MECHANISMS OF DEOXYNIVALENOL-INDUCED ANOREXIA AND ITS IMPACT ON WEIGHT IN THE FEMALE MOUSE By Brenna Montague Flannery A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Food Science - Environmental Toxicology 2012 ABSTRACT MECHANISMS OF DEOXYNIVALENOL-INDUCED ANOREXIA AND ITS IMPACT ON WEIGHT IN THE FEMALE MOUSE By Brenna Montague Flannery Each year in the United States, 38 million people suffer from acute gastroenteritis caused by unidentified agents. Deoxynivalenol is a mycotoxin elaborated from Fusarium species that might be a contributing agent to the large number of cases with unknown etiology. In humans, symptoms of acute DON intoxication include vomiting, diarrhea and abdominal pain, but lack of biomarker of effect for DON in humans has made evaluating the chronic adverse physiological consequences of DON impossible. Consequently, experimental animals have been utilized to predict DON’s chronic effects in humans. In experimental animals, constant low-level DON exposure results in anorexia, weight suppression and growth hormone dysregulation, of which weight suppression serves as the foundation for the tolerable daily intake established by the Joint FAO/WHO Expert Committee on Food Additives. A primary mechanism by which DON causes weight suppression is through anorexia, yet the mechanisms of DONinduced anorexia are not well established. This impedes 1) comprehensive risk assessment of this mycotoxin, 2) potential mitigation efforts for weight suppression and 3) identification of particularly sensitive populations to DON’s adverse physiological effects. Using a characterized mouse model, this dissertation addresses potential mechanisms of DON-induced anorexia with the overall hypothesis that DON-induced anorexia results from the peripheral release of gut satiety hormones that work in conjunction with the central nervous system (CNS) to reduce food intake. Results suggested anorexia occurred in a rapid (30 min) and transient nature, a manner that was consistent with both peripheral and central mechanisms. Notably, DON administration increased plasma satiety hormones peptide YY (PYY) and cholecystokinin (CCK) as early as 15 min post exposure, with peripheral PYY contributing modestly to DON’s anorectic effects. In support of central action, DON was dose-dependently detected in the brain and hypothalamus beginning at 15 min. Intracerebroventricular injection (ICV) of DON resulted in decreased food intake, an effect previously reported by other researchers. Since PYY can act peripherally via the vagus nerve and centrally via central NPY2 receptors to reduce food intake, two additional studies were performed to confirm PYY’s role in DON’s anorectic effects. Results demonstrated that a subdiaphragmatic vagotomy could not attenuate anorexia induced by peripheral administered DON nor could centrally administered BIIE0246, a NPY2 receptor antagonist. Taken together, these results suggested the importance of peripheral PYY in DON-induced anorexia might be mediated by a mechanism other than the vagus nerve but that this mechanism was not linked to activation of central Y2 receptors. DEDICATION For my parents Mary and Patrick Flannery iv ACKNOWLEDGMENTS So many people have contributed to my positive learning experience here at Michigan State, really too many to thank here. Words of thanks do not do justice for the support I have received throughout my Ph.D career. Foremost, I would like to thank my mentor Dr. Jim Pestka for accepting me into his lab and serving as my Ph.D advisor. I appreciate the guidance, trust and responsibility you have given me. You are the perfect role model for those who wish to be successful in whatever they seek. Next, I would like to thank my dissertation committee members Dr. Dale Romsos, Dr. Jenifer Fenton, Dr. Patricia Ganey and Dr. Jim Galligan. Thank you for your guidance and constructively challenging me. You have taught me the importance of asking questions about my research. As for laboratory colleagues, Dr. Dozie Amuzie really set the tone for my graduate studies. I learned from the best! Thank you to my other fellow laboratory colleagues, Dr. Hui-Ren Zhou, Dr. Kaiyu He, Laura Vines, Xiao Pan, Erica Clark, Melissa Bates, Wenda Wu and Victoria Minton, who have aided me in mental and physical tasks. I was so lucky to work with such supportive people. I would also like to thank Mary Rosner for her positive attitude and keeping the lab organized. People from the Linz Lab, Gardner Lab (especially Dr. Gardner) and Fenton Lab have always been refreshing and provided leisurely conversation about research, which was always so helpful. I would not have been able to complete my education and this dissertation without funding support from sponsors. The Center for Integrative Toxicology, Food Science and Human Nutrition and Dr. Pestka have all provided financial support for my graduate career. I sincerely value my education and this opportunity at MSU. It would v not have been possible if it were not for your departments’ hard work to obtain funding. Thank you. I was taught the physical skills and assays needed to complete this dissertation from many people and facilities. Thank you to the IACUC and ULAR for teaching me how to handle mice and taking such great care of our animals. I feel very privileged to work with experimental animals. Thank you to my teachers who took time to disperse their knowledge to me. I would like to acknowledge many people in my personal life as well. These people were crucial for my mental well being during graduate school. Thank you to my best friends Katie O’Rielly, Angie Apsey, Sally Hanks, Lisa Harlow and Eileen Dickson for your constant support and willingness to go have fun. My family has served the foundation for any of my success throughout my life. I was lucky to be born to parents who emphasized excellent work ethic and a positive attitude. Both of my parents have always led by example and completed what they started. Thank you for instilling the importance of these qualities in me. My significant other Stephen DiFranco has been my best friend throughout this process- always giving me encouragement and advice. I appreciate the encouraging attitudes of my sister and the DiFrancos as well. Finally, I am very thankful for my education, which allowed me to complete this dissertation. vi TABLE OF CONTENTS LIST OF TABLES .......................................................................................................... xi LIST OF FIGURES........................................................................................................ xii LIST OF ABBREVIATIONS .......................................................................................... xv INTRODUCTION ............................................................................................................. 1 Overall Hypothesis .................................................................................................... 4 Chapter Summaries ................................................................................................... 4 Significance ............................................................................................................... 7 CHAPTER 1: LITERATURE REVIEW ............................................................................ 9 Introduction ................................................................................................................ 9 Deoxynivalenol ........................................................................................................ 10 Prevalence.......................................................................................................... 10 DON Toxicology ....................................................................................................... 12 Human Exposure ................................................................................................ 12 The Use Of Experimental Animals to Assess DON Toxicity ............................... 13 Reported Mechanisms of DON-Induced Weight Suppression ............................. 14 Relationship of Weight Suppression to Anorexia ................................................ 14 Relationship of Weight Suppression to Growth Hormone Dysregulation ............ 17 Mechanisms of DON-Induced Anorexia................................................................. 20 Contribution of Taste to DON-Induced Anorexia ................................................ 20 Contribution of Proinflammatory Cytokines to DON-induced Anorexia ............... 21 Contribution of 5-HT to DON-induced Anorexia ................................................. 21 Central Nervous System Control of Food Intake .................................................. 23 Hypothalamic Signaling ...................................................................................... 23 Vagus Nerve ....................................................................................................... 24 5-HT ................................................................................................................... 26 Gastrointestinal Satiety Hormone Release: Cholecystokinin and Peptide YY ... 26 Cholecystokinin .................................................................................................. 27 Peptide YY.......................................................................................................... 28 Alternative Hypothesis for DON-induced Anorexia .............................................. 31 Rationale for Proposed Mechanisms of DON-Induced Anorexia ........................ 33 Model Justification .................................................................................................. 34 CHAPTER 2: CHARACTERIZATION OF DEOXYNIVALENOL-INDUCED ANOREXIA USING MOUSE BIOASSAY ......................................................................................... 37 Abstract .................................................................................................................... 37 Introduction .............................................................................................................. 38 Materials and Methods ............................................................................................ 40 vii Laboratory animals and DON. ............................................................................ 40 Experimental design. .......................................................................................... 40 Statistics. ............................................................................................................ 43 Results...................................................................................................................... 44 Ip. and oral exposure to DON causes rapid and transient feed refusal .............. 44 Short-term (2 d) repeated DON exposure evokes partial tolerance to feed refusal effects ................................................................................................................. 45 Intermittent (7d) repeated exposure to DON does not evoke tolerance to feed refusal effects ..................................................................................................... 45 Discussion ............................................................................................................... 52 CHAPTER 3: ANOREXIA INDUCTION BY THE TRICHOTHECENE DEOXYNIVALENOL (VOMITOXIN) IS MEDIATED BY THE RELEASE OF THE GUT SATIETY HORMONE PEPTIDE YY (PYY) ................................................................... 59 Abstract .................................................................................................................... 59 Introduction .............................................................................................................. 60 Materials and Methods ............................................................................................ 63 Animals. .............................................................................................................. 63 DON administration. ........................................................................................... 64 DON-induced gut satiety hormone release. ........................................................ 64 Hormone analyses.............................................................................................. 65 DON- and hormone- induced anorexia. .............................................................. 66 Receptor antagonist effects on DON-induced anorexia...................................... 66 Statistics. ............................................................................................................ 67 Results...................................................................................................................... 68 DON exposure induces elevations in PYY, CCK but not other appetite-regulating hormones............................................................................................................ 68 DON exposure rapidly induces anorexia ............................................................ 69 Both orolingual and ip DON exposure induce PYY, CCK and anorectic responses ........................................................................................................................... 74 Exogenous PYY and CCK suppress food intake ................................................ 74 NPY2 receptor antagonist BIIE0246 interferes with PYY- and DON-induced anorexia. ............................................................................................................. 80 CCK1A receptor antagonist DEV ablates CCK- but not DON-induced anorexia 81 Discussion ............................................................................................................... 81 CHAPTER 4: CENTRAL MECHANISMS OTHER THAN PYY CONTRIBUTE TO DONINDUCED ANOREXIA .................................................................................................. 87 Abstract .................................................................................................................... 87 Introduction .............................................................................................................. 88 Materials and Methods ............................................................................................ 91 Chemicals. .......................................................................................................... 91 Laboratory animals. ............................................................................................ 92 Experimental Designs ......................................................................................... 92 Perfusion. ........................................................................................................... 95 DON ELISA. ....................................................................................................... 95 viii Vagotomy. .......................................................................................................... 96 ICV Cannulation and Injection. ........................................................................... 96 Statistics. ............................................................................................................ 97 Results...................................................................................................................... 97 Peripheral DON administration rapidly reduces food intake ............................... 97 DON distributes to peripheral and central tissue in a dose-dependent manner .. 97 DON-induced feed refusal is not attenuated in vagotomized mice ..................... 98 Central DON administration minimally depresses food intake ............................ 98 The central Y2 receptor is not necessary for DON-induced anorexia. ................ 99 Discussion ............................................................................................................. 107 CHAPTER 5: EVALUATION OF INSULIN-LIKE GROWTH FACTOR ACID-LABILE SUBUNIT AS A BIOMARKER OF EFFECT FOR THE MYCOTOXIN DEOXYNIVALENOL ................................................................................................... 111 Abstract .................................................................................................................. 111 Introduction ............................................................................................................ 112 Materials and Methods .......................................................................................... 115 Laboratory Animals and diet. ............................................................................ 115 Study Design 1: Sub-chronic dietary DON exposure. ....................................... 116 Study Design 2: Restricted Dietary DON Exposure. ......................................... 116 ELISA for IGFALS. ........................................................................................... 117 Quantitative Real-Time PCR. ........................................................................... 117 Statistics. .......................................................................................................... 118 Benchmark Dose Modeling............................................................................... 118 Results.................................................................................................................... 119 Study 1. DON consumption dose-dependently suppresses weight gain and IGFALS ............................................................................................................. 119 Study 2. Plasma IGFALS depression is specific to DON exposure .................. 124 Discussion ............................................................................................................. 129 CHAPTER 6: DON- INDUCED WEIGHT LOSS AND ANOREXIA IS REVERSIBLE IN THE DIET-INDUCED OBESE MOUSE ....................................................................... 135 Abstract .................................................................................................................. 135 Introduction ............................................................................................................ 136 Materials and Methods .......................................................................................... 138 Mice. ................................................................................................................. 138 Diet. .................................................................................................................. 138 Food Intake Measurements. ............................................................................. 139 Experimental Design Study 1. .......................................................................... 139 Experimental Design Study 2. .......................................................................... 139 Statistics. .......................................................................................................... 140 Results.................................................................................................................... 140 Study 1. Dietary DON removal results in complete weight compensation in dietinduced obese mice.......................................................................................... 140 Study 2. Dietary DON removal simulates appetite in diet-induced obese mice 143 Discussion ............................................................................................................. 147 ix CHAPTER 7: CONCLUSIONS ................................................................................... 150 APPENDICES ............................................................................................................. 155 Appendix A: The Role of the 5-HT -1B, -2C, and -3 Receptors in DeoxynivalenolInduced Anorexia in the Mouse ............................................................................... 156 Appendix B: Model Considerations for Toxicological Endpoint Measurements in the Mouse ...................................................................................................................... 175 Appendix C: Protein Kinase R is Not Critical for Deoxynivalenol-Induced Weight Suppression ............................................................................................................. 191 REFERENCES ............................................................................................................ 203 x LIST OF TABLES Table 1.1. The reported no observed adverse effect level (NOAEL) for feeding studies using weight suppression and anorexia as the adverse effect in growing DONexposed experimental animals. .............................................................................. 15 Table 1.2. NOAELS for feeding studies using weight suppression and anorexia as the adverse effect in mature DON-exposed experimental animals. ............................. 16 Table 1.3. Hormones that control food intake................................................................ 30 Table 3.1. Comparison of effects of orolingual and intraperitoneal exposure to DON on food intake and gut satiety hormone release. ........................................................ 76 Table 6.1. Statistics for Fig. 6.1. .................................................................................. 141 Table A.A.1. Doses and volues for experimental groups (Exp.) groups during the evaluation of 5-HT -3, -1B and -2C receptor inhibitors in agonist and DON-induced anorexia ............................................................................................................... 163 Table A.B.1. The no observed adverse effect level using plasma IGF1, IGFALS and hepatic mRNA expression of SOCS3, IGF1 and IGFALS as biological endpoints. ............................................................................................................................. 188 xi LIST OF FIGURES Figure 1.1. The chemical structure of deoxynivalenol…………………………………......11 Figure 1.2. Simplistic representation of satiety signaling upon food consumption…......25 Figure 1.3. Symptoms experienced in humans upon consuming increasing amounts of food or given increasing amounts of PYY 3-36……………...................…………............32 Figure 1.4. Potential mechanisms of DON-induced weight suppression and anorexia..........................................................................................................................35 Figure 2.1. Experimental design for mouse feed refusal assay………………….............42 Figure 2.2. Intraperitoneal DON exposure in mice induces an early anorexigenic response followed by a delayed orexigenic response……………..................................46 Figure 2.3. Oral DON exposure in mice induces an early anorexigenic response followed by a delayed orexigenic response………………………...............…….............47 Figure 2.4. Experimental design for determining the duration of DON-induced feed refusal.............................................................................................................................48 Figure 2.5. DON-induced feed refusal is transient…………………………………...........49 Figure 2.6. Short-term repeated DON exposure results in partial tolerance to DONinduced feed refusal…………….............…………………………………………...............50 Figure 2.7. DON-induced feed refusal tolerance is short-lived…………………..............51 Figure 2.8. Comparison of feed refusal induction by ip. injection and oral gavage of DON….