of variance ATP adenosine triphosphate AUC area under the plasma concentration-time curve β-ZAL β-zearalanol β-ZEL β-zearalenol BEA beauvericin BEMEFA Belgian Association of Feed Manufacturers BLAST basic local alignment search tool BW body weight Caco-2 human colorectal adenocarcinoma cell CAST Council for Agricultural Science and Technology cDNA copy-deoxyribonucleic acid Cl clearance CLDN claudin Cmax maximum plasma concentration Ct threshold cycle CYP cytochrome P450 d day Da dalton DAcDON di-acetyldeoxynivalenol DAcNIV di-acetylnivalenol DAS diacetoxyscirpenol DNA deoxyribonucleic acid DOM-1 deepoxy-deoxynivalenol DON deoxynivalenol dsDNA double stranded DNA EC Ethical Committee EDTA ethylenediaminetetra-acetic acid EFSA European Food Safety Authority ELISA enzyme-linked immunosorbent assay 1 Abbreviation key ERK extracellular signal-regulated kinases F absolute oral bioavailability FA fusaric acid FB fumonisin B FUC fusarochromanone FUP fusaproliferin FUS fusarenone-X g g-force GAP Good Agricultural Practice GAPDH glyceraldehydes-3-phosphate dehydrogenase GCY glucose, yeast extract and peptone GI(T) gastro-intestinal (tract) GOIs genes of interest h hour H6PD hexose-6-phosphate dehydrogenase HcK hematopoietic cell kinase HCT human cell-line derived from colon-carcinoma microtissues HIF-1α hypoxia-inducible factor 1 subunit alpha HKG housekeeping gene HMOX heme-oxygenase HPLC high-performance liquid chromatography HPRT hypoxanthine-guanine phosphoribosyl transferase HT-2 HT-2 toxin IPEC intestinal porcine epithelial cell IS internal standard iv intravenous(ly) JAMs junctional adhesion molecules JNK c-Jun N-terminal kinases kel elimination rate constant kg kilogram LC liquid chromatography LD50 lethal dose for 50% of subjects LOD limit of detection LOQ limit of quantification μg microgram μl microliter μm micrometer MAPK mitogen-activated protein kinase MAS monoacetoxyscirpenol 2 Abbreviation key MDR1 multiple drug resistance 1 MDZ midazolam mg milligram min minute ml milliliter MON moniliformin mRNA messenger RNA MRP2 multidrug resistance-associated protein 2 MS/MS tandem mass spectrometry MUCL Mycothèque de l’Université Catholique de Louvain n number NADPH nicotinamide adenine dinucleotide phosphate NCBI National Center for Biotechnology Information NEO neosolaniol ng nanogram NIV nivalenol 1-OH-midazolam 1-hydroxy-midazolam 4-OH-midazolam 4-hydroxy-midazolam