………………………………………………………………….................................54 Figure 3.1. DON induces plasma PYY elevation………………………………………......70 Figure 3.2. DON induces plasma CCK elevation…………………………………….........71 Figure 3.3. Kinetics of DON-induced plasma PYY elevation…………………………......72 Figure 3.4. Kinetics of DON-induced plasma CCK elevation…………………………......73 Figure 3.5. Kinetics of DON-induced anorexic response……………………………….....75 Figure 3.6. Kinetics of the PYY-induced anorexic response………………………….......77 xii Figure 3.7. Kinetics of the CCK-induced anorexic response………………………..........78 Figure 3.8. Effect of BIIE0246 on PYY and DON-induced anorexic response………….79 Figure 3.9. Effect of DEV on CCK and DON-induced anorexic response……………….82 Figure 4.1. Food intake in mice administered DON by intraperitoneal injection………100 Figure 4.2. Dose-dependent DON distribution in peripheral and central tissues……...101 Figure 4.3. Short-term kinetics of DON distribution in the brain and hypothalamus of saline perfused mice…....…………………………………………..……………………….102 Figure 4.4. Short-term kinetics of DON distribution to the small intestine, spleen and liver of saline perfused mice………………………………………………...................…..103 Figure 4.5. Food intake of vagotomized mice upon DON administration………………104 Figure 4.6. Food intake in mice upon intracerebroventricular injection of DON……….105 Figure 4.7. The effect of the Y2 receptor antagonist BIIE0246 DON-induced anorexia........................................................................................................................106 Figure 5.1. DON consumption suppresses weight gain………………………………….121 Figure 5.2. DON consumption suppresses plasma IGFALS concentrations…………..122 Figure 5.3. Plasma IGFALS depression correlates with decreased weight gain……...123 Figure 5.4. Effect of DON on hepatic mRNA expression of Socs-3 and Igfals………..125 Figure 5.5. Comparison of food intakes during restricted feeding……………………...126 Figure 5.6. Comparison of body weight changes during restricted feeding…………...127 Figure 5.7. Comparison of plasma IGFALS levels in following restricted feeding…….128 Figure 5.8. Proposed pathways for DON-induced weight reduction…………………....130 Figure 6.1. Weights of diet-induced obese mice upon addition and removal of dietary DON………………………………………………………………………………..................142 Figure 6.2. Weights of diet-induced obese mice upon addition and removal of dietary DON……………………………………………………………................………………..…144 xiii Figure 6.3. Food intake and weights of mice before and after dietary DON addition………………………………………………….………………………….........…...145 Figure 6.4. Food intake and weights of mice before and after dietary DON removal……………………………………………………………………………………......146 Figure 7.1. Suggested model of DON-induced anorexia based on data collected within this dissertation……………………….........................………………….……........152 Figure 7.2. Alternative hypotheses for DON-induced anorexia................................... 154 Figure A.A.1. Plasma 5-HT levels upon DON administration……………………….......165 Figure A.A.2. Evaluation of the 5-HT3 receptor in 5-HT induced anorexia…………....166 Figure A.A.3. Evaluation of the 5-HT3 receptor in DON-induced anorexia……………167 Figure A.A.4. Evaluation of the 5HT-1B receptor in m-CPP and DON-induced anorexia……………………......................………………………………………………....170 Figure A.A.5. Evaluation of the 5HT-2C receptor in m-CPP and DON-induced anorexia…………………………………......................…………………………………....171 Figure A.B.1. Relationship of diet consistency to DON-induced weight suppression…………………...........……………………………………………………......183 Figure A.B.2. Effect of diet consistency on plasma IGF1 and IGFALS upon DON Exposure in mice fed pellet diet……......................……………………………………....184 Figure A.B.3. Effect of diet consistency on plasma IGF1 and IGFALS upon DON Exposure in mice fed powder diet……...................……………………………………....185 Figure A.B.4. Effect of diet consistency on hepatic mRNA expression of IGF1 and IGFALS upon DON exposure……………………………………………..........................186 Figure A.B.5. Food consumption relative to animal handler………………………….....187 Figure A.C.1. Weights of WT and PKR-KO mice fed 10 ppm DON…………………....196 Figure A.C.2. Cumulative weight gain of WT and PKR-KO mice fed10 ppm DON……………….............……………………………………………………………….....197 Figure A.C.3. Body fat percentage of WT and PKRKO mice fed 0 or 10 ppm………...198 xiv LIST OF ABBREVIATIONS α-MSH α-melanocyte stimulating hormone 5-HIAA 5-hydroxyindoleacetic acid 5-HT 5-hydroxytryptamine Ad-lib Ad-libitum AgRP Agouti-related peptide ANOVA Analysis of variance ARC Arcuate Nucleus CART Cocaine-amphetamine regulated transcript CCK Cholecystokinin CNS Central nervous system CSF Cerebral Spinal Fluid d Day DEV Devazepide DG Deoxynivalenol-glucuronide DIO Diet-induced obesity DMSO Dimethyl sulfoxide DON Deoxynivalenol ELISA Enzyme-linked immunosorbent assay xv FAO/WHO Food and agriculture organization/world health organization GI Gastrointestinal tract GIP Gastric inhibitory peptide GLP-1 Glucagon-like peptide 1 HFD High fat diet hr Hour IGF1 Insulin-like growth factor 1 IGFALS Insulin-like growth factor acid-labile subunit IL-1β Interleukin - 1β IL-6 Interleukin – 6 i.v. Intravenous ip. Intraperitoneal JAK/STAT Janus kinase/signal transducer and activator of transcription JECFA Joint expert committee on food additives KO Knock-out LHA Lateral hypothalamus MCR4 Melanocortin – 4 receptor min Minute ND Not determined NOAEL No observed adverse effect level NPY2 Neuropeptide Y2 receptor xvi n.s. Not significant PFA Perifornical area PKR Protein kinase R PNS Peripheral nervous system POMC Pro-opiomelanocortin PVN Paraventricular nucleus PYY Peptide YY RT-PCR Real-time polymerase chain reaction SH2 Src homology 2 SOCS Suppressor of cytokine signaling TDI Tolerable daily intake TNF –α Tumor necrosis factor – α VHFD Very high fat diet wk Week xvii INTRODUCTION Various human populations may be differentially sensitive to environmental toxins, particularly 1) children who may have not fully developed toxin metabolizing enzymes, 2) the obese who exhibit metabolic disturbances that could worsen a toxin’s adverse effects, 3) people ill with cancer whose immune systems are weaker and 4) finally, people in the general population who are genetically predisposed because they lack efficient toxin metabolizing enzymes. Deoxynivalenol (DON) is an environmental toxin produced from Fusarium species that causes illness in humans and animals, and may target the potentially susceptible populations described above. DON remains stable throughout heat processing resulting in chronic low levels contaminating the food supply worldwide (Lauren and Smith 2001). Depending on demographics, DON consumption ranges from 0.74 to 2.4 µg/kg bw/day in adults (Canady et al. 2001). Because children consume more grains per kilogram body weight than adults (Lin and Yen 2007), children are likely to have higher DON exposure per kilogram of body weight making them a population of concern for DON’s adverse effects. In humans, DON is reported to cause symptoms of acute gastroenteritis such as vomiting, diarrhea, abdominal pain and fever upon acute high dose exposure (Canady et al. 2001). Low dose chronic DON exposure in humans has been frequently assessed using the biomarker of exposure DON-glucuronide (Turner et al. 2011a; Turner et al. 2011b); however, long-term effects of low dose DON exposure have been impossible to assess due to lack of biomarker of effect. As a result, experimental animals are often 1 used to decipher DON’s chronic, low-level effects. DON exposure results in robust proinflammatory cytokine induction, growth hormone dysregulation, anorexia, weight suppression and weight loss in experimental animals (Pestka 2010b). Since children are considered particularly susceptible to weight suppression, this adverse effect of DON was the foundation of DON’s regulatory limit established by the Joint FAO/WHO Expert Committee on Food Additives (Canady et al. 2001; Pieters et al. 2002). The majority of previous scientific investigations into DON-induced weight suppression in experimental animals have determined that weight changes likely result from anorexia (Amuzie et al. 2011; Forsell et al. 1986; Iverson et al. 1995; KobayashiHattori et al. 2011; Prelusky 1997). Not surprisingly, researchers have been investigating how DON causes anorexia for the past 25 years. Initially, palatability of dietary DON was assessed in rats through preference testing and it was found that DON did not alter palatability (Clark et al. 1987). Then, Prelusky and coworkers studied the role of the neurotransmitter 5-hydroxytryptamine (5-HT; serotonin) in DON-induced anorexia with variable results. Conclusions from these studies were varied as 5-HT was not detected in the plasma of DON exposed pigs (Prelusky 1993), yet its metabolite 5hydroxyindoleacetic acid (5-HIAA) was detected in the cerebral spinal fluid of DON exposed pigs (Prelusky 1994). In addition, DON-induced anorexia in the mouse was blocked using cyproheptadine, a highly non-specific 5-HT-2 receptor antagonist (Prelusky et al. 1997). Finally, investigators determined that DON caused mice and rats to exhibit the ‘fed pattern’ of gut motility, a motility pattern associated with fullness, and these effects on gut motility were blocked using 5-HT-3 receptor inhibitors (Fioramonti 2 et al. 1993). Though 5-HT signaling appears to play a role in some of DON’s adverse effects, its role in DON-induced anorexia has yet to be established. Recently our lab demonstrated both the rapid (30 min) and transient nature (3 – 6 hr) of DON-induced anorexia in the mouse upon intraperitoneal (ip.) administration (Flannery et al. 2011). Other researchers have confirmed this result in pigs and mice upon intravenous infusion and oral gavage, respectively (Girardet et al. 2011b; Prelusky 1997). The rapid and transient nature of DON-induced anorexia suggests two potential mechanisms. First, food consumption resulted in the ‘fed pattern’ of gut motility, an effect observed in DON exposed animals (Fioramonti et al. 1993). Upon food consumption, the ‘fed pattern’ results from the release of gut satiety hormones (Allen et al. 1984; Liddle et al. 1986b). Therefore, DON could be causing the release of gastrointestinal (GI) satiety hormones resulting in anorexia. Second, direct administration of DON into the lateral ventricle of the brain at a dose less than that which causes feed refusal when injected into the periphery, resulted in feed refusal (Girardet et al. 2011b). Additionally, DON administration peripherally and centrally resulted in increased anorexigenic signaling molecules in the hypothalamus (Girardet et al. 2011b). Consequently, it is likely DON-induced anorexia results from signaling changes within the central nervous system (CNS), an effect also consistent with the release of gut satiety hormones (Challis et al. 2003; McMinn et al. 2000). This dissertation employs a mouse model to determine a role of gut satiety hormones and the CNS in DON-induced anorexia. Additionally, this information will be applied to assess the susceptibility of growing and obese mice to DON’s anorectic and weight suppressive effects. 3 Overall Hypothesis Weight suppression serves as the foundation for risk assessment of DON, an effect primarily attributed to anorexia, yet the mechanisms by which DON produces anorexia are not well understood. The objective of this dissertation was to use a standardized mouse model to determine the mechanisms of DON-induced anorexia and weight suppression for the sake of identifying particularly susceptible populations to DON’s adverse effects and contributing much needed information for comprehensive risk assessment for this mycotoxin. The guiding hypothesis of this research is that DONinduced anorexia results from the peripheral release of gut satiety hormones that work in conjunction with the central nervous system (CNS) to reduce food intake. This dissertation tests this hypothesis through four specific aims 1) evaluate the contribution of satiety hormones peptide YY (PYY) and cholecystokinin (CCK) to DON-induced anorexia, 2) determine the role of the CNS in DON-induced anorexia 3) evaluate the susceptibility of growing mice to dietary DON using plasma IGFALS and weight as endpoints and 4) evaluate the susceptibility of obese mice to dietary DON using weight and anorexia as endpoints. Chapter Summaries Chapter 1 is a literature review that provides background information for the research problems addressed in this dissertation. It will begin by explaining the problem with DON in our food system and the resultant human exposure rates. DON toxicity in humans and animals will then be described with specific focus on DON’s capacity to cause gastrointestinal illness in humans and weight suppression and anorexia in 4 experimental animals. Previously published studies will be presented that will associate DON-induced weight suppression with primarily anorexia but also growth hormone dysregulation. The focus of Chapter 1 will then shift to anorexia. First, previously investigated mechanisms of DON-induced anorexia will be described. These mechanisms will include palatability of DON contaminated food, upregulation of proinflammatory cytokines and the role of the neurotransmitter 5-HT. Other potential mechanisms of DON-induced anorexia will be introduced with focus on central nervous system signaling and gut satiety hormone release, specifically CCK and PYY. Following this background information an alternative hypothesis for DON-induced anorexia will be offered with the suggestion that DON exposed animals could be satiated and thus, not eat. The alternative hypothesis will be founded on gut satiety hormone signaling and the central nervous system. Finally, the rationale for experiments performed in this dissertation and mouse model justification will be given. Chapter 2 will describe a mouse model developed to assess DON’s acute anorectic effects. Data in this chapter have been published in Food and Chemical Toxicology and can be found at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3124119/. Data will be presented that demonstrate DON induced anorexia upon ip. and oral exposure in a rapid and transient fashion. This model will be used in Chapters 3 and 4 to relate dose and timing of DON-induced anorexia to potential mechanisms. In Chapter 3, the capacity of DON to induce release of the gut satiety hormones PYY and CCK will be evaluated. Plasma levels of these two hormones will be determined using a Milliplex® Map Bead Array and an enzyme-linked immunosorbent 5 assay (ELISA), respectively. To confirm the role of PYY and CCK in DON-induced anorexia the Y2 receptor inhibitor BIIE0246 and the CCKA1 receptor inhibitor devazepide will be given to mice before DON exposure and food intake evaluated. The data will indicate that DON increases plasma levels of PYY and CCK, though inhibitor data will suggest PYY is more important than CCK in causing DON-induced anorexia. Dose and timing of DON-induced anorexia will be related to food intake which will lead to the suggestion the animals exposed to DON may exhibit anorexia because they feel full. These findings will also lead to suggestions of potentially susceptible populations of anorexia caused by DON. Data in this chapter have been published online in Toxicological Sciences and can be found at http://toxsci.oxfordjournals.org/content/early/2012/08/16/toxsci.kfs255.long. In Chapter 4, the role of the CNS in DON-induced anorexia will be addressed by first establishing that DON enters the brain in a time and dose consistent with DONinduced anorexia. Then surgically altered mice will be used to determine the role of the vagus nerve and central Y2 receptors in DON-induced anorexia. Data will suggest that mechanisms of anorexia not requiring the vagus nerve likely contribute to DON’s anorectic effects. Furthermore, intracerebroventricular injection of the Y2 receptor BIIE0246 will establish that peripheral PYY, rather than central PYY, contributes to DON-induced anorexia. Chapter 5 will assess the susceptibility of growing mice to DON’s weight suppressive and anorectic effects resulting from dietary DON exposure. Susceptibility will be evaluated using weight, decreased plasma IGFALS, and hepatic IGFALS mRNA expression as endpoints. A dietary restriction study will demonstrate that DON’s ability 6 to depress IGFALS is not solely due to DON-induced anorexia. These data will lead to the suggestion that growing mice could be a susceptible population for DON’s growth effects and finally that plasma IGFALS could be used as a biomarker of effect for DON. In Chapter 6, the susceptibility of obese mice to dietary DON will be investigated. Weights will be determined in diet-induced obese (DIO) mice before DON, after addition of dietary DON and upon dietary DON removal. Food intake during these dietary transition times will be associated with DON’s weight effects. Data will reveal that upon dietary DON exposure DIO mice lose a significant amount of weight, which will correspond to anorexia; however, upon dietary DON removal, data will demonstrate the reversibility of DON’s weight effects, which will correspond to orexigenic intake behavior. This information will lead to the conclusion that obese mice are not more susceptible to DON’s weight loss effects than their lean counterparts. In the conclusion, the importance and application of dissertation findings will be presented. Mechanisms of DON-induced anorexia will be deduced from data presented within the dissertation. Finally, alternative mechanisms of DON-induced anorexia will be addressed and future studies proposed. Significance The results described in this dissertation are novel because they are the first to develop a mouse model to demonstrate the integration of peripheral satiety signaling with CNS control in DON-induced anorexia. This research is significant to human health because it demonstrates previously unknown adverse effects of DON, information that could serve to inform risk assessment of this mycotoxin. In the future, DON-induced gut 7 satiety hormone release can be used to identify susceptible populations to DON’s anorectic effects, populations such as children, the obese, the elderly or people in diseased states. Additionally, DON’s capacity to induce gut satiety hormone release could be valuable in deciphering mechanisms of DON-induced vomiting. 8 CHAPTER 1: LITERATURE REVIEW Introduction Each year in the United States 47.8 million illnesses are caused by foodborne factors, with 80% of those from unspecified agents (Scallan et al. 2011). Unspecified agents are defined by the Centers for Disease Control as those causing acute gastroenteritis as characterized by vomiting lasting more than one day and diarrhea of three or more episodes (Kubota et al. 2011). These agents can be characterized into both infectious and non-infectious with examples of non-infectious being natural toxins, metals and inorganic toxins. Since foodborne illness from unspecified factors accounts for 71,878 hospitalizations and 1,686 deaths per year, identifying these agents, their prevalence and negative physiological effects need to become a greater priority for public health officials (Scallan et al. 2011). A group of agents likely contributing to unspecified foodborne illness in the United States are mycotoxins. Mycotoxins are secondary metabolites elaborated from fungi that most often contaminate cereal grains, groundnuts, tree nuts and fruit worldwide (Tanaka et al. 1988). The genera responsible for producing the majority of mycotoxins are Aspergillus, Penicillum, Claviceps and Fusarium (Bennett and Klich 2003). Fusaria produce a structurally related group of toxins known as the trichothecenes, of which ingestion has been extensively linked with symptoms of acute gastroenteritis (Pestka and Smolinski 2005). The trichothecene mycotoxin deoxynivalenol (DON), also known as vomitoxin, is of specific interest because of its 9 prevalence in grains, its resistance to processing and its ability to rapidly cause acute gastroenteritis symptoms. Deoxynivalenol Fusarium graminearum and F. culmorum are the two fungal species most known to produce DON, a small molecule with molar mass of 296.3 (Fig. 1.1) (Vesonder et al. 1973, 1977). Depending on weather conditions, the Fusarium infection can lead to Fusarium head blight (head scab) and the production of DON. In the United States, DON contamination has led to an estimated $657 million in economic losses each year (Richard et al. 2003). Prevalence DON is the most commonly found trichothecene worldwide and is extremely resistant to heat and chemical processing (Abouzied et al. 1991; Lombaert et al. 2003; Schothorst and van Egmond 2004). Even up to extrusion temperatures of 180° DON C, remained stable in wheat grits with a reduction rate of only 31.9% (Wu et al. 2011). An extensive study performed by Voss and coworkers demonstrated DON reductions of 30%, 39%, 56%, 70% and 100% in crackers, cookies, donuts, breads and pretzels under standardized processing procedures, respectively (Voss and Snook 2010). Interestingly, in this same study, processed flaked wheat cereal exhibited a higher DON level than the starting wheat material suggesting DON may have been released from DON conjugates or DON glucosides during processing. Additionally, in twelve European countries, 57% of 11,022 cereal samples tested positive for DON with 6% of these products testing above 5 ppm, a contamination level that has been reported to cause adverse effects in experimental animals (Schothorst and van Egmond 2004). In 10 the United States, a survey of cereal grains and ready-to-eat cereal products revealed a 50% positive rate with DON values ranging from 1.2 to 19 ppm (Abouzied et al. 1991) thus confirming DON’s prevalence and the potential for human exposure. Figure 1.1 The chemical structure of deoxynivalenol. 11 DON Toxicology Human Exposure In order for DON to adversely affect the human population, exposure must occur. Evaluation of human exposure to DON has been performed through food surveys and through an established biomarker of exposure for DON, urinary DON and DONglucuronide. In humans, acute DON exposure results in symptoms such as nausea, vomiting, diarrhea and gastrointestinal pain, symptoms often occurring within 30 minutes of exposure. For example, in China between 1961 – 1985 there were 35 reported outbreaks affecting 7818 victims who reported these symptoms after consuming Fusarium infected grain (Canady et al. 2001). Assuming 560 g grain consumption per day and 50 kg body weight, an outbreak in Xingtai province resulted in DON exposure up to 1 mg/kg bw (Canady et al. 2001), a dose known to cause vomiting and anorexia in experimental animals. Using urinary DON and its metabolites, Turner and colleagues demonstrated that 94% of adults from the United Kingdom were exposed to DON and exposure was reduced to 51% upon implementation of a wheat-restricted diet (Turner et al. 2010). In fact, the major source for DON in the diet is wheat with humans exposed to 0.77 – 2.4 ug/kg bw/day depending on diet (Canady et al. 2001). A tolerable daily intake (TDI) of 1 µg/kg bw/day was established in Europe from a two-year murine study showing no significant weight suppression at dietary concentrations of 1 ppm DON (Schothorst and van Egmond 2004). Of those humans exposed, children are of highest concern for DON’s adverse effects because of their 12 high level of grain intake relative to body weight. It has been estimated that 20% of the one-year old children in The Netherlands were exposed to more than twice the TDI (Pieters et al. 2002). Therefore, the potential for DON to cause growth retardation in developing children is the primary concern surrounding DON exposure amounts. The Use Of Experimental Animals to Assess DON Toxicity Due to lack of epidemiological data for DON’s adverse effects in humans, experimental animal models are often used to evaluate the acute, sub-chronic and chronic health effects of both high- and low-level exposure to DON. In experimental animals, acute oral high-dose DON administration resulted in detectable DON tissue concentrations within 5 min and peak plasma DON concentrations within 30 min (Pestka et al. 2008). Upon acute exposure, DON results in robust proinflammatory cytokine induction, diarrhea, gut motility alterations, anorexia and vomiting in susceptible species within 2 hr post exposure while sub-chronic and chronic low-level DON exposure results in weight suppression and dysregulation of the growth hormone axis (Pestka 2010b). Again, of these adverse long-term effects, weight suppression is of highest concern because children are exceptionally susceptible (Canady et al. 2001). Child growth is measured using weight for height (WHZ), weight or age (WAZ) or height for age (HAZ) (WHO 2008). Therefore, weight serves as one parameter of growth; thus DON-induced weight suppression and growth suppression are synonymous when described in this dissertation. 13 Reported Mechanisms of DON-Induced Weight Suppression In the United States, nearly $2,000,000 a year is lost due to DON’s negative effects on livestock (Richard et al. 2003). One of the most common adverse effects of DON contributing to economic loss in livestock is decreased weight gain. Indeed, dietary DON has been extensively linked to weight suppression of young and mature livestock and experimental animals (Table 1.1 and 1.2). In most reported cases, DON-induced weight suppression has been attributed to feed refusal (Table 1.1 and 1.2). Exposed animals consume less food over lengths of time leading to suppressed or decreased weight. Food and weight changes caused by DON differ among species, sex, age, diet type, dose and method of exposure. For example, pigs are the most sensitive species with DON impacting weight gain at 0.06 ppm dietary DON (Canady et al. 2001) while ruminant animals and poultry are less sensitive with no changes in food intake or weight observed at 5.6 (Harvey et al. 1986) and 20 ppm dietary DON, (Morris et al. 1999) respectively. Lack of significant food and weight changes from DON in ruminant animals and poultry is likely due to the efficient metabolism of DON to de-epoxy DON by microorganisms in the ruminant and gastrointestinal tract, respectively (Swanson et al. 1987; Young et al. 2007). Relationship of Weight Suppression to Anorexia Most of the of non-ruminant animal studies where food intake was measured suggest that DON caused anorexia in mature (Table 1.1) and growing animals (Table 1.2) over short-term and long-term depending on the method of DON exposure (Awad et al. 2006; Drochner et al. 2004; Kubena and Harvey 1988; Trenholm et al. 1985). Long-term DON-induced anorexia could result in weight decreases by disrupting the 14 balance between calories consumed and calories used leading to a long-term energy deficit (Berthould and Seeley 1999). Long-term dietary DON in pigs has been reported to cause vomiting, anorexia and weight suppression as doses as low as 0.8, 0.6 and 0.06 ppm DON, respectively (Canady et al. 2001). Table 1.1 The reported no observed adverse effect level (NOAEL) for feeding studies using weight suppression and anorexia as the adverse effect in growing DON-exposed experimental animals. Author Year Species Diet Type Young, LG et al. 1983 pig natural 0.14 NOAEL (ppm) Food Intake 0.14 Forsell, JH et al. 1986 mouse purified 0.5 10 8 wk Harvey, RB et al. 1986 sheep natural 5.6* 5.6* 4 wk Kubena, LF et al. 1988 chicken natural 18* 18* 12 wk Bergsjo, B et al. 1992 natural 1 2 8 wk purified 1 1 52 wk purified 1 5 52 wk purified 20* 20* 3 wk NOAEL (ppm) Weight Study Length 3 wk Morris, CM et al. 1999 pig mouse male mousefemale turkey Drochner, W et al. 2004 pig purified 1.2* 1.2* 8 wk Accensi, F et al. 2006 pig natural 0.84* 0.84* 4 wk Iverson, F et al. 1992 * indicates only dose evaluated part per million (ppm) 15 Table 1.2 NOAELS for feeding studies using weight suppression and anorexia as the adverse effect in mature DONexposed experimental animals. Author Year Species Diet Type NOAEL (ppm) Weight Trenholm, HL et al. 1985 Arnold, DL et al. 1986 Hughes, DM et al. 1999 Awad, WA et al. Amuzie, CJ et al. 2006 2011 cow rat male rat female cat dog chicken mouse natural purified purified natural natural purified purified 1.5* 0.5 0 ND ND 10* 2 * indicates only dose evaluated ND = Not determined 16 NOAEL (ppm) Food Intake 1.5* ND ND 8 8 10* ND Study Length 3 wk 9 wk 9 wk 2 wk 2 wk 6 wk 8 wk In a 52 wk rodent study, the no observed adverse effect level (NOAEL) for weight suppression and anorexia in female mice was 1 and 10 ppm, respectively, while male mice were more sensitive with the NOAELS of 1 and 5 ppm, respectively (Iverson et al. 1995). Similar to the aforementioned study, Forsell and coworkers demonstrated weight suppression was a more sensitive marker of DON’s effects than anorexia (Forsell et al. 1986). Here, the NOAEL for weight suppression was 0.5 ppm, while the NOAEL for anorexia was 10 ppm in female mice given dietary DON for 8 wk. In conclusion, anorexia caused by DON is a consistent phenomenon associated with DON-induced weight suppression. Previous experiments have shown short-term anorexia caused by oral gavage and intraperitoneal (ip.) injection of DON was transient, lasting only hours depending on the dose (Flannery et al. 2011; Girardet et al. 2011b). Furthermore, full food intake compensation was observed in DON-exposed animals within 16 hr leading to acute DON-induced anorexia having no effect on weight suppression in those experiments. However, long-term regulation of food intake can be expressed in short-term events that lead to an overall pattern of food intake and weight changes (Berthould and Seeley 1999). Thus, discrete, transient episodes of anorexia could lead to weight loss if constant overlapping exposures occur. Relationship of Weight Suppression to Growth Hormone Dysregulation Currently, discrepancies in scientific literature exist between the dietary DON dose that causes decreased weight and the dose that causes decreased food intake. For example, Iverson and colleagues determined that female mice given varying 17 concentrations of dietary DON exhibited significantly suppressed weight at 5 ppm, but food intake was not significantly depressed up to 10 ppm (Iverson et al. 1995). Furthermore, mice fed DON for 8 wk exhibited significant weight depression at 2 ppm or greater but only significant feed refusal at 25 ppm dietary DON (Forsell et al. 1986). These data suggest another mechanism in addition to anorexia likely contributes to DON-induced weight suppression. An alternative mechanism by which DON is hypothesized to decrease weight gain is through reductions of the growth factor insulin-like growth factor acid-labile subunit (IGFALS) via excessive DON-induced proinflammatory cytokine induction. Invivo DON targets the macrophage through ‘ribotoxic stress response’ leading to the robust induction of proinflammatory cytokines such as interleukin-1β (IL-1β), interleukin6 (IL-6) and tumor necrosis factor alpha (TNF-α) (Pestka 2010a). Upon gavage of 12.5 mg/kg bw DON, mice exhibited significant increases in plasma IL-1β, IL-6 and TNF-α, which peaked 2 h after administration but returned to basal levels by 4 hr (Amuzie et al. 2009). Furthermore, increased mRNA expression of these three cytokines has been reported in the spleen, liver and lung (Azcona-Olivera et al. 1995; Zhou et al. 1997). Upregulation of IL-1β, IL-6 and TNF-α has been extensively linked to weight suppression in experimental animals (Borish and Steinke 2003). Overexpression of IL-6 in mice resulted in a 50 – 70% growth reduction as compared to control animals (De Benedetti et al. 1997). Sub-lethal doses of TNF-α have been linked to weight loss in mice (Tracey et al. 1988), while IL-1β knock-out mice were resistant to weight loss caused by turpentine induced inflammation and sickness (Leon et al. 1996). 18 One process by which proinflammatory cytokines could lead to decreased weight is through suppressor of cytokine signaling (SOCS) impairment of growth hormone signaling. SOCS proteins serve the body to reduce and prevent sickness and organ damage caused by undue cytokine responses (Starr et al. 1997). Specifically, SOCS3 was shown to impair growth hormone signaling by reducing growth hormone-induced transcription of Igfals in primary hepatic liver cells (Boisclair et al. 2000). IGFALS is an important binding partner to insulin-like growth factor - 1 (IGF1) and without its binding partner, plasma IGF1 is diminished and unable to reach target tissues to have its growth effects (Dai and Baxter 1994). Indeed, IGFALS has been shown to be critical for proper growth in both humans and experimental animals (Domene et al. 2010; FofanovaGambetti et al. 2010; Heath et al. 2008; Ueki et al. 2009). Mice orally gavaged with 12.5 mg/kg bw DON (Amuzie and Pestka 2010; Amuzie et al. 2009) demonstrated SOCS3 impairment of IGFALS and therefore may serve as an alternative mechanism by which DON decreases growth. It should be noted that another common effect of DON, anorexia, also reduces IGFALS (Fukuda et al. 1999; Oster et al. 1996). Future research needs to consider whether DON causes plasma IGFALS reduction directly or whether plasma IGFALS is reduced from anorexia caused by DON. The specificity of plasma IGFALS reduction to DON is an important consideration for fully validating the hypothesis that DON causes weight suppression via directly targeting the growth hormone system. 19 Mechanisms of DON-Induced Anorexia Contribution of Taste to DON-Induced Anorexia Since DON is a toxin, and likely tastes bitter (Glendinning 2007), animals could refuse DON-contaminated food because of palatability. However, when given a choice between uncontaminated chow diet and chow containing 8 ppm DON, rats did not show a preference for consuming uncontaminated chow suggesting palatability did not contribute to DON consumption (Clark et al. 1987). Given that humans and animals have differing sensitivities to taste, humans might not exhibit the same refusal response observed in DON-exposed animals (Wooding et al. 2006). Furthermore, animals exposed to DON via intraperitoneal (ip.) injection and oral gavage consistently exhibited acute, significant feed refusal suggesting that anorexia caused by DON was not solely due to taste (Table 1.3) (Flannery et al. 2012). Typically bitter taste is thought of as sensory perception from the oral cavity; however, the gastrointestinal tract also contains bitter taste receptors, termed T2Rs (Chandrashekar et al. 2000; Wu et al. 2002). T2Rs are G-coupled protein receptors that upon activation serve an organism to decrease ingestion of noxious and potentially poisonous substances (Ueda et al. 2003). T2R activation is thought to decrease food intake via gut satiety hormone receptor activation of neurons in the nucleus tractus solitarius (NTS), a substructure in the brainstem that frequently communicates with the hypothalamus (Hao et al. 2008). Therefore, it is plausible that upon injection or oral gavage of DON, DON reaches the gut and activates bitter taste receptors, though no evidence for this conjecture currently exists. 20 Contribution of Proinflammatory Cytokines to DON-induced Anorexia One of DON’s initial adverse physiological effects that may be linked to anorexia in experimental animals is robust generation of proinflammatory cytokines, more specifically IL-1β, IL-6 and TNF-α. Anorexia is one reported mechanism by which these three cytokines cause weight suppression. For example, both central and peripheral injection of IL-1β and TNF-α caused anorexia in experimental animals as well as central administration of IL-6 (Johnson 1998). Despite the implication of IL-1β, TNF-α and IL-6 in weight loss and anorexia, TNF-α and IL-6 knockout mice did not display any attenuation of feed refusal or weight suppression upon dietary DON concentrations of 10 ppm (Pestka and Zhou 2000, 2002) suggesting these proinflammatory cytokines might not have a critical role in DON-induced feed refusal and weight changes. A previous study has also demonstrated that TNF-α mRNA expression in the numerous tissues evaluated was not increased until 60 min post DON exposure, though significant feed refusal has been reported to occur as early as 30 min (Flannery et al. 2012; Pestka and Amuzie 2008). These data suggest that while increased proinflammatory cytokines could be responsible for longer term feed refusal, they may not be responsible for DON’s initial anorectic response. Contribution of 5-HT to DON-induced Anorexia The rapidity of food intake changes and gastrointestinal distress caused by DON would suggest a biological mechanism(s) that can affect the body immediately. Gastrointestinal hormones are peptides or chemicals that are rapidly released and distributed to both the central nervous system (CNS) and peripheral nervous system (PNS) in response to external and internal stimuli. The majority of scientific 21 investigations into DON-induced hormone release have focused on 5hydroxytryptamine (5-HT; serotonin) as the candidate hormone for animals’ rapid anorexic response (Prelusky 1993, 1994; Prelusky et al. 1997). 5-HT is released from enterochromaffin cells in the gut upon ingestion of food or noxious substances. When released, 5-HT acts within the gastrointestinal tract and CNS to decrease food intake (Kidd et al. 2008; Simon and Ternaux 1990). Despite its mechanistic link to decreases in food intake, investigators have failed to detect increased plasma 5-HT levels after intragastric or intravenous administration of 100 µg/kg bw or 300 µg/kg bw DON to pigs, respectively (Prelusky 1994). However, a chemical metabolite of 5-HT, 5 – hydroxyindoleacetic acid (5-HIAA), was increased in cerebral spinal fluid (CSF) of pigs upon intragastric and intravenous low-level DON administration suggesting serotonergic signaling within central nervous system may play a role in DON-induced food intake changes (Prelusky 1993). Conversely, radiolabeled DON exhibited minimal affinity for 5-HT receptors in pig brain slices (Prelusky 1996), indicating DON could not act directly on central 5-HT receptors. Despite earlier negative results, Prelusky and coworkers were able to attenuate DON-induced anorexia in mice using the 5-HT -2 receptor antagonist cyproheptadine (Prelusky et al. 1997). It should be noted that during these experiments cyproheptadine alone increased food intake indicating this inhibitor may have inhibited basal action of 5HT on 5HT -2C receptors. Additionally, cyproheptadine also antagonizes the receptors histamine -1, dopamine - 3, 5-HT- 2A, -2B and muscarinic acetylcholine – 1 through 5, implying the inhibition of DON-induced anorexia may have occurred through these receptors (Bonhaus et al. 1997; Eltze et al. 1989; Moguilevsky et al. 1994; Peroutka 22 1988; Stanton et al. 1993; Toll et al. 1998). Thus, more specific receptor inhibitors should be utilized to decipher the role of different 5-HT receptor subtypes on DONinduced anorexia. Central Nervous System Control of Food Intake Hypothalamic Signaling The hypothalamus is the area within the brain most often associated with the control of food intake. It is centrally located atop the brainstem with most neuronal inputs connected to the paraventricular nucleus (PVN) which originate from nucleus tractus solitarius (NTS) of the brainstem. Within the arcuate nucleus (ARC), a structure neuronally located within the hypothalamus, two opposite neuronal sets communicate to stimulate or halt food intake. One neuronal group is termed orexigenic because it contains proteins (neuropeptide Y, NPY; agouti-related protein, AgRP) that increase food intake and the other set termed anorexigenic because these proteins (proopiomelanocortin, POMC; α-melanocyte stimulating hormone, α-MSH; cocaineamphetamine transcript, CART) that decrease food intake (Schwartz et al. 2000). Other structures of the hypothalamus such as the perifornical area (PFA) and the lateral hypothalamic area (LHA) are thought to control food intake as well because these areas exhibit connectivity to orexigenic and anorexigenic expressing neurons (Schwartz et al. 2000). There are many signaling mechanisms by which orexigenic and anorexigenic peptides exert control over food intake. For example, POMC is an agonist of the melanocortin – 4 receptor (MC4R), of which leads to decreased food intake via meal termination (Adan et al. 2006). AgRP serves as an inverse agonist of MCR4 leading to 23 an increase in food intake upon MC4R activation (Morton et al. 2006). The balance between NPY, AgRP, POMC, α-MSH and CART within the hypothalamus occurs via brain penetrable gut satiety hormones and via peripheral neuronal signals relayed by the vagus nerve (Fig 1.2). Interestingly, mice orally gavaged with 12.5 mg/kg bw DON exhibited increased mRNA expression of POMC, CART and MCR4 in the hypothalamus (Girardet et al. 2011b) indicating this could be a mechanism by which DON acts to decrease food intake. Furthermore, direct DON injection into the brain at 0.1 mg/kg bw, a dose lower than that which decreases food intake by peripheral injection, significantly decreased food intake, thus confirming DON’s capacity to act centrally to decrease food intake (Girardet et al. 2011b). However, the mechanisms by which DON changes hypothalamic balance of orexigenic and anorexigenic signaling proteins remain unknown. Vagus Nerve One process by which satiety signals are transferred from the gut to the brain is through the vagus nerve. The vagus nerve is the tenth cranial nerve that contains primarily afferent sensory neurons and runs from the gastrointestinal tract, more specifically the stomach, to its termination point in the brainstem, the NTS. Once mechanoreceptors and satiety hormone receptors on the vagus nerve are activated, neurons within the NTS are activated. Subsequently, these NTS neurons communicate with the hypothalamus to change food intake by altering the balance of orexigenic and anorexigenic peptides (Berthoud 2008; Kakei et al. 2002; Phillips and Powley 2000). 24 Figure 1.2 Simplistic representation of satiety signaling upon food consumption. Upon food consumption, gut satiety hormones are released from the gastrointestinal tract. These satiety signals reduce food intake by altering hypothalamic peptides within the arcuate nucleus to favor increased anorexigenic peptides and/or decreased orexigenic peptides. Satiety hormone signaling is transmitted to the hypothalamus via the peripheral vagus-NTS-hypotha hypothalamic pathway or centrally via the bloodstreambloodstream circumventricular organ (eg. Area postrema, median eminence) – NTS/ARC pathway. For interpretation of the references to color in this and all other figures, the reader is referred to the electronic version of this dissertation. 25 Girardet and investigators have demonstrated in mice that upon oral gavage, DON increases c-fos expression, a marker of neuronal activation, in the NTS (Girardet et al. 2011b). Interestingly, upon cervical vagotomy, DON-induced activation of c-fos expression went unaffected. These results, suggest the vagus nerve may not be important for DON-induced food intake changes, though food intake was not measured in these mice (Girardet et al. 2011b). 5-HT Based on previously performed studies, DON administration does not lead to a measurable release of 5-HT from enterochromaffin (EC) cells with in the GI tract; however, DON may still affect serotonergic signaling within the brain. Serotonergic neurons are mostly located within the raphe nuclei of the brain, but have ascending and descending projections to many areas of the brain including the hypothalamus (Lam et al. 2009). To affect food intake, 5-HT is released from presynaptic neuronal terminals and next activates the 5-HT – 2C and 5HT – 1B receptors located on postsynaptic neuron terminals (Lam et al. 2010). Activation of these receptors leads to increased anorexigenic neuronal signaling (POMC) and decreased orexigenic signaling (AgRP) through the MC4R receptor (Heisler et al. 2006) (Lam et al. 2008). Gastrointestinal Satiety Hormone Release: Cholecystokinin and Peptide YY Enteroendocrine cells located inside the gastrointestinal tract release numerous hormones (Table 1.3) that serve to control food intake including ghrelin, which increases food intake and cholecystokinin (CCK), peptide YY (PYY), glucagon-like peptide-1 and others, which decrease food intake. CCK and PYY are primary hormone candidates 26 potentially contributing to DON-induced anorexia because 1) they have been extensively linked to decreases in food intake, 2) they are released from the GI tract, a primary target of DON (Kolf-Clauw et al. 2009; Pinton et al. 2010), 3) release, timing and duration of these two hormones upon food consumption correspond to the timing and duration of DON’s effects on food intake (Flannery et al. 2011; Moran et al. 2005)and 4) both hormones shift the balance of hypothalamic anorexigenic and orexigenic peptides to favor decreased food intake (Challis et al. 2003; de Lartigue et al. 2007). Cholecystokinin Cholecystokinin octapeptide (CCK) is released from I-cells within the small intestine in response to nutrient intake and it serves to decrease amount and duration of food consumption (McLaughlin et al. 1983). In humans consuming a fatty meal, CCK peaked at 45 min and was back to baseline levels by 105 min indicating the rapidity by which CCK has its satiating effects (Reeder et al. 1973). CCK acts through peripheral mechanisms to cause its satiety effects, which is evidenced by 1) experiments showing that upon IP administration, CCK did not enter the cerebral spinal fluid and 2) results illustrating that a vagotomy attenuated CCK-induced feed refusal in rats (Lorenz and Goldman 1982; Smith et al. 1981; Zhu et al. 1986). Since DON works through the brain, as demonstrated by ICV injection of DON causing feed refusal (Girardet et al. 2011b), a hormone working solely through the vagus nerve such as CCK does, may not be critical for DON-induced feed refusal to occur. 27 Peptide YY As early as 15 min after meal consumption, both peptide YY1-36 and PYY3-36, which serve to terminate food consumption, are released from the L-cells of the ileum and colon (Adrian et al. 1985). Of the two main forms of PYY, PYY3-36 is more effective in decreasing food intake (Chelikani et al. 2005). Therefore, the use of the abbreviation PYY throughout this dissertation will refer specifically to PYY3-36. PYY has been extensively linked to reductions in food intake in experimental animals and humans. For example, ip. injection of PYY significantly decreased food intake and body weight of rats while a PYY infusion in obese humans resulted in a 33% reduction in food intake over 24 h as compared to the control treatment group (Abbott et al. 2005a; Batterham et al. 2003). PYY controls food intake primarily though the Y2 receptor (Y2R) located within the arcuate nucleus of the hypothalamus, brainstem and vagal afferents (Abbott et al. 2005a; Dumont et al. 1995; Koda et al. 2005; Suzuki et al. 2010). Due to the wide distribution of Y2Rs and to the ability of PYY to penetrate the blood brain barrier freely (Nonaka et al. 2003), PYY has the capacity to act both peripherally and centrally to reduce food intake. Peripherally, PYY acts through Y2 receptors on the vagus nerve, which in turn signals satiety through the NTS-hypothalamic pathway (Suzuki et al. 2010). The importance of vagus-NTS-hypothalamic pathway was demonstrated in rats that exhibited an attenuated feed refusal response upon a vagotomy or transsectioning between NTS and the hypothalamus (Abbott et al. 2005a). Upon the PYY signal 28 reaching the hypothalamic arcuate nucleus, hypothalamic mRNA expression of the orexigenic peptide NPY was reported to decrease and expression of the anorexigenic peptide POMC increase, thereby decreasing food intake (Acuna-Goycolea and van den Pol 2005; Challis et al. 2003). Because PYY penetrates the BBB, peripheral administration can result in this hormone reaching the brainstem to decrease food intake. The ability of PYY to signal directly within the brain occurs through the circumventricular organs, more specifically the median eminence, which has access to the hypothalamus and the area postrema, which has access to the NTS (Suzuki et al. 2010). Central administration of PYY to the arcuate nucleus decreased food intake in mice, an effect which likely occurred from activation of POMC neurons, increased αMSH and decreased NPY expression (Batterham et al. 2002). In sum, PYY is a candidate hormone by which DON may have its feed-refusal effects, because PYY acts both centrally and peripherally to decrease food intake. 29 Table 1.3 Hormones that control food intake. This list is not exhaustive and sources of this information came from (Bado et al. 1998; Chance et al. 1993; Cummings and Overduin 2007). Effect on Food Intake Peripheral Location Receptor of Action for Food Intake Enterochromaffin cells, GI tract 5-HT- 1A, -1B, -2C Amylin β-cells, pancreas Calcitonin, ReceptorActivity Modifying Protein Cholecystokinin (CCK) I cells, GI tract CCK1 Glucagon-like Peptide -1 (GLP-1) L cells, GI tract GLP1 Leptin Adipocytes, adipose P/D1 cells, stomach Leptin Pancreatic Polypeptide (PP) F cells, pancreas NPY4, NPYY5 Peptide YY (PYY) L cells, GI tract oral cavity NPY2 Ghrelin P/D1 cells, stomach, GI tract Growth hormone secretagogue Hormone 5-HT 30 Alternative Hypothesis for DON-induced Anorexia Food intake is a complex behavior involving physiological, emotional, social and environmental factors. Biologically, the gastrointestinal tract and the brain, more specifically the hypothalamus, work together to control beginning, duration and end of food intake. An alternative hypothesis for DON-induced anorexia is that DON leads animals to feel satiated due to gut satiety hormone release and the action of these hormones within the CNS (Fig. 1.2), thus leading to decreased food consumption. This hypothesis is evidenced by 1) DON causes the intestinal tract to enter a ‘fed pattern’ of motility, a pattern which is characteristic upon food consumption (Fioramonti et al. 1993; Krantis and Durst 2001a), 2) animals capable of vomiting have experienced decreased food intake without vomiting (Prelusky 1997), and 3) the rapidity and duration of food intake inhibition by DON mimics meal consumption (Flannery et al. 2011). Too much food intake could lead to feelings of bloating, nausea and even vomiting, feelings associated with DON-consumption in humans (Fig. 1.3). These ill feelings are also associated with administration of the gut hormone peptide YY (PYY). For example, in fasted male humans, the average PYY plasma level was 126 pg/mL and upon meal consumption raised to 190 pg/mL (Degen et al. 2005). However, when an IV infusion of PYY was administered at 0.4 pmol/kg/min, plasma levels rose to 325 pg/mL with subjects experiencing fullness, abdominal discomfort and nausea (Degen et al. 2005). With plasma levels raised to 550 pg/mL due to a PYY IV infusion of 0.8 pmol/kg/min, one subject experienced vomiting (Degen et al. 2005). Indeed, PYY was classified as the most powerful emetic peptide known based from experiments in dogs (Harding and McDonald 1989). 31 Figure 1.3 Symptoms experienced in humans upon consuming increasing amounts of food or given increasing amounts of PYY 3-36. Bloating, nausea and emesis have been reported in humans u upon increasing DON exposure. 32 Rationale for Proposed Mechanisms of DON-Induced Anorexia Growing children are considered particularly susceptible to DON’s negative weight effects and as a result, the European TDI (1 µg/kg bw/d) was established based on the NOAEL for growth suppression in mice over a 2-year period. Though DON’s weight effects are mostly attributed to its capacity to cause anorexia in experimental animals, the mechanisms by which DON triggers anorexia are not well understood. This lack of knowledge hinders complete risk assessment of this mycotoxin in food products and also delays any potential mitigation efforts for DON’s growth effects. Furthermore, understanding how DON causes anorexia may give insight into the mechanisms by which DON causes acute gastroenteritis symptoms in humans. Therefore, understanding how DON causes anorexia is critical for comprehensive risk assessment of DON. Many complicated biological processes control food intake, though it primarily involves the GI tract and brain working in conjunction. More specifically, the gut communicates about the composition and amount of food to the hypothalamic center of the brain, a signal that then alters the balance of anorexigenic and orexigenic signaling peptides to decrease food intake. The toxin DON causes the GI tract to enter the ‘fed pattern’ of motility and increases anorexigenic signaling within the hypothalamus, which are both characteristics of meal consumption. Thus, DON may cause decreases in food intake because of satiation. The objective of this dissertation is to use a standardized mouse model to determine the mechanisms of DON-induced anorexia and weight suppression for the sake of identifying particularly susceptible populations to DON’s adverse effects and 33 contributing much needed information for comprehensive risk assessment for this mycotoxin. The guiding hypothesis of this dissertation is that DON-induced anorexia results from the peripheral release of gut satiety hormones that work in conjunction with the CNS to reduce food intake (Fig 1.4). This dissertation addresses four specific aims 1) evaluate the contribution of satiety hormones PYY and CCK to DON-induced anorexia, 2) determine the role of the CNS in DON-induced anorexia 3) evaluate the susceptibility of growing mice to dietary DON using plasma IGFALS and weight as endpoints and 4) evaluate the susceptibility of obese mice to dietary DON using weight and anorexia as endpoints. Model Justification For in-vivo studies, the B6C3F1 mouse was chosen. The B6C3F1 mouse is a hybrid of a C3H and a C57BL6 mouse. This strain of mouse has responded favorably in toxicological studies. For example, evaluation of multiple carcinogenicity studies performed by the National Toxicology Program demonstrated that mice survived longer than rats (Haseman et al. 1998). Particularly the female mouse was chosen for these experiments because of their ease of handling over male mice. Since studies performed within this dissertation measure food intake and hormone levels, both which can be influenced by stress (Abbott et al. 2006; Vallee et al. 1996), ease of handling will lead to decreased stress and thus, decreased experimental variability. The purpose of using the mouse was that rodents cannot physically vomit when exposed to DON (Ueno et al. 1987), thereby allowing assessment of anorexia as the main adverse endpoint. 34 Figure 1.4 Potential mechanisms of DON induced weight suppression and anorexia. DON-induced Weight suppression is likely caused from both anorexia and growth hormone dysregulation, with anorexia being the primary cause. Anorexia may be caused by satiety hormones originating in the gut, which signal through the vagus nerve or the hrough brain through the blood. 35 The route of administration in all acute mechanistic exposure studies was ip. injection. This route was chosen because it is more easily reproduced than oral gavage and placing volume in the stomach by gavage could affect gut hormone release. More details describing the acute mouse model are found in Chapter 2. For sub-chronic experiments assessing growth and weight, DON was administered via the diet (chapters 5 and 6). The Institute for Animal Care and Use Committee at Michigan State University approved all animal experiments performed. 36 CHAPTER 2: CHARACTERIZATION OF DEOXYNIVALENOL-INDUCED ANOREXIA USING MOUSE BIOASSAY Data in this chapter has been published in Flannery B.M., Wu, W., and Pestka J.J. Characterization of deoxynivalenol-induced anorexia using mouse bioassay. Food and Chemical Toxicology 2011 Aug;49(8):1863-9. Abstract A short-term mouse model was devised to investigate induction of food refusal by the common foodborne trichothecene deoxynivalenol (DON). DON dose-dependently induced anorexia within 2 h of exposure when administered either by intraperitoneal (ip.) injection or by oral gavage. The no observed adverse effect and lowest observed adverse effect levels in this assay were 0.5 mg/kg bw and 1 mg/kg bw for ip. exposure and 1 mg/kg bw and 2.5 mg/kg bw for oral exposure, respectively. DON’s effects on food intake were transient, lasting up to 3 h at 1 mg/kg bw and up to 6 h at 5 mg/kg bw. Interestingly, a dose-dependent orexigenic response was observed in the 14 h following the initial 2 h food intake measurement. Toxin-treated mice exhibited partial resistance to feed refusal when exposed to DON subsequently after 2 d, but not after 7 d suggesting that this modest tolerance was reversible. The short-term mouse bioassay described here was useful in characterizing DON-induced anorexia and should be applicable to elucidating mechanisms underlying this adverse nutritional effect. 37 Introduction The frequent presence of deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium sp., in cereal grains and processed grain products (Lauren and Smith 2001; Schothorst and van Egmond 2004; Vesonder et al. 1973) is of public health concern worldwide (Pieters et al. 2002; Schothorst and van Egmond 2004; Vesonder and Hesseltine 1980). Current regulatory standards for DON in foods are based on its ability to cause growth suppression- a primary adverse effect observed in experimental animals chronically exposed to the toxin via diet (Amuzie and Pestka 2010; Canady et al. 2001; Forsyth et al. 1977). DON-induced growth suppression results largely in part from its capacity to inhibit food intake (Arnold et al. 1986b; Forsell et al. 1986; Forsyth et al. 1977; Trenholm et al. 1984). The observation that pigs refuse food even when the toxin is infused intraperitoneally suggests that other mechanisms unrelated to palatability are contributory to anorexia evoked by this trichothecene (Prelusky 1997). Notably, several prior research studies have provided evidence supporting a role for neuroendocrine factors such as 5-HT (Fioramonti et al. 1993; Girish et al. 2008; Prelusky 1993; Prelusky and Trenholm 1993). Furthermore, DON rapidly activates the innate immune response evoking release of proinflammatory cytokines that are widely recognized to cause anorexia (Pestka 2010a). To better predict DON’s effects on growth in humans and animals, particularly the young, it is critically important to better understand how this toxin causes anorexia. While investigations of DON-induced feed refusal have typically employed sub-chronic and chronic exposure of mice and rats to the toxin via diet (Forsyth et al. 1977; Pestka 38 and Zhou 2000, 2002; Trenholm et al. 1984) such approaches are largely incompatible with relating transient changes in neuroendocrine and cytokine factor levels in the gut and brain to immediate feed refusal caused by DON because mechanisms of immediate DON-induced feed refusal would occur before feed refusal takes place. A further complication is that mouse and rat feeding studies often involve the use powdered diet which can lead to overestimation of the amount of food consumed because of spillage from a food jar. Even if DON were included in pelleted diet, rodents have the potential to grind brittle food pellets leaving tiny fragments (“orts”) that can cause up to 30 percent overestimation of food intake (Cameron and Speakman 2010). Finally, it has been recently observed that prolonged exposure to DON in a feeding study might cause tolerance through a variety of metabolic and hormonal compensatory mechanisms (Kobayashi-Hattori et al. 2011). Thus, initial feeding responses to this trichothecene might be quite different from those occurring hours or days later. Recent studies in our laboratory have demonstrated that reduced food intake is detectable in mice within the first day that DON is included in an experimental diet (Amuzie et al. 2011; Kobayashi-Hattori et al. 2011) suggesting that it might be feasible to develop a short-term feeding assay amenable to mechanistic exploration. The goal of this research was to test the hypothesis that acute administration of DON to the mouse causes a rapid, measurable and reproducible reduction in food intake. The results presented herein indicate that DON’s anorexigenic effects in this novel model were (1) observable within 2 h, (2) transient, (3) detectable following exposure by intraperitoneal (ip.) injection or oral gavage, and (4) succeeded by both a robust orexigenic response and modest short-lived tolerance to further DON challenge. 39 Materials and Methods Laboratory animals and DON. Mice were maintained according to National Institutes of Health guidelines as overseen by the All University Committee on Animal Use and Care at Michigan State University (Approval No. 01/08-007-00). Female, B6C3F1 mice (10 – 11 wk) were purchased from Charles River Breeding (Portage, MI) were housed singly in polycarbonate cages with aspen bedding sifted to uniform size with a flour sifter. Temperature (21° – 24° and relative humidity (40 – 55%) remained constant and C C) room lights were on a 12 h light (6:00-18:00h) /dark (18:00-6:00h) cycle. Mice were fed pelleted High Fat Diet, (45 kcal% fat diet, Research Diets Inc, New Brunswick, NJ) in 2” high glass jars for 1 wk prior to initiating experiments. HFD was chosen because it is (1) softer than 10 kcal% fat pellet thus facilitating rapid feeding (Ford 1977) and (2) less easily shattered than very high fat diet (60 kcal% fat) making it easier to recover fragments from cage bottom during intake measurements. DON was obtained from Dr. Tony Durst (University of Ottawa, Canada) and its purity verified by H1NMR. For exposure studies, mice received an injection volume of 100 µl of DON dissolved in phosphate buffered saline (PBS) or PBS vehicle. Experimental design. On the day prior to the experiment, mice were distributed into experimental groups based on body weight. On the day of the experiment, mice were fasted from 10:00 h to 18:00 h, but provided free access to water (Fig. 2.1). For dose response 40 studies, mice were treated at initiation of the dark cycle (18:00 h), by (1) ip. injection of 0, 0.1, 0.5, 1.0, or 2.5 mg/kg bw DON (n = 13/gp) in 100 µl using a sterile 27G, 0.5” needle or (2) oral gavage with 0, 0.5, 1.0, 2.5 or 5.0 mg/kg bw DON (n = 8/gp) in 100 µl using a sterile 22G, 1.5” disposable feeding tube (Instech Solomon; Plymouth Meeting, PA). Following DON exposure, the mice were immediately given two pre-weighed food pellets (≈ 7 g). Lights were turned off and mice were allowed to eat for 2 h. At 20:00 h, lights were turned on for 30 m, food was removed from the food jar and combined with shredded food in the sifted bedding on the cage bottom. The combined food was weighed and replaced back into the feed jar until a second intake measurement (16 h) at 10:00 h on day 2. All procedures were performed quickly and quietly to minimize stress in the animals. To determine the duration of DON-induced feed refusal, mice were given an ip. injection of 0, 1 or 5 mg/kg bw DON at 0, 1, 2 or 4 h (n = 4 – 5/gp) just prior to the onset of the dark cycle (18:00 h) (Fig. 4A). The remaining food was measured 2 h later. These equated to total DON exposure times of 2, 3, 4 or 6 h upon the completed food intake measurement. To assess the effect of short-term repeated DON exposures on feed refusal, naïve mice were given an ip. injection of 0, 0.1, 0.5, 1.0 and 2.5 mg/kg bw DON (n = 5/gp) just prior to the onset of the dark cycle (18:00 h). The remaining food was measured 2 h later. 41 Figure 2.1 Experimental design for mouse feed refusal assay Mice are fasted from 10:00 h on Day 1. At 18:00 h, the assay. onset of the dark cycle, mice are treated with DON or PBS vehicle and then provided with food. At 20:00 h lights were turned on for 30 min and food intake was measured an measured again at 10:00 h on Day 2. and 42 Mice were given the same doses 48 h after the previous DON injection and food intake measured after 2 h (exposure 2). 48 h between exposures was chosen to allow for a 1-day rest period, free of DON exposure. This latter protocol was again repeated a second time (exposure 3). To ascertain the effect of intermittent repeated DON exposure on food intake, mice were exposed to DON by ip. injection or oral gavage twice, with intervening 7 d rest periods. Briefly, naïve mice were given an ip. injection of 0, 1.0 and 2.5 mg/kg bw DON (n = 8/gp) and food intake measured. For the second exposure, mice were randomized, given an ip. injection of 0, 1.0 and 2.5 mg/kg DON (n = 8/gp) and 2 h food intake measured. Similar experiments were performed using oral gavage, except mice were exposed to 0, 0.5, 1.0, 2.5 or 5.0 mg/kg bw DON (n = 8/gp). Statistics. All statistics were performed using SigmaPlot 11 (Jandel Scientific; San Rafael, CA) with α= 0.05 and significance established when p < 0.05. The specific statistical tests performed are described in the relevant figure legends. A One-way ANOVA using Dunnett’s Test was used to assess significant differences in food intake between doses and the control. Two-Way ANOVA and Holm-Sidak post hoc tests were employed to determine the effects of dose and time on food intake. Data from one mouse was consistently deemed a statistical outlier using Grubb’s Test for Outliers (www.graphpad.com) and not included in the statistical analyses. 43 Results Ip. and oral exposure to DON causes rapid and transient feed refusal When the effects of ip. injection of DON on food intake were measured using the short-term mouse bioassay (Fig. 2.1), DON was observed to cause significant reduction in food intake within 2 h (Fig. 2.2). Two hours after exposure to 1 and 2.5 mg/kg bw DON, food intake was reduced by 41% and 69%, respectively, whereas doses of 0.1 and 0.5 mg/kg bw DON were without effect. Conversely, in the 14 h following the initial food intake measurement, 1 and 2.5 mg/kg bw DON caused significant increases in food consumption, eating 21% and 26% more than the control, respectively. When cumulative intakes were compared over the entire 16 h period, no differences were observed in the DON-exposed mice, suggesting that the mice effectively compensated for the initial suppression of food intake. Following administration with 2.5 and 5 mg/kg bw DON by oral gavage, food consumption was reduced by 68% and 77%, respectively, while doses of 0.5 and 1.0 mg/kg bw DON had no effect (Fig. 2.3). In the succeeding 14 h, the 2.5 and 5 mg/kg bw DON groups consumed 32% and 19% more than the control mice, respectively. Again, when cumulative intakes were compared over the entire 16 h period, no differences were observed in the DON exposed mice. The duration of DON-induced feed refusal was ascertained by dosing mice with the toxin at various intervals prior to onset of the dark phase and measuring food intake (Fig. 4). At 0 mg/kg bw food intake did not significantly change over time (Fig. 5). At 1 mg/kg bw DON the effects of feed refusal were no longer evident beginning at 3 h post DON exposure, while at 5 mg/kg bw, feed refusal was no longer evident at 6 h. 44 Thus, as time after toxin exposure increased, food intake increased, suggesting again that DON-induced feed refusal was transient. Short-term (2 d) repeated DON exposure evokes partial tolerance to feed refusal effects To assess the effect of short-term repeated DON exposure on feed refusal, mice were exposed to DON three separate times, 2 d apart (Fig. 2.6). Mice exposed to 2.5 mg/kg bw DON developed tolerance to DON-induced feed refusal as exposure number increased; In contrast, the opposite was seen for mice exposed to 0.1 mg/kg bw as food intake decreased over multiple exposures. Intermittent (7d) repeated exposure to DON does not evoke tolerance to feed refusal effects The effects of a longer-term, rest period between ip. injections of DON on food intake were assessed using randomized mice (Fig. 2.7A). There was a modest increase in food intake after the second DON exposure for 0, 1 and 2.5 mg/kg DON group. However, mice exposed to 1 and 2.5 mg/kg bw exhibited significantly reduced food intake capacity. For oral exposure, as with ip. injection, orally exposed mice significantly reduced food intake as DON dose was increased, independently of exposure number (Fig. 2.7B). For both exposure methods, DON - treated mice significantly decreased food intake upon the second DON exposure. Thus DON-induced feed refusal persisted after a 7 d rest period. 45 Figure 2.2 Intraperitoneal DON exposure in mice induces an early anorexigenic response followed by a delayed orexigenic response Mice were given an ip. injection of response. DON immediately before the dark cycle. Food intake was measured at 2 h and 16 h post injection time and graphically depicted. Data are mean ± SEM (n = 13/gp). Asterisks indicate statistically significant differences in food consumption as compared to the control (p < 0.05). 46 Figure 2.3 Oral DON exposure in mice induces an early anorexigenic response followed by a delayed orexigenic response. Mice were orally gavaged with DON immediately before the dark cycle. Food intake was m measured at 2 h and 16 h post exposure and graphically depicted. Data are mean ± SEM (n = 8/gp). Asterisks indicate statistically significant differences in food consumption compared to the control (p > 0.05). ferences 47 DON-induced Figure 2.4 Experimental design for determining the duration of DON induced feed refusal. Mice were given an ip. injection of DON either 0, 1, 2 or 4 hours before the dark cycle as indicated by gray inverted arrows. Food intake was measured 2 hr after the injection for a total exposure time of 2, 3, 4 and 6 h. or 48 induced transient. Figure 2.5 DON-induced feed refusal is transient At 1 mg/kg bw DON feed refusal is attenuated within 3 h and at 5 mg/kg bw DON, feed refusal is attenuated within 6 h after exposure. Data are mean ± SEM (n = 4 5/gp). Symbols: *Food consumption for a given ean 4–5/gp). treatment is significantly different from treatment control at specified time (p < 0.05); ŧ Food consumption for a given time is significantly different from 2 hr exposure time at specified dose (p < 0.05). 49 term DONFigure 2.6 Short-term repeated DON exposure results in partial tolerance to DON induced feed refusal. Mice were given an ip. injection of DON immediately before the dark cycle. Food intake was measured 2 h after injection time. This was repeated twice, 48 h apart. Data are mean ± SEM (n = 5/gp). Symbols: *Food consumption for a given treatment is significantly different from treatment control at specified exposure number control (p < 0.05). ŧFood consumption for a given exposure number is significantly different from Food the first exposure at the specified dose (p < 0.05). 50 Figure 2.7 DON-induced feed refusal tolerance is short lived. Mice were treated with induced DON via (A) ip. injection and (B) oral gavage and food intake measured after 2 h. After randomization, mice were exposed to DON, 7 d later. Data are mean ± SEM (n = 8/gp). Symbols are as described in Figure 2.6. 51 Discussion The results presented herein demonstrate, for the first time, the efficacy of using an acute mouse bioassay to measure DON-induced anorexia and further show that this response is rapid and transient. Specifically, DON-induced feed refusal occurred within 2 h and the duration of the effect was dose-dependent. Mice exposed to DON exhibited increased food intake in the 14 h following the initial food intake measurement thus confirming the transient and reversible nature of DON’s feed refusal effects. The initial 2 h decrease in food intake may initiate compensatory mechanisms that increase food intake in the 2 – 16 h following DON exposure. Fasted rats compensate for lack of food intake by decreasing the anorexic hormone leptin and by increasing hypothalamic mRNA expression of the orexigenic peptide neuropeptide Y (NPY) (Vuagnat et al. 1998). Recently, we have shown that upon subchronic DON exposure, a decrease in both food intake and weight is correlated to a decrease in serum leptin levels and increased hypothalamic mRNA expression levels of the orexigenic peptide, agouti- related protein (AgRP) (Kobayashi-Hattori et al. 2011). Therefore, it is possible that the compensatory increase in food intake exhibited by mice acutely exposed to DON, may be due to changes in leptin and mRNA expression of orexigenic peptides in the hypothalamus. In addition to exposing mice to DON by ip. injection, oral gavage was employed as a complementary method because humans are primarily exposed to DON though consumption of food. It was therefore notable that the NOAEL for DON was 0.5 and 1.0 mg/kg bw following ip. and oral exposure, respectively. Linear regression analysis revealed that ip. injection was slightly more effective than gavage at causing 50% feed 52 refusal (Fig. 2.8). A 50% reduction in food intake occurred at 1.66 mg/kg bw DON (r2 = 0.88; p < 0.05) for ip. administration and 1.83 mg/kg bw DON for oral administration (r2 = 0.946; p < 0.05). Consistent with our findings here, the minimum emetic dose in swine for DON ip. injection is lower than the minimum emetic dose for the toxin when administered by oral gavage (Forsell et al. 1987; Forsyth et al. 1977). Not only might these differences relate to the different bioavailabilities of the toxin when administered by the two different methods, but also to the sensitivity of the pig versus the mouse to DON exposure and the respective endpoint measurements (emesis versus feed refusal). Humans are exposed to DON through a variety of grain products (Turner et al. 2008b). Since people consume grain products regularly throughout a lifetime, they are likely to be exposed to DON multiple times. Our data suggest that multiple exposures to DON 48 hr apart resulted in partial tolerance to DON-induced feed at the highest dose tested. In contrast, at the lowest DON dose examined, mice exhibited increased sensitivity to DON-induced feed refusal. Thus mice became more or less susceptible to DON’s feed refusal effects depending on dose and exposure number. Since these findings might have implications in assessing the risk posed to humans by DON in food, further research is needed to clarify the effects of dose and exposure number on DONinduced anorexia. 53 Figure 2.8 Comparison of feed refusal induction by intraperitoneal (ip.) injection and oral gavage (gav) of DON. To determine the dose at which mice exhibit 50% reduction in food intake relative to the control, a linear regression was calculated for ip. injection was (Fig. 2.2) (Fig. 2.2) (y = 94.852 – 26.961x; r2 = 0.94; p < 0.05) and for gavage (Fig. 2.3) ) (y = 94.741 – 24.496x; r2 = 0.88 p < 0.05) with x being dose and y being percent food 0.88; ) consumption relative to the control. 54 Although the mechanisms for DON’s anorectic effects are not well-understood, they potentially involve altered neuroendocrine and cytokine signaling within the gutbrain axis. The brain, particularly the hindbrain (including the area postrema) and hypothalamus, can signal immediate changes in food intake (Schwartz et al. 2000). DON exposure has also been reported to cause changes in brain neurochemistry in turkeys, pigs and chickens (Girish et al. 2008; Swamy et al. 2004). Moreover, DONinduced conditioned taste aversion to saccharin in rats can be attenuated by area postrema ablation, suggesting the role of the hindbrain in DON-induced food intake changes (Ossenkopp et al. 1994). Indeed, DON is distributed to brain tissue rapidly (5 min) following oral exposure in the mouse (Pestka et al. 2008) and it might be speculated that this could contribute to altered neuronal signaling that evokes anorexia. DON is known to rapidly induce in mice the systemic production of interleukin-1β, interleukin-6 and tumor necrosis factor-α (Amuzie et al. 2008; Amuzie et al. 2009; Azcona-Olivera et al. 1995). These proinflammatory cytokines reduce food intake when administered into the brain (Lawrence and Rothwell 2001; Plata-Salaman et al. 1988; Plata-Salaman et al. 1996; Sonti et al. 1996; Wallenius et al. 2002). Since circulating proinflammatory cytokines can reach the brain (Banks et al. 1994) it could be speculated that the systemic induction of proinflammatory cytokines by DON leads increased cytokine levels in the brain and subsequent feed refusal. In addition to the ability of proinflammatory cytokines to cause anorexia, increased proinflammatory cytokines can lead to altered growth hormone signaling and growth suppression through the activation of suppressors of cytokine signaling 3 (SOCS3) (Boisclair et al. 2000). Acute exposure to DON has been shown to increase 55 hepatic SOCS3 mRNA expression and decrease hepatic mRNA expression of the binding partner of insulin-like growth factor 1 (IGF-1), insulin-like growth factor acid labile subunit (IGFALS) (Amuzie et al. 2009); though decreases in the plasma levels of IGF-1 and IGFALS have not been seen until 7 d DON exposure (Amuzie and Pestka 2010). DON capacity to decrease food intake may cause decreases in plasma levels of IGF-1 and IGFALS and subsequent growth suppression as it has been well documented that anorexia leads to decreases in plasma IGF-1 and IGFALS (Fukuda et al. 1999; Merimee et al. 1982). 5-HT (5-HT), a neurotransmitter produced primarily in the GI tract, has been implicated in DON-induced feed refusal and vomiting. Increased levels of the 5-HT metabolite, 5-hydroxyindoleacetic acid (5-HIAA) are found in the cerebral spinal fluid of DON-exposed pigs (Prelusky 1993). It was also observed that the 5-HT2 receptor inhibitor, cyproheptadine inhibits DON-induced feed refusal in mice (Prelusky et al. 1997). Interestingly, oral administration of DON causes slowing of gastrointestinal motility in mice and rats, which can be reversed by the 5-HT3 receptor antagonists granisetron and ondansetron (Fioramonti et al. 1993). Although detectable changes in plasma concentrations of 5-HT have not been observed in DON-exposed animals (Prelusky 1994), the potential exists for this neurotransmitter to act locally at the intestinal level by stimulating receptors on vagal afferents, which can signal the hindbrain and ultimately cause changes in appetite. It is further possible that the presence of DON alters oral-sensory properties of a food. In support of this contention, pigs exposed to DON-amended food exhibited greater feed refusal than those given unamended food and administered DON by 56 infusion (i.v.) (Prelusky 1997).This feed refusal seen with DON-contaminated food may be due to an inherent objectionable sensory property of DON being found with in food. In support of this contention, it has been reported that many toxic compounds may taste bitter, though the degree of bitterness does not always relate directly to the degree of toxicity (Glendinning 2007). Even though the route of exposure for DON was ip. injection and oral gavage in experiments described herein, DON might still activate bitter taste receptors (T2Rs) found throughout the GI tract (Wu et al. 2002) and thus cause feed refusal of even uncontaminated food. Although mice and other rodents are incapable of vomiting (Ueno et al. 1987), similarities exist between anorexia induction observed here and emetic responses to DON relative to their rapidity and transient nature. For example pigs vomit within 30 min of being injected or gavaged with DON and the response lasts up 1 h (Forsyth et al. 1977; Pestka et al. 1987; Young et al. 1983). Indeed, even though mice are not capable of vomiting, they have acquired the ability to (1) rapidly develop conditioned taste aversions to toxins, (2) delay gastric emptying and (3) decrease food intake to prevent further delivery of the known emetic stimuli (Andrews and Horn 2006). Thus, DONinduced feed refusal in the mouse, a non-emetic species, might serve as a surrogate for emesis in non-capable species. To summarize, using a short-term (2 h) mouse bioassay, we have demonstrated that DON causes robust, but transient feed refusal followed a delayed orexigenic response. Additionally, while prior toxin exposures partially attenuated anorexia induced by DON, no such tolerance was evident after 7 d rest from toxin exposure. This shortterm model for DON-induced anorexia should be applicable to determining the putative 57 roles of neuroendocrine factors, proinflammatory cytokines and bitter taste receptors in DON-induced feed refusal. Such studies could enable an improved, mechanism-based understanding of the nutritional risks posed to humans and animals from acute and chronic DON exposure. 58 CHAPTER 3: ANOREXIA INDUCTION BY THE TRICHOTHECENE DEOXYNIVALENOL (VOMITOXIN) IS MEDIATED BY THE RELEASE OF THE GUT SATIETY HORMONE PEPTIDE YY (PYY) Data in this chapter has been published in Flannery B.M., Clark E.S. and Pestka J.J. Anorexia induction by the trichothecene deoxynivalenol (vomitoxin) is mediated by the release of the gut satiety hormone peptide YY (PYY). Toxicological Sciences. 2012 Dec;130(2):289-97. Abstract Consumption of deoxynivalenol (DON), a trichothecene mycotoxin known to commonly contaminate grain-based foods, suppresses growth of experimental animals, thus raising concerns over its potential to adversely affect young children. While this growth impairment is believed to result from anorexia, the initiating mechanisms for appetite suppression remain unknown. Here, we tested the hypothesis that DON induces the release of satiety hormones and that this response corresponds to the toxin’s anorectic action. Acute intraperitoneal (ip.) exposure to DON had no effect on plasma glucagonlike peptide-1, leptin, amylin, pancreatic polypeptide, gastric inhibitory peptide or ghrelin, however, the toxin was found to robustly elevate peptide YY (PYY) and cholecystokinin (CCK). Specifically, ip exposure to DON at 1 and 5 mg/kg bw induced PYY by up to 2.5-fold and CCK by up to 4.1-fold. These responses peaked within 15 to 120 min and lasted up to 120 min (CCK) and 240 min (PPY), corresponding with depressed rates of food intake. Direct administration of exogenous PYY or CCK similarly caused reduced food intake. Food intake experiments using the NPY2 receptor antagonist BIIE0246 and the CCK1A receptor antagonist devazepide, individually, suggested that PYY mediated 59 DON-induced anorexia but CCK did not. Orolingual exposure to DON induced plasma PYY and CCK elevation as well as anorexia comparable to that observed for ip exposure. Taken together, these findings suggest that PYY might be one critical mediator of DON-induced anorexia and, ultimately, growth suppression. Introduction Fungal infection of crops in the field and during storage often leads to the production of mycotoxins, harmful secondary metabolites that can cause adverse health consequences in humans and animals. Deoxynivalenol (DON; vomitoxin), a trichothecene mycotoxin produced by Fusarium sp., is of particular public health concern because it contaminates grains with high frequency, is resistant to cooking processes and has been associated with both animal and human illnesses (Pestka 2010b). The mouse and the pig have been most widely used to study DON toxicity. Following oral DON exposure in these species, the toxin is absorbed extremely rapidly and distributed throughout body including the gut (Azcona-Olivera et al. 1995; Pestka et al. 2008; Prelusky et al. 1988). In mice, which are incapable of vomiting, acute DON exposure is associated with anorexia and proinflammatory cytokine induction while subchronic and chronic DON exposure causes growth suppression and weight loss (Canady et al. 2001). In pigs, which have an emetic response, DON elicits the aforementioned effects as well as vomiting (Forsyth et al. 1977; Pestka et al. 1987). Interestingly, the Joint Expert Committee on Food Additives of the WHO and FAO established a human tolerable daily intake (TDI) of 1 µg/kg bw/d for DON based on 60 growth suppression in mice (Canady et al. 2001). This TDI now serves as the basis for current regulatory tolerances for DON in grain-based foods. Although experimental animal studies strongly suggest that DON-induced growth suppression results from reduced food intake (Arnold et al. 1986a; Goyarts et al. 2005; Hughes et al. 1999), the mechanisms by which the toxin causes anorexia still remain poorly understood. The regulation of food intake is multifaceted and reflects both central and peripheral neuroendocrine control mechanisms. Centrally, the balance of orexigenic (eg. Neuropeptide Y [NPY], agouti-related protein [AGRP]) and anorexigenic signaling molecules (eg. pro-opiomelanocortin [POMC], cocaine- and amphetamine-regulated transcript [CART]) produced within hypothalamic neurons are critical for food intake control (Morton et al. 2006). Based on a recent elegant study, it has been established that DON exposure upregulates hypothalamic mRNA expression of signaling molecules CART, POMC and one of the receptors through which they have their action, the melanocortin 4 receptor (MC4R) (Girardet et al. 2011b). However, it remains to be established how DON modulates expression of these anorexigenic signaling molecules. One possible mechanism for the initiation of anorexia induction by DON is through upregulation of proinflammatory genes. DON can stimulate expression of cyclooxgenase-2, microsomal prostaglandin synthase-1, tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), intereukin-6 (IL-6) in many tissues in mice including the brain (Girardet et al. 2011a; Pestka et al. 2008) and our laboratory has established that the underlying mechanism for this upregulation is ribotoxic stress in mononuclear phagocytes (Pestka 2010a). These proinflammatory gene products are well-known to mediate anorexia through classic sickness behavior mechanisms (Kelley et al. 2003) 61 and potentially can impair insulin-like growth factor expression (Amuzie and Pestka 2010). However, the dose thresholds and the kinetics for proinflammatory gene upregulation in vivo (2 h) (Zhou et al. 1997) are not congruent with the rapid initiation of anorexia (Flannery et al. 2011). Moreover, food intake and growth are unaffected by dietary DON in mice deficient in these genes, suggesting that DON might additionally act through other mechanisms different from those associated with endotoxin exposure or infection (Girardet et al. 2011a; Jia et al. 2006; Pestka and Zhou 2000, 2002). An alternative unexplored possibility is that satiety-regulating hormones produced by enteroendocrine cells in the gastrointestinal system might serve as early mediators of DON-induced anorexia. Secretion of satiety hormones represents an important mechanism for peripheral control of the balance of hypothalamic orexigenic and anorexigenic signaling. Although 5-hydroxytryptamine (5-HT; serotonin), which can be produced at the gut level, is one obvious candidate gut hormone, changes in plasma levels of this neurotransmitter were not detectable in DON-exposed pigs (Prelusky 1994). Other possible gut hormones that affect appetite include glucagon-like peptide-1 (GLP-1), leptin, amylin, pancreatic polypeptide (PP), gastric inhibitory peptide (GIP), ghrelin, peptide YY (PYY) and cholecystokinin (CCK). The latter two peptides are particularly attractive candidates for mediating DON’s anorectic actions because the rapidity by which they decrease food intake (Gibbs et al. 1976; Moran et al. 2005) mimics that observed following acute DON exposure (Flannery et al. 2011). PYY is a 36-amino acid protein released from the L-cells of the colon and ileum that signals reductions in food intake by increasing expression of anorexigenic peptides and decreasing orexigenic signaling peptides within the hypothalamus (Challis et al. 2003). 62 CCK is a peptide hormone that is secreted by the small intestine that acts on vagal afferent neurons to increase expression of anorexigenic peptides including CART, though recent research suggests CCK can act directly within the brain as well (Brown et al. 2011; de Lartigue et al. 2007). Thus, DON-induced release of PYY and CCK from the gastrointestinal tract could promote signaling changes observed within the hypothalamus (Girardet et al. 2011b). The purpose of this study was to test the hypothesis that DON induces the release of gut satiety hormones in the mouse and that this response corresponds to the toxin’s anorectic action. The results demonstrate that acute exposure to DON at levels of 1 to 5 mg/kg bw caused release of PYY and CCK and that kinetics of these responses corresponded to onset and duration of feed refusal. While direct administration of both hormones similarly evoked transient anorexia, experiments using receptor inhibitors suggest that PYY might play a more dominant role in DON-induced anorexia. Accordingly, aberrant release of gut satiety hormones might be one critical underlying mechanism for DON-induced anorexia and ultimately growth suppression. Materials and Methods Animals. Naïve female B6C3F1 mice (9 – 11 w) were purchased from Charles River Breeding (Portage, MI) and allowed to acclimate at least 1 wk prior to beginning experiments. During acclimation, mice were adapted to high fat diet (45% kcal from fat; Research Diets Inc., New Brunswick, NJ) and conditioned by frequent handling and sham injections to mimic experimental protocols. Mice were maintained at constant 63 temperature and humidity (21 – 24° and 40 – 55%, r espectively) under 12 h light/dark C cycles with free access to food and water. All experiments performed and were approved by the Institute of Animal Care and Use Committee at Michigan State University and in accordance with the National Institutes of Health guidelines for animal use. DON administration. DON was obtained from Dr. Tony Durst (University of Ottawa, Canada) and purity verified using 1HNMR and carbon and hydrogen elemental analysis (Galbraith Labs, Knoxville, TN). The intraperitoneal (ip) route of DON exposure was used for most experiments to ensure accurate and reproducible delivery of the toxin as well as to minimize handling stress that might artifactually raise plasma hormone concentrations. DON was dissolved at appropriate concentrations in 100 µl phosphate buffered saline (PBS) and administered via ip injection using sterile 31G, ¼ inch syringes. For a comparative study using orolingual exposure, DON was dissolved in 10 µl of PBS and administered to the mouth and tongue using a 20 µl sterile filter pipette tip. DON-induced gut satiety hormone release. For preliminary studies, groups of mice were treated with 2.5 and 10 mg/kg bw DON for 15 and 60 min. For the initial study on PYY and CCK, groups of mice were given an ip. injection of 0, 0.05, 0.25, 1 or 5 mg/kg bw DON and sacrificed at 15 min and 240 min after DON exposure. For kinetic studies, groups of mice were given an ip. injection of 0, 1 or 5 mg/kg bw and sacrificed at 15, 60, 120 and 180 min for 1 mg/kg bw dose and at 30, 120, 240 and 360 min for 5 mg/kg bw DON exposure. The control group was given 64 PBS and sacrificed at 0, 15, 30 and 120 min to confirm that PYY and CCK levels remain stable. These time points were chosen based on data from Flannery et. al. (2011) showing that feed refusal was attenuated by 180 min at 1 mg/kg bw and by 360 min at 5 mg/kg bw DON (Flannery et al. 2011). For ip versus orolingual studies, mice were given 2.5 mg/kg bw DON or PBS and sacrificed at 30 min. For all experiments, mice were euthanized by ip injection with 56 mg/mL bw sodium pentobarbital (100 µl) or 100 mg/ml ketamine (40 µl). Blood was removed from the inferior vena cava using sterile BD syringes with 25 µl of 1% (vol/vol) EDTA (pH 7.5) and placed into EDTA-coated tubes. Plasma was separated by centrifugation at 3500 x g for 10 min at 4° and then frozen in aliquots at -80° until hormone analyses. C C Hormone analyses. Plasma PYY (1-36), GLP-1, leptin, amylin, PP, GIP and ghrelin were measured using a Milliplex® MAP Kit according to manufacturer’s instructions (Mouse Gut Hormone Panel, #MGT-78K; Millipore, St. Louis, MO). Plates were read using a BioRad Bioplex 100 system and data analyzed from a standard curve using Bioplex Manager 5.0 (Bio-Rad Life Science, Hercules, CA). CCK was analyzed using a Cholecystokinin Octapeptide (26-33, non-sulfonated) Extra Sensitive EIA Kit (# EKE069-04; Phoenix Pharmaceuticals Inc., Burlingame, CA). Plate absorbance was read at 450 nm using an ELISA plate reader (Molecular Devices, Menlo Park, CA) and data were analyzed using Softmax software. 65 DON- and hormone- induced anorexia. DON-induced anorexia was assessed in conditioned mice by measuring food intake at 1, 2, 4, 6, and 16 h after ip exposure and 2 h after orolingual exposure under red light conditions according to our previously described protocol (Flannery et al. 2011). For hormone-induced anorexia, PYY (3-36) was purchased from Tocris Biosciences (Ellisville, MO) and CCK (23-33, sulfonated) from Sigma-Aldrich (St. Louis, MO) and dissolved in PBS for use. Food intake was assessed at 1, 2 and 6 h after ip injection with 100 µg/kg bw PYY or 15 30, 60 and 120 min after ip injection with 8 µg/kg bw CCK. Measurement times were based on reports that anorectic effects of PYY last longer than CCK (Adrian et al. 1985; Liddle et al. 1986a). Doses were chosen based on previously reports that these consistently induce robust anorexia (Parkinson et al. 2008; Weatherford et al. 1992). Receptor antagonist effects on DON-induced anorexia. The effect of receptor antagonists on DON-induced anorexia was assessed as described above with the exception that fresh food was given for the first two measurements to minimize the time animals were without food. Also, to reduce animal usage, a cohort of mice was used for experiments with the respective hormone and inhibitor and then the same cohort rested at least 5 d, randomized and used in inhibitor plus DON experiments. Doses of receptor antagonists were chosen based on 1) their reported capacity to abolish hormone-induced anorexia in rats (Reidelberger et al. 2003; Reidelberger et al. 1991; Scott et al. 2005) and 2) lack of adverse motor effects in mice as determined by a preliminary toxicological study. 66 To confirm that the NPY2 receptor antagonist BIIE0246 (Tocris Bioscience, Minneapolis, MN) attenuated PYY-induced anorexia, 100 μl of 7% DMSO in PBS (control) or 1.67 mg/kg bw BIIE0246 in 7% DMSO was given via ip. injection 30 minutes before the ip administration of 50 μl of PBS or 100 μg/kg bw PYY. Food intake was measured at 30, 60 and 120 min. To establish the role of PYY in DON-induced anorexia, mice were given an ip injection of 100 μl of 7% DMSO in PBS (control) or 1.67 mg/kg bw BIIE0246 in 7% DMSO 30 minutes before the administration of 50 μl PBS or 1 mg/kg bw DON in PBS. Food intake was subsequently measured at 30, 60 and 120 min. This PYY experiment was performed twice to confirm findings and data combined. To verify that the CCK1A receptor antagonist devazepide (DEV; Tocris Bioscience, Minneapolis, MN) attenuated CCK induced anorexia, 50 μl of either 3% DMSO in PBS (control) or 0.6 mg/kg bw DEV in 3% DMSO was given via ip. injection. Thirty minutes later mice were injected ip with 8 µ/kg bw CCK in PBS. Food intake was measured at 15, 30, 60 and 120 min. To ascertain a potential role of CCK in DON-induced anorexia, mice were given an ip. injection of 50 μl of either 3% DMSO in PBS (control) or 0.6 mg/kg bw DEV in 3% DMSO. Thirty minutes later mice were injected ip with 50 μl PBS or 1 mg/kg bw DON in PBS. Food intake was then measured at 30, 60 and 120 min. Statistics. Data were analyzed using SigmaPlot 11.0 (Systat Software, San Jose, CA). Student’s t test was used to establish statistical significance between two independent 67 groups (p ≤ 0.05). To determine statistical significance for multiple groups, one-way ANOVA with Holm-Sidak multiple comparisons test was used (p ≤ 0.05). If normality failed, a Kruskal-Wallis One-Way ANOVA was executed with a Mann-Whitney U test, including a Bonferroni correction for multiple comparisons. A one or two factor repetition two-way ANOVA with Holm-Sidak multiple comparisons procedures was employed to establish statistical significance among groups and time points (p ≤ 0.05). Samples receiving two treatments (receptor antagonist plus DON) were analyzed for additivity, potentiation, or antagonism by randomly combining single treatment replicates to calculate a predicted mean additive response with variance as described previously (Zhou et al., 2000). This calculated value was compared to actual co-treated samples using a Mann Whitney Rank Sum test. Results DON exposure induces elevations in PYY, CCK but not other appetite-regulating hormones. Preliminary studies using a gut hormone panel revealed that acute ip exposure to DON at 2.5 and 10 mg/kg bw had no effect on plasma GLP-1, leptin, amylin, pancreatic polypeptide, GIP or ghrelin at 15 and 60 min, however, these treatments markedly increased plasma PYY and CCK (data not shown). Based on these findings the effects of DON doses of 0, 0.05, 0.25, 1.0 and 5 mg/kg on plasma levels of PYY and CCK were assessed (Fig. 3.1 and Fig. 3.2). Exposure to 5 mg/kg DON significantly increased plasma PYY relative to the vehicle control value at 15 min and this elevation was still evident after 240 min. At 15 min, a similar trend towards elevated PYY was observed for 68 1 mg/kg bw DON, compared to vehicle control (p = 0.053). Administration of 1 and 5 mg/kg DON also significantly increased plasma CCK, respectively, as compared to vehicle-treated controls at 15 min but not 240 min. Based on the observed induction of PYY and CCK by DON exposure at 1 and 5 mg/kg bw, the kinetics of responses to these two doses were further investigated. At 1 mg/kg bw, plasma PYY was approximately twice that of the zero time control from 15 to 180 min after DON treatment (Fig. 3.3). Following exposure to 5 mg/kg bw, plasma PYY remained significantly elevated (2.0- to 2.5-fold) through 240 min but not at 360 min compared to the zero time control. Plasma CCK increased rapidly in response to both 1 and 5 mg/kg bw DON (Fig. 3.4). At 1 mg/kg bw DON plasma CCK was 2.6- and 2.7-fold the zero time control at 15 and 60 min, respectively and returned to zero time control levels by 120 min. At 5 mg/kg bw DON plasma CCK increased by nearly 4-fold at 30 min and 120 min, but returned to zero time control levels by 240 min. Finally for both hormones, vehicle treatment had no effect at 15, 30 and 120 min suggesting that their initial induction during the first 2 h was not related to handling or the injection procedure (Fig. 3.3 and Fig. 3.4). DON exposure rapidly induces anorexia To relate the PYY and CCK responses to DON-induced anorexia, food consumption was monitored over intervals for 16 h after ip exposure to 1 and 5 mg/kg bw DON. Total food intake in mice exposed to 1 mg/kg bw DON was reduced by 58 and 42% as compared to control mice at 1 and 2 h, respectively but recovered by 4 h (Fig. 3.5). 69 Figure 3.1 DON induces plasma PYY elevation. Mice were treated ip with various doses of DON and plasma analyzed for PYY after 15 and 240 min. Data (n = 5 – 6/gp) are mean ± SEM. Symbols: * indicates a statistically significant difference relative to the control (p ≤ 0.05); # indicates p = 0.053. 70 Figure 3.2 DON induces plasma CCK elevation. Mice were treated ip with various doses of DON and plasma analyzed for CCK after 15 and 240 min. Data (n = 5 – 6/gp) are mean ± SEM. Symbols: * indicates a statistically significant difference relative to the control (p ≤ 0.05). 71 induced Figure 3.3 Kinetics of DON-induced plasma PYY elevation. Mice were treated ip with 0, 1 and 5 mg/kg bw DON and plasma analyzed for PYY at various time intervals. Data are mean ± SEM (n = 6/gp). Symbols: * indicates a statistically significant difference relative to the control at 0 min (p ≤ 0.05). 72 induced Figure 3.4 Kinetics of DON-induced plasma CCK elevation. Mice were treated ip with 0, 1 and 5 mg/kg bw DON and plasma analyzed for CCK at various time intervals. Data d are mean ± SEM (n = 6/gp). Symbols: * indicates a statistically significant difference relative to the control at 0 min (p ≤ 0.05). 73 Food intake by mice exposed to 5 mg/kg bw at 1, 2, and 4 h was reduced by 86%, 92% and 87%, respectively, relative to the control. By 6 h, mice began eating more relative to earlier measurements, but cumulative intake was still depressed by 51%. By 16 h cumulative food intake returned to control levels. Both orolingual and ip DON exposure induce PYY, CCK and anorectic responses The effects of orolingual exposure to DON at 2.5 mg/kg bw on hormone release and anorexia induction were measured and compared to those caused by ip exposure. Treatment with DON by the orolingual route elevated plasma PYY and CCK by 2.1- and 2.5- fold, respectively after 30 min as well as suppressed food intake by 67% (Table 3.1). These responses were comparable to effects observed following ip exposure to identical DON dose. Taken together, both orolingual and ip exposure similarly affected gut satiety hormone release and anorectic responses and this might relate to DON’s capacity to be absorbed and distributed rapidly by either route of exposure. Exogenous PYY and CCK suppress food intake Mice were treated with exogenous PYY and CCK to confirm that these hormones could indeed evoke satiety responses in the female B6C3F1 mouse. The doses chosen were consistent with those previously established to consistently evoke anorexia in mice. At 1, 2 and 6 h, PYY significantly reduced food intake by 42%, 39% and 11%, respectively, relative to control levels (Fig. 3.6). 74 Figure 3.5 Kinetics of DON-induced anorexic response. Mice were treated ip with 0, 1 induced and 5 mg/kg bw DON at initiation of dark period and food consumption measure at measured various time intervals. Data are mean ± SEM (n = 5/gp). Symbols: * indicates a statistically significant difference relative to the control dose at that specific time point (p atistically ≤ 0.05); ŧ indicates a statistically significant difference relative to the 1 h time point within a given dose (p ≤ 0.05). 75 Table 3.1 Comparison of effects of orolingual and intraperitoneal exposure to DON on food intake and gut satiety hormone release. Orolingual exposure Intraperitoneal exposure Vehicle DON Vehicle DON 0.87 ± 0.26 0.29 ± 0.05 * 0.76 ± 0.05 τ 0.24 ± 0.05 * τ PYY (pg/mL) 108 ± 15.1 229 ± 25.3 * 105 ± 17.5 276 ± 55.9 * CCK (pg/mL) 260 ± 41.4 741 ± 41.6 * 303 ± 24.3 753 ± 21.5 * Endpoint Food intake over 2 h (g) Mice were exposed to 2.5 mg/kg bw DON by orolingual and ip routes. Data are mean ± SEM (n = 5, food intake; n = 3 for hormones). Symbol * indicates a statistically significant difference relative to the control for route of exposure (p ≤ 0.05). Intraperitoneal food intake data obtained from Flannery et. al. (2011). 76 τ Figure 3.6 Kinetics of the PYY PYY-induced anorexic response. Mice were injected ip with 100 µg/kg bw PYY and food consumption measure at various time intervals. Data are measured mean ± SEM (n = 15/gp). Symbols: * indicates a statistically significant difference between the PYY treatment group and the control group at that time point (p ≤ 0.05); ŧ indicates a statistically significant difference relative to the 1 h time point within a given dose (p ≤ 0.05). 77 Figure 3.7 Kinetics of the CCK CCK-induced anorexic response. Mice were injected ip with 8 µg/kg bw CCK and food consumption measure at various time intervals. Data are g/kg measured mean ± SEM (n = 5/gp). Symbols: * indicates a statistically significant difference between the CCK treatment group and the control group at that time point (p ≤ 0.05); ŧ indicates a statistically significant difference relative to the 15 min time point w within a given dose (p ≤ 0.05). 78 DON-induced Figure 3.8 Effect of BIIE0246 on PYY and DON induced anorexic response. A. Mice were pretreated with 1.67 mg/kg bw BII0246 or DMSO and 30 min later treated with PBS or 100 µg/kg bw PYY. Food intake was then measured at various intervals. Data YY. then D are mean ± SEM (n = 5-7/gp) B. Mice were pretreated with 1.67 mg/kg bw BIIE0246 or 79 (Figure 3.8 cont’d) DMSO and 30 min later treated with PBS or 1 mg/kg bw DON. Food intake was then measured after. Data are mean ± SEM (n = 14-15/gp). For both graphs, different letters indicate a statistically significant difference between treatment groups for that time point (p ≤ 0.05). Percent food consumption in animals receiving DON and receptor antagonist were further compared to the predicted mean additive food intake response as described in methods (p=0.059). CCK’s anorectic effects did not last as long as those of PYY. Relative to the control, CCK significantly reduced food intake by 90% at 15 min and 46% at 30 min with food intake was no longer statistically different from control values by 60 min (Fig. 3.7). NPY2 receptor antagonist BIIE0246 interferes with PYY- and DON-induced anorexia. The effects blocking the NPY2 receptor in PYY and DON-induced food refusal were evaluated. PYY decreased food intake by 54, 41 and 35 % relative to the control at 30, 60 and 120 min, respectively (Fig. 3.8A). These effects were attenuated with BIIE0246 thus confirming the action of the inhibitor. When mice were treated with DON or BIIE0246 alone, food consumption over 60 min was reduced by 56 and 17 percent, respectively (Fig. 3.8B). Although the predicted reductions in food intake from DON and BIIE0246 cotreatment over this time period was 71%, the actual observed reduction was 33%. This strong trend (p= 0.059) towards reversal of DON-induced anorexia in mice upon NPY2 receptor antagonism suggests that PYY might play an important role in this effect. 80 CCK1A receptor antagonist DEV ablates CCK- but not DON-induced anorexia Mice treated with CCK exhibited a significant reduction in food intake at 15 and 30 min with similar trend being evident at 60 min (Fig. 3.9A). CCK-induced food reduction was ablated by prior to DEV administration. To ascertain a possible role for CCK in DON-induced anorexia, mice were pretreated with DEV before toxin exposure. Mice exposed to DON consumed 25% less over 30 min whereas mice receiving DEV consumed 34% more food over the same period (Fig. 3.9B). DON-exposed mice that were pre-treated with DEV exhibited a trend of increased food intake compared to mice exposed to DON alone. However, when the predicted combined effects of the antagonist and DON were compared to the actual response, no differences were evident. Therefore, the attenuation exhibited in DEV treated DON-exposed mice appeared to result from DEV blocking basal CCK from interacting with CCK1R. Discussion DON’s capacity to impair food intake and suppress growth has long been of major concern for both the human and animal health fields, however, the underlying mechanisms for these clinical effects are still relatively poorly understood. Our findings here demonstrate, for the first time, that DON rapidly induces plasma levels of two major gut satiety hormones PYY and CCK in the mouse and that this induction occurs in a manner remarkably consistent with anorexia induction. In addition, like DON, administration of exogenous PYY and CCK, DON caused immediate food refusal. 81 DON-induced Figure 3.9 Effect of DEV on CCK and DON induced anorexic response. A. Mice were pretreated with 0.6 mg/kg bw DEV or DMSO and 30 min later treated with PBS or 8 µg/kg bw CCK. Food intake was than measured at various intervals. Data are mean ± g/kg SEM (n = 6-7/gp). B. Mice were pretreated with 0.6 mg/kg bw DEV or DMSO and 30 7/gp). pretreated 82 (Figure 3.9 cont’d) min later treated with PBS or 1 mg/kg bw DON. Food intake was then measured after 60 min. Data are mean ± SEM (n = 9-10/gp). For both graphs, different letters indicate a statistically significant difference between treatment groups for that time point (p ≤ 0.05). Comparison of percent food consumption in animals receiving DON and receptor antagonist were to the predicted mean additive percent food consumption indicated the difference was not significant (n.s.). While NPY2 receptor antagonism attenuated DON-induced anorexia, CCK1 receptor antagonism did not, suggesting PYY might play a more important role than CCK in DON-induced anorexia. These findings have public health significance because they suggest DON’s anorectic effects might, in part, be hormone-driven and linked to an aberrant postprandial satiety response. Satiety is a complex process involving 1) release of hormones such as PYY and CCK from the gastrointestinal tract, 2) changes in the expression of orexigenic and anorexigenic signaling peptides within the arcuate nucleus of the hypothalamus and 3) alterations in gut motility and secretion. Since DON caused a release of satiety hormones, it might be speculated that the toxin caused mice to be satiated and thus, prevented food intake. These findings are consistent with those of two research groups who have shown that DON exposure leads to the “fed pattern” of gut motility in rats (Fioramonti et al. 1993; Krantis and Durst 2001b). Thus, DON exposed mice might refuse food in part, because they feel full. Upon consuming a meal, both PYY and CCK increase rapidly with circulating plasma levels elevated within 15 min for PYY in humans and 5 min for CCK in rats and lasting 6 h and 1 to 2 h, respectively (Adrian et al. 1985; Liddle et al. 1986a). As 83 demonstrated here, DON mimicked these effects by causing rapid increases in plasma PYY and CCK. The rapid release of PYY and CCK (<15 min) upon DON administration coincided with the onset of DON-induced anorexia (<30 min). Interestingly, CCK returned to zero time levels by the time mice recovered from DON-induced anorexia, whereas plasma PYY concentrations remained elevated even after cessation of feed refusal. It is possible that elevated plasma PYY no longer impacted food intake because of the desensitization, saturation and rate of cycling of NPY2 receptors or because other hormones modulated sensitivity of NPY2 receptors to PYY (Parker and Balasubramaniam 2008). While modest food consumption results in satiation, overconsumption could lead to excessive fullness, nausea and even vomiting (in susceptible species). Interestingly, PYY is extremely potent in its ability to cause dogs to vomit and its elevation in plasma has also been associated with cisplatin-induced emesis (Harding and McDonald 1989; Perry et al. 1994). Furthermore, humans given exogenous PYY to suppress appetite as an obesity treatment exhibit, at the highest doses, adverse effects that include excessive fullness, abdominal discomfort, nausea, and vomiting (Degen et al. 2005). Postprandially, lean mice given a 2.6% fat meal exhibited approximately a 20 percent increase over basal plasma levels of PYY after 120 min (le Roux et al. 2006). We observed here that unfed mice exposed to DON exhibited a remarkably greater (~ 2fold) increase in plasma PYY levels over control values at both 1 and 5 mg/kg bw. Thus, the PYY response in DON-exposed mice was far greater than that reported previously from meal consumption. It might be speculated that, in emesis-capable species, such a large increase in PYY (ie. relative to meal-induced) might lead to vomiting. Further 84 studies are therefore warranted on the role of PYY in DON-induced emesis in an appropriately sensitive species. Here, the use of the antagonists of the NPY2 and CCK1 receptors revealed that PYY plays a more important role than CCK in DON-induced anorexia. Unlike CCK, PYY can penetrate the blood brain barrier to affect signaling changes within the hypothalamus while CCK acts through the vagus nerve to evoke changes in food intake (Moran et al. 1997). Consistent with this mechanism, Girardet and coworkers showed that upon a cervical vagotomy, mice dosed with oral DON still exhibited changes in cFos expression within the brainstem (Girardet et al. 2011b). While food intake was not measured in that particular experiment, these data suggest the vagus nerve was not necessary for C-Fos activation and may not be necessary for hypothalamic signaling or food intake changes caused by DON. The observation that the NPY2 receptor antagonist could not completely inhibit DON-induced anorexia might relate to the distribution kinetics of BIIE0246 as well as the complexity of appetite regulation. Administration of BIIE0246 by the ip route in the mouse resulted in observed brain concentrations being 2% of those found in the plasma (Brothers et al. 2010). Therefore, only small amounts of BIIE0246 could have entered the brain of DON-exposed mice, leading to only a modest effect of BIIE0246 on DONinduced anorexia. Higher doses of BIIE0246 were not used in these experiments because we observed these doses caused ataxia in preliminary experiments. It should be emphasized that food intake is so critical for survival that many redundant and complex, pathways exist to regulate appetite. Furthermore, food intake behavior is affected by social factors, such as housing, or by emotional factors, such as stress of 85 handling, which do play a role in food intake changes (Schwartz et al. 2000). Thus suppression of food intake inhibition as observed with BIIE0246 on DON induced feed refusal would likely be of biological significance. Further experiments are needed to elucidate the role of the brain in the peripheral release of gut satiety hormones in the DON feed refusal model. In conclusion, the heretofore unreported findings presented here highlight a potential new mechanism by which DON causes anorexia and, as a result, growth suppression. Secretion of PYY may serve as a defense mechanism for preventing continued ingestion of toxic DON by suppressing appetite and decreasing gut motility. Aberrant release of PYY could have further potential to contribute to DON-induced emesis in animal species capable of a vomiting response. Thus, important future considerations will be to determine whether the release of PYY contributes to the reported vomiting caused by DON. In addition, gut satiety hormones are reportedly elevated in eating disorders related to conditions such as old age (Silver et al. 1988), chronic gastrointestinal illnesses (Chua et al. 2006; Khoo et al. 2010; Van Der Veek et al. 2006), cancer cachexia (Moschovi et al. 2008), and anorexia/bulimia (Germain et al. 2007; Lawson et al. 2011). It will therefore be of interest to discern if these disease states and other susceptibility factors that might further heighten an individual’s sensitivity to DON or other trichothecenes. Finally, the exact mechanisms for robust PYY and CCK responses to DON are not known. A plausible hypothesis is that these gut satiety hormones are released from enteroendocrine cells as an evolutionary response to toxic substances (Glendinning 2007), to diminish and prevent further ingestion of the agent. Future work should focus on the role of specific receptors in mediating this response. 86 CHAPTER 4: CENTRAL MECHANISMS OTHER THAN PYY CONTRIBUTE TO DONINDUCED ANOREXIA Abstract Deoxynivalenol (DON) is a mycotoxin found in grains products that was reported to cause acute gastroenteritis in humans and anorexia, weight loss and growth suppression in animals. Regulatory limits of DON in food are based on its capacity to cause growth suppression in mice, a likely effect of anorexia. Both central and peripheral mechanisms of DON-induced anorexia have been identified recently. More specifically, DON has the capacity to cause anorexia and alter hypothalamic signaling upon direct administration. Peripherally, DON administration results in increased plasma PYY levels, an effect which could also alter hypothalamic signaling through the vagus nerve or by direct action on circumventricular organs. Using a mouse model, the objective of this research was to evaluate DON’s potential to act centrally. Results suggested 1) DON entered the brain and hypothalamus, 2) the vagus nerve was not important for DON’s anorectic effects and 3) central Y2 receptors were not critical for anorexia suggesting PYY’s anorectic effects originate from the periphery. Therefore, central factors likely play a role in DON-induced anorexia, though not central PYY. 87 Introduction Trichothecene mycotoxins are a group of structurally related metabolites produced from fungi that cause adverse health effects in humans and animals. Deoxynivalenol, produced from Fusarium species, is a trichothecene mycotoxin of particular public health concern because of its high worldwide contamination rate and because it is impervious to processing methods resulting in persistent contamination of grain products (Lauren and Smith 2001; Rodrigues and Naehrer 2012). Excessive acute DON consumption by humans results in gastroenteritis while acute exposure in experimental animals causes vomiting (susceptible species), anorexia, and excessive proinflammatory cytokine induction (Canady et al. 2001; Pestka 2010b). While sub-chronic low-level DON exposure has not been well studied in humans, experimental animals exhibit anorexia, growth hormone dysregulation and weight suppression (Amuzie and Pestka 2010; Flannery et al. 2011). Of these harmful effects, the no observed adverse effect level for growth suppression was used by the Joint FAO/WHO Committee on Food Additives (JECFA) as the foundation for the current established tolerable daily intake (TDI) (Canady et al. 2001; Pieters et al. 2002). Consumption of grains relative to body weight is greater in children than adults rendering children a particularly vulnerable population for DON’s potential growth effects (Lin and Yen 2007). Experimental animal data consistently demonstrates that DONinduced growth suppression is caused, in part, by anorexia yet the mechanisms by which DON causes anorexia are not completely understood (Accensi et al. 2006; Bergsjo et al. 1992; Forsell et al. 1986; Harvey et al. 1986; Iverson et al. 1995). 88 The redundancy of the biological pathways controlling food intake make determining mechanisms of anorexia extremely complex. Peripherally, food intake is regulated by, though not limited to, the release of gut satiety hormones that signal to hypothalamic neurons through the vagus – nucleus tractus solitarius (NTS) - hypothalamic pathway or through these hormones reaching circumventricular organs via release into the blood stream (Suzuki et al. 2010). Centrally, the brain organ most associated with controlling food intake is the hypothalamus via a balance of neurons expressing both orexigenic (neuropeptide Y, NPY; agouti related peptide, AGRP) and anorexigenic peptides (alphamelanocyte stimulating hormone, α-MSH; pro-opiomelanocortin, POMC; cocaine amphetamine regulated transcript, CART), a balance that is affected by peripheral satiety signals (Schwartz et al. 2000). Since DON rapidly distributes to both the gastrointestinal (GI) tract and the brain within 5 min of oral exposure, (Pestka et al. 2008) it has the potential to elicit food intake changes through both central and peripheral mechanisms. In support of DON’s central action, Girardet and coworkers have gracefully demonstrated an intracerebroventricular injection (ICV) of 2 µg/mouse of DON, a dose lower than that which causes feed refusal when administered peripherally, resulted in significantly depressed food intake between 2 to 12 h post exposure. (Girardet et al. 2011b) These authors also showed that an ICV injection of DON resulted in increased c-fos expression in pro-opiomelanocortin (POMC) positive neurons at 3 h. Since c-fos expression is a marker of neuronal activation (Day et al. 2008; Dragunow and Faull 1989), these data suggested the anorexigenic neurons positive for POMC was activated by direct DON exposure. Oral exposure of 12.5 mg/kg bw DON similarly resulted in 89 activation of c-fos with in anorexigenic signaling neurons (Girardet et al. 2011b). Upon cervical vagotomy, oral DON administration still resulted in increased c-fos expression within the NTS of the brainstem, further confirming DON’s capacity to act directly within the central nervous system. DON was reported to compromise GI tract integrity in pigs though decreasing expression of claudin, a protein critical for tight junction permeability (Pinton et al. 2010). Since DON exerts toxic action on the GI tract, DON could cause a release of GI satiety hormones. 5-hydroxytryptamine (5-HT, serotonin), cholecystokinin (CCK) and peptide YY (PYY) are candidate targets for DON-induced release because the rapidity by which they are released upon nutrient consumption corresponds to the timing by which DON causes anorexia in experimental animals (Blum et al. 1992; Gibbs et al. 1976; Moran et al. 2005). 5-HT is a neurotransmitter released from endochromaffin cells of the gut as well as located within the brain while CCK and PYY are peptide satiety hormones released from the L and I cells of the GI tract, respectively. 5-HT acts on the 5-HT1B and 2C to reduce food intake while CCK and PYY act on the CCK1 and Y2 receptors, respectively (Batterham et al. 2002; Dourish 1995; Miesner et al. 1992). Investigations by Flannery and coworkers demonstrated that DON exposure increases plasma levels of both CCK and PYY in the mouse (Flannery et al. 2012). Though, food intake experiments with the CCK1 receptor antagonist devazepide and the Y2 receptor antagonist BIIE0246 administered in the periphery suggest PYY was more important than CCK in DON-induced anorexia in the mouse (Flannery et al. 2012). Peripherally released PYY has the capacity to cause anorexia by acting directly within the brain via the area postrema, a circumventricular organ, or by acting 90 peripherally by signaling through the vagus – NTS – hypothalamic pathway (Suzuki et al. 2010). PYY reduces food intake by decreasing NPY expression and increasing POMC expression, both of which exert their action through the melanocortin receptor 4 (Challis et al. 2003). Despite DON’s capacity to cause peripheral PYY release, it is not yet known if PYY acts through the peripheral or central nervous system to cause DONinduced anorexia. Therefore, the goal of these experiments was to ascertain the capacity of DON to act within the CNS to cause anorexia more specifically, through the Y2 receptor. We hypothesize that DON enters the brain at a dose and time consistent with anorexia and that the peripheral PYY released by DON likely acts centrally to have its anorectic effects. To assess this hypothesis, we first confirmed DON entered the brain in a dose and time that is consistent with DON-induced anorexia. Then we assessed the necessity of the vagus nerve in DON-induced anorexia. Finally, we used ICV injection of BIIE0246, a Y2 receptor antagonist with low brain penetrability, to establish the function of the central Y2 receptor in DON-induced anorexia.(Brothers et al. 2010) Materials and Methods Chemicals. DON was obtained from Dr. Tony Durst (University of Ottawa, Canada) and purity confirmed using HPLC and elemental analysis (Galbraith Laboratories, Knoxville, TN). DON was diluted using sterile phosphate-buffered saline (PBS). BIIE0246 was purchased from Tocris Bioscience (Minneapolis, MN) and dissolved in 2% dimethyl 91 sulfoxide (DMSO) and sterile nanopure water. PYY was also purchased from Tocris Bioscience and dissolved in PBS for use. Laboratory animals. Details of mouse sex, age and acclimation period are listed under respective experiments. Mice were housed under constant temperature and humidity with a 12 hr light/dark cycle. Prior to performing experiments, experimental approval was obtained from the Institutional Animal Use and Care Committee at Michigan State University. Experimental Designs Effects of peripheral DON injection on food intake. Naïve, B6C3F1 female mice ages 9 – 10 wks were purchased from Charles River Breeding (Portage, MI). Mice were acclimated to environment and high fat diet (45% kcal from fat; D12451; New Brunswick, NJ) for 1 wk before beginning experiments. Food intake was measured according to Flannery and coworkers (Flannery et al. 2011) under red light conditions. Mice were given an ip. injection of 0, 1 or 5 mg/kg bw DON and food intake measured at 0.5, 1 and 2 hr. Effects of dose and time on DON distribution. Since DON is metabolized within 24 to 48 hrs (Pestka et al. 2008), non-naïve, female, B6C3F1 age 12 – 13 wks mice used once for food intake measurements from a previously published study (Flannery et al. 2011) were allowed to rest for at least 2 wks, randomized and used for DON toxicokinetic studies. Two experiments were performed to establish dose and kinetics of DON entering the brain and other tissues. For dose studies, food was removed and 92 randomized mice were given an ip. injection of 0, 1, 2.5, 5 or 10 mg/kg bw DON dissolved in 100 µl PBS. After 3 h, mice were sacrificed by lethal injection of sodium pentobarbital and prepared for perfusion when reflexes were no longer present. For kinetics studies, food was removed and randomized mice were given an ip. injection of 0, 1 or 5 mg/kg bw DON dissolved in 100 µl PBS. The control group was sacrificed at 0 m, while treated mice were sacrificed at 15, 30, 60 and 120 m. Saline perfusion was then executed, tissues were removed and immediately frozen in liquid nitrogen for subsequent tissue DON concentration analyses. The role of the vagus nerve in DON-induced feed refusal. For vagotomy studies, naïve, B6C3F1 adult male mice were necessary because mice needed be 25 g to receive surgery from Charles River Breeding making female B6C3F1 too small at the age desired. A repeated measures design was employed to determine the role of the vagus nerve in DON-induced feed refusal. To establish a baseline measurement, naïve mice were injected with 100 µl saline and food intake measured at 2 and 16 h. After resting 1 wk, both sham and vagox mice were given an ip. injection of 1 mg/kg bw DON dissolved in 100 µl PBS and food intake measured. Finally, after another 1 wk rest, both surgical groups were given an ip. injection (100 µl) of 0.5 mg/kg bw DON and food intake measured. The role of central DON administration and central Y2 receptors in DON-induced feed refusal. Female, B6C3F1 mice ages 9 – 11 wks with intracerebroventricular (ICV) cannulas were used for experiments. Mice were received 2 d post surgery and acclimated to environment and HFD for 3 d before beginning experiments. A shorter acclimation period was chosen as compared to other experiments because cannula 93 integrity decreases over time. All food intake measurements are according to previously published studies (Flannery et al. 2012; Flannery et al. 2011) and performed under red light. Experiments were split over 4 nights (n = 8) with 2 mice per dose group each night. Four groups of mice (n = 2/gp) were given either a 2 µl ICV injection of 1.8 µg/mouse BIIE0246 dissolved in 2% DMSO and nanopure water or 2 µl ICV injection of 2% DMSO dissolved in nanopure water (control). An 50 µl intraperitoneal injection of either PBS or 1 mg/kg bw DON dissolved in PBS was given 30 min after the ICV injection. Mice then received food and food intake measured at 60, 120, 240 and 360 min. The dose of BIIE0246 administered via ICV injection was chosen based on Abbott and coworkers demonstrating central BIIE0246 injection abolished PYY induced anorexia in the rat (Abbott et al. 2005b). Once these experiments were completed, mice were rested 3 d and randomized for a final experiment. Four groups of mice (n = 5-8/gp) received at ICV injection of 0, 9.2, 10.7 or 18.4 or 20 µg/mouse of DON (≈ 0, 0.4, 0.5, 0.8 mg/kg bw). These doses are lower than that which have been reported to cause significant feed refusal in female mice administered ip. injection or oral gavage of DON (Flannery et al. 2011). Food intake was measured for 1 hr in accordance with the parameters previously published (Flannery et al. 2012; Flannery et al. 2011). The role of the brain in DON-induced anorexia. Immediately after 1 hr food intake measurement of mice treated with ICV DON, mice were then sacrificed using 56 mg/kg bw sodium pentobarbital. Upon sacrifice, 2 µl Evans Blue Dye was injected into the ICV cannula and the brain removed. The brain was placed in a cryomold with OCT 94 compound and frozen on dry ice. Brain tissue was sliced and dye placement compared with a mouse brain atlas to assure correct placement of the cannula into the ventricle. Results from 4 mice were excluded from experimental results due to incorrect cannula placement. Blood was removed from the inferior vena cava using syringes containing 1% EDTA solution. Blood was then put in purple top tubes containing EDTA, centrifuged at 3600 x g for 10 min at 4° and plasma removed fo r PYY measurements C Perfusion. After mice were properly anesthetized, the abdominal cavity was opened and rib cage cut to expose the heart. A 20 G needle connected to a perfusion apparatus containing 0.9% sterile saline solution and heparin (10 IU/mL saline) was inserted into the apex of the left ventricle and allowed to flow. Immediately, the inferior vena cava was cut to allow for the removal of blood and saline from the body until the liquid was clear (approximately 3 min). Tissues were then removed and prepared for further analyses according to a previously described protocol (Pestka et al. 2008). Two animals with failed perfusions were not included in the DON tissue analyses. DON ELISA. Tissue DON concentrations were measured using a Veratox High Sensitivity (HS) ELISA (Neogen, Lansing, MI) according to a previously published protocol (Amuzie et al. 2009) with modifications for tissue dilutions. Absorbance was read at 690 nm using an ELISA plate reader (Molecular Devices, Menlo Park, CA) and sample concentration determined using a standard curve. DON concentrations are given as 95 DON equivalents per gram of tissue because the DON ELISA may also detect the DON metabolite 3-acetyl-deoxynivalenol. Vagotomy. Vagotomized and sham treated male mice were purchased from Charles River Breeding. Sub-diaphragm vagotomies and sham surgeries were performed and verified according to Charles River Breeding surgical protocols. Mice were checked for infection and monitored for 14 d after arrival and mice exhibiting improper healing were excluded from experiments. ICV Cannulation and Injection. Experienced surgeons at Charles River Breeding performed ICV cannulation on adult female B6C3F1 mice. Cannulas were placed in the left lateral ventricle with the coordinates – 0.22 mm anterior-posterior, -1.00 mm medial-lateral and – 2.3 mm dorsal – ventral to the bregma. The Institutional Animal Care and Use Committee at Charles River Breeding approved the standardized procedure for ICV cannulation. ICV injections were performed using aseptic technique. Mice were anesthetized using isoflurane. A 10 µl syringe (Hamilton Syringes; Reno, NV) was used to inject treatments at a rate of 1 µl/min over 2 min. To prevent aspiration, a 30 sec time period was allowed before removing the syringe after injection. 96 Statistics. A One-Way ANOVA with the Holm-Sidak method for multiple comparisons was used to establish statistical significance and a One-Way ANOVA on Ranks with Dunn’s multiple comparisons produce was used with normality failed. A Two-way ANOVA was employed for each time point to determine statistically significant differences among treatment and surgical groups for vagotomized mice. Analyses were performed using SigmaPlot 11 (Jandel Scientific; San Rafael, CA). Statistical significance was considered established when p < 0.05. Results Peripheral DON administration rapidly reduces food intake DON-induced anorexia was detectable at 30 min with food intake significantly decreased by 67% and 85% at 1 and 5 mg/kg bw, respectively (Fig. 4.1). Food intake remained depressed through 2 hr in mice exposed to 5 mg/kg bw DON. At 2 hr, 1 mg/kg bw DON depressed food intake by 53%, though this reduction was not statistically significant. DON distributes to peripheral and central tissue in a dose-dependent manner DON dose-dependently distributed to liver, small intestine and brain tissue of saline perfused mice at 3 h (Fig. 4.2). Notably, DON was detectable in the brain at 1 mg/kg bw, a dose which we have previously reported to elicit significant feed refusal in the mouse (Flannery et al. 2011). 97 Concurrent with the hypothesis that DON acts centrally to cause food intake changes, DON was detected in the brain and hypothalamus of saline perfused mice at 15 m and remained constant up to 120 m (Fig. 4.3). DON was found in the brain and hypothalamus at lower concentrations relative to the liver, spleen and small intestine, but in contrast to the brain, DON was cleared rapidly from these other tissues (Fig. 4.4). DON-induced feed refusal is not attenuated in vagotomized mice Both sham and vagox mice exhibited significant feed refusal at 2 h after an ip. injection of 0.5 and 1 mg/kg bw DON, though no significant differences in food intake between surgical groups was detected (Fig. 4.5). At 2 h, sham mice ate 31% less at 0.5 mg/kg bw and 39% less at 1 mg/kg bw as compared to the no treatment group while vagox mice at 36% and 47% less, respectively. These data suggest that an intact vagus nerve is not necessary for DONinduced feed refusal to occur; thus, likely indicating a central mechanism for DONinduced food intake changes. Central DON administration minimally depresses food intake The capacity of centrally administered DON to decrease food intake at 1 hr was assessed using ICV cannulated mice (Fig. 4.6). DON doses used were 9.2, 10.7 and 18.4 µg/mouse, which were equivalent to 0.4, 0.5 and 0.8 mg/kg bw, respectively. Though not statistically significant, mice administered 10.7 and 18.4 µg/mouse ate 27 and 22% less, respectively. Mice administered 9.2 µg/mouse consumed 33% more food than the control at 1 hr. 98 The central Y2 receptor is not necessary for DON-induced anorexia. To assess the necessity of central Y2 receptors in DON-induced anorexia, mice were administered either vehicle or BIIE0246 by ICV injection 30 min prior to peripherally administration of PBS or 1 mg/kg bw DON (Fig. 4.7). Initially at 60 min ICV injection BIIE0246 alone raised food intake by 11% relative to the control, though this effect was not statistically significant. This increase in food intake was likely because inhibiting the Y2 receptor leads to increased expression of the orexigenic peptide NPY within the hypothalamus (Abbott et al. 2005b). In mice administered DON, BIE0246 pretreatment had no significant effect on attenuating DON-induced anorexia. Mice administered by ICV with vehicle and then peripheral DON ate significantly less than the PBS control with food intake being depressed by 52, 61,39 and 21% at 60, 120, 240 and 360 min respectively. Mice administered ICV injection of BIIE0246 before peripheral DON exhibited similar food intake depressions relative to the control, depressions that were statistically significant relative to the control. Taken together, data demonstrated DON did not act through the central Y2 receptor to reduce food intake. 99 Figure 4.1 Food intake in mice administered DON by intraperitoneal injection Data are injection. mean ± SEM (n = 4-5). Different letters indicate a statistically significant difference between treatments (p < 0.05). 100 Figure 4.2 Dose dependent DON distribution in peripheral and central tissues. Mice were given and IP injection of 0, 1, 2.5, 5 or 10 mg/kg bw DON and A) small intestine, B) liver and C) brain DON concentrations measured by ELISA. Data are mean ± SEM (n ELISA. = 4-5). 101 Figure 4.3 Short-term kinetics of DON distribution in the brain and hypothalamus of cs hypothala saline perfused mice. Mice were exposed to DON via IP injection. Tissue DON was . measured in the A) brain and B) hypothalamus using a DON ELISA. Data are mean ± SEM (n = 4-6) except hypothalamic measurements (n =2 of pooled samples 6) samples). 102 Figure 4.4 Short-term kinetics of DON distribution to the small intestine, spleen and liver s of saline perfused mice. Mice were exposed to DON via IP injection and DON in the A) small intestine, B) spleen and C) liver was measured using a DON ELISA. Data are mean ± SEM (n = 4-6). 103 Figure 4.5 Food intake of vagotomized mice upon DON administration. Mice were administered 0, 0.5 or 1 mg/kg bw DON and food measured at 2 h and 16 h. Data are mean ± SEM (n = 7-8). An asterisk indicates a statistically significant difference between 8). a dose group and the saline control with p < 0.05. No significant differences were se detected between vagox and sham treated mice at either time point point. 104 Figure 4.6 Food intake in mice upon intracerebroventricular injection of DON. Mice were M administered 0, 9.2, 10.75 or 18.4 µg/mouse of DON and food intake measured at 1 hr. g/mouse No significant differences were detected among groups p < 0.05. 105 receptor induced Figure 4.7 The effect of the Y2 recep antagonist BIIE0246 DON-induced anorexia. Mice were pretreated ICV with ve vehicle or 1.8 µg/mouse BIIE0246 30 min before PBS or g/mouse DON. Food intake was measured at 60, 120, 240 and 360 min. Data are mean ± SEM (n = 8/gp). Different letters indicate a statistically significant difference at the specified time point (p < 0.05). 106 Discussion Given that DON causes weight suppression in experimental animals, an effect partly attributable to anorexia, and its potential to adversely affect growth in children, it is of utmost importance to understand the mechanisms that govern DON-induced anorexia. Recently Girardet and coworkers demonstrated ICV administration of DON resulted in anorexia confirming that DON worked centrally to cause anorexia (Girardet et al. 2011b). Additionally, Flannery and colleagues showed that DON caused anorexia through the release of the gut satiety hormone PYY (Flannery et al. 2012; Girardet et al. 2011b). The purpose of these studies presented herein was to elucidate central mechanisms of DON-induced anorexia. Results suggested DON was able to enter the brain at a dose and time consistent with feed refusal; however, both a vagotomy and central administration of the Y2 receptor antagonist BIIE0246 were unable to attenuate DON’s anorectic effects. These data suggest that PYY’s capacity to attenuate DONinduced anorexia likely originated from the periphery and that mechanisms other than PYY, mechanisms that do not rely on the vagus nerve, are mediators of DON-induced anorexia. PYY and its Y2 receptor are expressed in the hindbrain, brainstem, NTS and dorsal motor nucleus of the vagus nerve (Batterham et al. 2002; Gelegen et al. 2012). The importance of central Y2 receptors in PYY-induced anorexia was confirmed by Abbott and investigators who demonstrated that ICV injection of BIIE0246 into the arcuate nucleus partially attenuated PYY-induced anorexia in the rat (Abbott et al. 2005b). Other scientific investigations into the capacity of PYY to reduce food intake have mainly focused on the role of the vagus nerve, NTS and circumventricular organs. 107 Baraboi and coworkers were able to decipher the signaling pathway of peripheral PYY by conjugating PYY to albumin thereby making PYY unable to cross the blood brain barrier (Baraboi et al. 2010). By administering this conjugate into the periphery of rats, they were able to show that a vagotomy reduced PYY’s anorectic effects as did ablation of circumventricular organs, the area postrema (AP) and the subfornical organ. These data suggest that the vagus-NTS pathway is critical for anorexia induced by peripheral PYY. The role of PYY in DON-induced anorexia was demonstrated by data showing DON elevated plasma PYY and the Y2 receptor antagonist BIIE0246 slightly attenuated DON-induced anorexia in the mouse (Flannery et al. 2012). Since BIIE0246 has poor brain penetrability (Brothers et al. 2010), this attenuation likely occurred from BIIE0246 inhibiting peripheral Y2 receptors on the vagus nerve and circumventricular organs. However, the studies presented herein suggest that the vagus nerve was not necessary for DON-induced anorexia indicating the capacity of DON-induced PYY to act on the vagus nerve was not necessary for DON-induced anorexia. These contradictory results may be due to 1) the capacity of the vagal afferents to regenerate making the vagotomy ineffective (Phillips et al. 2003), 2) the BIIE0246 was not given in vagotomized mice, therefore brain and peripheral signaling of PYY were not negated at the same time and 3) other compensatory mechanisms could also play a critical role in DON-induced anorexia. The area postrema (AP) could likely play a role in DON-induced anorexia. The AP is a circumventricular organ located in the brain at the base of the fourth ventricle that has access to both the blood and the brain thereby allowing communication of 108 blood contents, including toxins, to the brain (Miller and Leslie 1994). The AP contains the chemoreceptor trigger zone, an area responsible for signaling vomiting upon ingestions of noxious substances in species capable of vomiting (Miller and Leslie 1994); however, rodents are not capable of vomiting and as a result, development of a conditioned taste aversion (CTA) may indicate nausea/malaise from ingestion of a poisonous substance (Ossenkopp and Eckel 1995). CTAs occur when the taste of a substance is associated with adverse symptoms, a result of pairing this taste with a noxious substance. Some CTAs are mediated by the AP and can lead to anorexia and weight loss (Eckel and Ossenkopp 1993). DON administration in rats has been reported to cause a CTA to saccharin suggesting DON may cause malaise in rodents, an effect that may serve as a surrogate for DON-induced vomiting (Ossenkopp et al. 1994). Interestingly, DON-induced CTA was abolished upon AP ablation suggesting the AP could mediate DON-induced anorexia (Ossenkopp et al. 1994). Another possible mechanism by which DON decreases food intake is through the increase of central nesfatin-1. Nesfatin-1 is a potent anorexigenic peptide found in adipose tissue, gastrointestinal mucosa and the brain, more specifically in central areas critical for food intake such as the ARC, paraventricular nucleus and the lateral hypothalamic area (Oh et al. 2006; Ramanjaneya et al. 2010; Stengel et al. 2009; Zhang et al. 2010). Nesfatin-1 is expressed both in POMC/CART and NPY/AgRP neurons and through its capacity to decrease NPY/AgRP expression in the ARC, reduces food intake (Palasz et al. 2012). Recently, Girardet and coworkers demonstrated that ICV and oral DON administration resulted in increased expression of nesfatin-1 on hypothalamic POMC positive neurons suggesting that DON could have its 109 anorectic effects though increasing nesfatin-1 (Girardet et al. 2011b). Therefore, nesfatin-1 may serve as another mechanism by which DON causes its anorectic effects, a mechanism that would be unaffected by vagotomy or central BIIE0246 administration. The results presented herein indicate that DON rapidly and dose-dependently entered the central nervous system, notably the hypothalamus, in a manner consistent with the dose effects and timing of feed refusal. However, neither a vagotomy nor ICV administration of BIIE0246 attenuate DON-induced feed refusal. Taken together, these results strengthen the hypothesis that DON acts centrally to cause feed refusal, though data suggest not through the Y2 receptor. Future investigations should focus on the role of other central acting satiety signals in DON-induced anorexia. 110 CHAPTER 5: EVALUATION OF INSULIN-LIKE GROWTH FACTOR ACID-LABILE SUBUNIT AS A BIOMARKER OF EFFECT FOR THE MYCOTOXIN DEOXYNIVALENOL Data from this chapter was submitted to Toxicology by Flannery B.M., Amuzie, C.J. and Pestka J.J. Abstract Consumption of the trichothecene deoxynivalenol (DON) suppresses growth in experimental animals - an adverse effect that was used to establish the tolerable daily intake for this toxin. DON ingestion has been recently found to suppress plasma insulinlike growth factor acid-labile subunit (IGFALS), a protein essential for growth. Studies were conducted to explore the feasibility of using plasma IGFALS as a biomarker of effect for DON. In the first study, weanling mice were fed 0, 1, 2.5, 5 and 10 ppm DON and weight and plasma IGFALS determined at intervals over 9 wk. Reduced body weight gains were detectable beginning at wk 5 in the 10 ppm dose and wk 7 at the 5 ppm dose. Plasma IGFALS was significantly depressed at wk 5 in the 5 and 10 ppm groups at wk 9 in the 10 ppm group. Depressed IGFALS significantly correlated with reduced body weight at wk 5 and 9. Benchmark dose modeling revealed the BMDL and BMD for plasma IGFALS reduction were 1.1 and 3.0 ppm DON and for weight reduction were 2.1 and 4.5 ppm DON, respectively. In the second study, it was demonstrated that mice fed 15 ppm DON diet had significantly less plasma IGFALS than mice fed identical amounts of control diet. Thus DON’s influence on IGFALS likely reflects the combined effects of reduced food intake as well as its physiological action involving suppressors 111 of cytokine signaling. Taken together, these findings suggest that plasma IGFALS might be a useful biomarker for DON’s adverse effects on growth. Introduction The Type B trichothecene mycotoxin deoxynivalenol (DON) is a frequent contaminant of grain products throughout the world and is an important public health concern because of its reported toxicological effects in humans and animals (Pestka 2010b). In experimental mice, DON’s acute effects (hours) include anorexia and robust proinflammatory gene expression and longer-term effects (days) include anorexia and growth suppression (Arnold et al. 1986a; Azcona-Olivera et al. 1995; Flannery et al. 2011; Forsell et al. 1986; Rotter et al. 1996). Children are likely to be exposed to DON to a greater extent than adults due to food consumption per body weight. A survey by the United States Department of Agriculture demonstrated that adults on average consume 189 g grains per day while children consume 191 g/day but because they weigh less they are exposed to a greater amount of grains per kg body weight (Lin and Yen 2007). Given children’s potential susceptibility to growth effects of DON, growth suppression was the primary adverse effect identified by The Joint FAO/WHO Expert Committee on Food Additives (JECFA) as the basis for establishing a tolerable daily intake (TDI) for this toxin (Canady et al. 2001; Iverson et al. 1995). During an exposure study in The Netherlands, it was estimated 80% of children exceeded the TDI for DON (Pieters et al. 2002). These findings raise questions whether DON might affect growth in children or other high consumers of wheat and other grains, and, furthermore, suggest the need for 112 epidemiological studies in linking exposure to this toxin to growth impairment in humans. To assess human exposure to xenobiotics and their resultant effects, biomarkers are often used. Biomarkers of exposure are typically metabolites that correlate with the amount of substance ingested. Relative to DON, Turner and coworkers have developed and validated the exposure biomarker DON-glucuronide (DG) (Turner et al. 2008a). Using urinary DG and free DON, these investigators determined that 98.7% of the UK adults evaluated had been exposed to DON and furthermore, increased DG and free DON were strongly correlated with high grain consumption (Turner et al. 2008b). While these exposure biomarkers are highly informative for quantifying DON consumption, they are not indicative of adverse effects that might result from exposure to this toxin. Biomarkers of effect are typically physiological indicators that predict the likelihood of harmful downstream biological consequences of xenobiotic exposure. Desirable characteristics of candidate effect biomarkers for implementation in epidemiological studies include 1) specificity for the xenobiotic of interest, 2) facile measurement, 3) presence after physiologically relevant doses and 4) validation in humans exposed to the toxin. No biomarker of effect has yet been established for DON, making it impossible to measure adverse physiological outcomes. One candidate effect biomarker for DON-induced growth perturbation is insulinlike growth factor acid-labile subunit (IGFALS) (Amuzie and Pestka 2010; Voss 2010). IGFALS is a plasma protein primarily produced in the liver that is an essential binding partner of the highly critical insulin like growth factor – 1 (IGF1). IGFALS functions to extend IGF1’s half-life and enables it to reach target tissues to modulate growth (Dai 113 and Baxter 1994; Dai et al. 1994; Domene et al. 2011). Mutations in the Igfals gene have been linked to short stature in humans (Domene et al. 2010; Heath et al. 2008). Based on studies in the mouse, our laboratory has recently proposed a mechanistic link between depressed plasma IGFALS and growth suppression that involves suppressor of cytokine signaling 3 (SOCS3)–induced growth hormone receptor signaling impairment (Amuzie and Pestka 2010; Amuzie et al. 2009). Prior investigations in mice have demonstrated that plasma IGFALS is critical for normal growth. For example, Igfals null mice exhibited significantly depressed growth with weight being of 13% less than controls at 10 wk of age (Ueki et al. 2000). IGFALS has been reported to control postnatal growth by affecting the ability of growth hormone to increase IGF1 and by affecting stabilization of IGF-1 and thus, the duration of IGF1 in the plasma (Ueki et al. 2009). Evidence of this was shown in null ALS mice, which exhibited decreased IGF1 levels that could not be normalized through exogenous administration of growth hormone (Ueki et al. 2009). Consistent with this premise, our laboratory has previously demonstrated the inability of exogenous growth hormone to rescue DON-induced reductions of hepatic Igfals in mice, suggesting that growth hormone signaling was impaired (Amuzie and Pestka 2010). There are two key considerations regarding the suitability of IGFALS as an effect biomarker for DON. First, it must be confirmed that decreased plasma levels of IGFALS occur at dietary DON concentrations that affect weight gain in experimental animals. Second, clarification is needed on whether DON-induced decreases in plasma IGFALS are simply the result of reduced food intake or originate from a toxin-mediated pathway such as that proposed involving SOCS3. We addressed these concerns here by testing 114 two hypotheses. The first hypothesis tested was that decreases of plasma IGFALS will correlate with DON-induced depression in weight gain. The second hypothesis tested was that DON-fed mice will exhibit lower plasma IGFALS concentrations than mice consuming identical restricted amounts of control diet. The results presented herein indicate that DON-induced depression of plasma IGFALS was 1) indicative of growth suppression relevant DON doses and 2) a likely result from reduced food intake and aberrant SOCS regulation. Accordingly, IGFALS merits future consideration as a biomarker of effect. Materials and Methods Laboratory Animals and diet. B6C3F1 female mice (3 wk) were purchased from Charles River Breeding (Portage, MI) and were housed on ventilated racks in plastic cages with kiln-dried aspen bedding and filter tops to prevent aerosolization of DON contaminated food. The room was maintained at constant humidity (40-55%) and constant temperature (21C) with a 12-h light/dark cycle. Mice were given fresh food daily placed in 2” high glass containers. Animal experiments were conducted according to the National Institutes of Health guidelines and approved by the Michigan State University Committee on Animal Use and Care. DON (>98% purity by elemental analysis) was obtained from Dr. Tony Durst (University of Ottawa). DON was added to powdered AIN-93G (Research Diets, New Brunswick, NJ) at appropriate concentrations as described previously (Forsell et al. 1986). DON concentration in food was confirmed using Veratox High Sensitivity (HS) 115 enzyme-linked immunosorbent assay (ELISA) (Neogen, Lansing, MI) according to the manufacturer’s instructions. Study Design 1: Sub-chronic dietary DON exposure. Mice (n = 12 per group) were fed AIN-93G powder diet amended with DON at concentrations of 0, 1, 2.5, 5, and 10 ppm. These concentrations were chosen to encompass the NOAEL (1 ppm) and LOAEL (5 ppm) for weight suppression reported in a 2-year chronic feeding study (Iverson et al. 1995). Mice were weighed weekly and 100 µl blood removed via the saphenous vein at wk 5. After 9 wk exposure to DON, mice were euthanized by intraperitoneal (ip) injection of 100 µl sodium pentobarbital (56 mg/kg bw). Blood was collected using heparinized syringes (50 IU, BD, Franklin Lakes, NJ) from the inferior vena cava, mixed and immediately put on ice. Livers were removed for RT-PCR analysis of Socs3 and Igfals mRNA. Blood was centrifuged at 3000 x g for 10 min and plasma collected and frozen at - 80◦C for later IGFALS determination. Study Design 2: Restricted Dietary DON Exposure. The daily consumption of AIN93G diet amended with 15 ppm DON by mice (n = 30) was initially estimated over the period of 1 wk. This dose was chosen based on its ability to both rapidly depress plasma IGFALS and reduce food intake (Amuzie and Pestka 2010). Following the 1 wk trial, the same mice were given a 2 d washout period in which they consumed only unamended control diet. A 2 d washout period was chosen based on data by Pestka and coworkers demonstrating that <2% of the initial 116 DON dose remained in plasma and other tissues of mice after 24 hr indicating rapid metabolism of DON (Pestka et al. 2008). Mice were then randomized into three groups: 1) control diet ad libitum (ad lib), 2) restricted control diet or 3) restricted control diet containing 15 ppm DON. Food intake and weights were measured daily. Mice were sacrificed at 9 d and blood collected for IGFALS measurement as described above. ELISA for IGFALS. IGFALS was assayed by ELISA as previously described (Amuzie and Pestka 2010). Absorbance at 450 nm was measured using a Molecular Devices ELISA Plate Reader (Menlo Park, CA) and IGFALS calculated from a standard curve using Softmax software (Molecular Devices). Quantitative Real-Time PCR. RNA was extracted using Tri-Reagent (Molecular Research Center Inc., Cincinnati, OH) according to the manufacturer’s protocol. For RT-PCR mRNA expression determination, Assays-on-Demand primers for Socs3 and Igfals were utilized along with Taq-man One-Step Real-time PCR Master Mix (Applied Biosystems, Foster City, NY). Amplification and relative Ct values were determined using an ABI PRISM 7900HT Sequence Detection System. Gene expression was quantified relative to β2-microglobulin and fold change determined using the 2(-∆∆Ct) method (Livak and Schmittgen 2001). 117 Statistics. All statistics were performed using Sigma Plot 11.0 (Jandel Scientific, San Rafael, CA). For Study 1, a One-Way ANOVA with Holm-Sidak multiple comparisons procedure was used to determine statistical significance when comparing multiple dose groups during a given week. When normality failed, a One-Way ANOVA On Ranks was used with Dunn’s or Mann-Whitney U test along with a Bonferroni correction. Linear regression analysis was used to determine statistically if a measurement was dosedependent, while a Pearson Product Moment Correlation was used to relate weight to plasma IGFALS. Two animals were not included in the plasma IGFALS wk 5 measurement due to lack of plasma. A Two-Way ANOVA on Ranks (normality failed) with Dunn’s Test or Mann-Whitney U test with Bonferroni correction was utilized to determine statistical significance among dose, weight or food intake and time relative to the control group for Study 2. No measurement of food intake for day 8 – 9 exists for control fed animals due to equipment failure for that measurement. Benchmark Dose Modeling. The benchmark dose is a particular amount of substance given that results in a pre-specified physiological effect. This pre-specified effect is known as the benchmark response (BMR). The United States EPA Benchmark Dose (BMD) Software Version 2.3.1 was utilized to compare benchmark doses (BMD). Wk 5 and wk 8 were chosen for plasma IGFALS and weight, respectively, because they were the most sensitive time points for each parameter based on dose dependency and absolute differences between the control and 10 ppm DON dose groups. Since data needed to be 118 monotonically increasing to be modeled, results were converted to percent reduction in plasma IGFALS or weight relative to the respective control group. Both data sets modeled were considered normal as determined by a Shapiro-Wilk Normality Test. Data were considered continuous with the Hill Model applied to raw dose and response data for both percent reductions of weight and plasma IGFALS. The Hill Model was chosen because it fit the data the best based on Global Goodness-of-Fit criteria, Akaike’s Information Criteron, Chi-squared residuals and graphical verification. Goodness-of-Fit measures determined the model exhibited a dose-response relationship (p<0.05), homogenous variance (p>0.1), equal variance (p>0.1) and the model sufficiently described the data (p>0.1). One standard deviation above the control mean was used to determine the benchmark dose response. The adverse direction of ‘up’ was chosen because increased percent reduction in weight and plasma IGFALS would be considered to negatively impact health. The benchmark dose 95% lower confidence limit (BMDL) was also determined. DON concentrations of 0, 1, 2.5, 5 and 10 ppm were utilized for both weight and IGFALS data. Results Study 1. DON consumption dose-dependently suppresses weight gain and IGFALS DON consumption dose-dependently suppressed weight gain of mice over a 9 wk period (R2= 0.254. p < 0.001). Mice fed control and 1 ppm DON diet gained 7.9 g and 8.3 g, respectively, during the course of the experiment while mice fed 2.5, 5 and 10 ppm gained 7.6 g, 7.3 g and 5.8 g, respectively (Fig. 5.1). Weights of mice fed the 5 119 ppm DON diet were significantly different than the control at 7 and 8 wk, while at 10 ppm DON, weights were significantly different at 5, 7, 8 and 9 wk. Benchmark dose modeling indicated that the BMD using percent reduction in plasma IGFALS at wk 5 was 3.0 ppm DON, while the BMD for percent weight reduction at 8 wk was 4.5 ppm DON. The BMDLs for plasma IGFALS and weight reduction were 1.1 ppm and 2.1 ppm, respectively. Plasma IGFALS at wk 5 was decreased by 20 percent and 29 percent relative to control values in mice fed 5 and 10 ppm DON, respectively (Fig. 5.2A). At wk 9, plasma IGFALS was decreased by 24 percent at 10 ppm DON (Fig. 5.2B). Interestingly, at wk 5, plasma IGFALS was significantly decreased in the 5 ppm but weight was not, suggesting that IGFALS depression preceded weight suppression. For both wk 5 (R = 0.29; p = 0.02) (Fig. 5.3A) and wk 9 (R = 0.41; p = 0.001) (Fig. 5.3B), weight was significantly correlated to plasma IGFALS. The no effect level for IGFALS over the course of the experiment was 2.5 ppm DON. RT-PCR analysis at wk 9 revealed that DON dose-dependently increased hepatic Socs3 mRNA expression (R2 = 0.12, p = 0.007) with 10 ppm DON exhibiting a significant 8-fold difference over control values (Fig. 5.4). A trend toward decreased Igfals mRNA was also evident at 10 ppm but not significant. These findings suggest that aberrant SOCS3 regulation corresponded to weight suppression. 120 Figure 5.1 DON consumption suppresses weight gain. Mice were fed diet containing 0, 1, 2.5, 5 or 10 ppm DON for 9 wk and weights taken weekly. Data (n = 12/gp) are mean ± SEM. * indicates a statistically significant difference relative to the control at that time point (p ≤ 0.05). 121 Figure 5.2 DON consumption suppresses plasma IGFALS concentrations. Mice were .2 fed diet containing 0, 1, 2.5, 5 or 10 ppm DON and plasma IGFALS measured at A) 5 wk and B) 9 wk by ELISA. Data (n = 11 12/gp) are mean ± SEM. 11-12/gp) statistically significant difference relative to the control (p ≤ 0.05). 122 * indicates a Figure 5.3 Plasma IGFALS depression correlates with decreased weight gain. Correlations were performed at A) wk 5 and B) wk 9 (Data n = 59). Correlations were statistically significant when (p ≤ 0.05). 123 Study 2. Plasma IGFALS depression is specific to DON exposure A food restriction study was employed to compare the extent to which DONinduced plasma IGFALS depression is attributable to reduced food intake versus another toxin-mediated mechanism such as aberrant SOCS3-regulation. To achieve this, mice were fed a control diet ad-lib, a control restricted diet and a 15 ppm DON restricted diet. Food consumption in both restricted groups was identical and both were significantly less than the ad-lib fed group (Fig. 5.5). Over the 9 d experimental period, restricted mice ate an average of 37% less than control mice with this translating into an average weight suppression of 7% for DON restricted mice and 6 percent for control restricted mice, respectively (Fig. 5.6). Mice fed restricted control and DON diets had significantly less plasma IGFALS than to those fed control diet ad lib (Fig. 5.7). Food restriction alone significantly decreased plasma IGFALS with control restricted mice exhibiting an 18 percent reduction in plasma IGFALS relative to the ad lib control. Remarkably, mice fed the restricted DON diet exhibited a 33 and 15% reduction in plasma IGFALS relative to the ad lib control and control restricted mice, respectively, indicating that part of DON’s capacity to suppress IGFALS was independent of its anorectic effect. 124 Figure 5.4 Effect of DON on hepatic mRNA expression of Socs-3 and Igfals Mice were gfals. fed diets containing 0, 1, 2.5, 5 or 10 ppm DON over 9 wk and hepati mRNA hepatic expression measured using real time PCR. Data (n = 12/gp) are mean ± SEM. real-time * indicates a statistically significant difference relative to the control (p ≤ 0.05). 125 adFigure 5.5 Comparison of food intakes during restricted feeding. Mice were fed 1) ad libitum (ad-lib) control diet, 2) restricted control diet or 3) restricted diet containing 15 ppm DON for 9 d and food intake measured daily. Data (n = 10/gp) are mean ± SEM. * indicates a statistically significant difference relative to the control at that time point (p ≤ icates 0.05). 126 Figure 5.6 Comparison of body weight changes during restricted feeding. Mice were fed as described in Fig. 6.5 legend. Data (n = 10/gp) are mean ± SEM. * indicates a 5 SEM. statistically significant difference relative to the control at that time point (p ≤ 0.05). 127 Figure 5.7 Comparison of plasma IGFALS levels in following restricted feeding. Mice were fed as described in Fig. 6.5. Terminal plasma IGFALS was measured using LS ELISA. Data (n = 10/gp) are mean ± SEM. significant difference between groups (p ≤ 0.05). 128 Different letters indicate a statistically Discussion Growth suppression in mice was identified by JECFA as the primary adverse effect on which to base the human TDI for DON (Canady et al. 2001). The data presented in this study are the first to demonstrate that dietary DON causes plasma reduction of plasma IGFALS in a dose-dependent manner and that depression of IGFALS precedes DON-induced weight suppression. This investigation further suggests that IGFALS suppression was likely to result from both DON-induced anorexia and a second mechanism, possibly involving Socs3 upregulation (Fig. 5.4). We have recently demonstrated that DON exposure rapidly induces release of the gut satiety hormones peptide YY and cholecystokinin (CCK) and this is likely to contribute to DON-induced anorexia (Flannery et al. 2012). The finding that dietary restriction per se depressed plasma IGFALS relative to the ad lib control is consistent with data from Oster and colleagues showing that consumption of a 40% calorically restricted diet reduced plasma IGFALS levels in rats by 14% and weight by 34% over a 20 d period (Oster et al. 1996). It is possible that DON-induced anorexia could lead to decreases in growth hormone receptors (Clemmons and Underwood 1991), a reported effect of anorexia in rats and thus, cause growth hormone signaling impairment. Anorexia-induced growth hormone impairment results in decreased plasma IGF1 as reported in mice (Al-Regaiey et al. 2005; Dunn et al. 1997) and could potentially lead to depressed IGFALS as well (Fig. 5.8). It should be noted that anorexia-mediated growth suppression is not exclusively linked to growth hormone signaling impairment but might involve other mechanisms. 129 Figure 5.8 Proposed pathways for DON DON-induced weight reduction. DON-induced growth suppression likely results from two parallel pathways based on this and prior studies (Amuzie et al., 2009; Amuzie and Pestka, 2010). First, anorexia resulting from DONinduced gut satiety hormone release could lead to decreases in growth hormone receptors (Clemmons and Underwood, 1991) and signaling, thus lowering plasma IGFALS. Second, growth hormone impairment caused by increased SOCS response to DON-induced proinflammatory cytokines, leads to depressed hepatic IGFALS mRNA and plasma IGFALS protein. Decreased plasma IGFALS destabilizes and reduces the half-life of binding partner IGF1 (Dai and Baxter, 1994), thus, leading to growth life suppression. 130 In our experiments, mice fed restricted DON diet exhibited even lower plasma IGFALS concentrations than did the restricted control indicating that toxin exposure can directly affect IGFALS by mechanisms other than anorexia. We postulate that DON’s additional effects on IGFALS could be a result of upregulated expression of proinflammatory cytokines and subsequently increased SOCS proteins that serve the purpose to reduce an excessive cytokine response. SOCS are group of 8 proteins containing an Src homology 2 (SH2) domain that can block cytokine signaling via the janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway by restricting JAK phosphorylation, specifically by interfering with STAT dimerization and by tagging important pathway signaling molecules with ubiquitin for proteasomal degradation (Rawlings et al. 2004). Notably, SOCS3 has been shown to affect growth of rat hepatoma cells and primary liver cells in a concentration dependent manner by preventing growth hormone stimulated increases in IGFALS mRNA expression (Boisclair et al. 2000). Early marked upregulation of proinflammatory cytokines in DON-exposed mice precedes both decreases in plasma IGFALS and growth, both of which correspond to increased Socs3 expression. Mice gavaged with DON exhibit increased plasma levels and hepatic mRNA IL-6 and TNF-α within 2 h after exposure, levels which rapidly decrease upon hepatic SOCS3 induction (Amuzie et al. 2009). At 5 h, maximum induction of hepatic SOCS3 corresponded to significant decreases in hepatic Igfals expression. Furthermore, a later subchronic mouse feeding study demonstrated DONinduced growth suppression corresponded to increased plasma levels of DON as well as decreased hepatic Igfals expression and plasma IGF1 and IGFALS (Amuzie and 131 Pestka 2010). Taken together, DON-mediated depression of plasma IGFALS likely results, in past, from growth hormone signaling impairment caused by increased SOCS3. The current TDI for DON, 1 µg/kg bw/d, was based on a 2 year mouse study demonstrating the NOAEL for weight suppression was 1 ppm DON. However, using the NOAEL to establish TDIs and reference doses is limited because it relies only the doses evaluated. Furthermore, the NOAEL does not account for the slope of the dose response curve. The EPA has addressed these issues by applying mathematical models to data to obtain a BMD. In our experiments, the BMD and BMDL were lower for percent reduction in plasma IGFALS than percent reduction in weight suggesting that the former may be an early and sensitive predictor of DON’s effects than weight reduction. It was notable that the lowest dose for IGFALS decrease was 5 ppm dietary DON at 5 wk, but increased to 10 ppm by wk 9. Though food intake was not measured in the present study, differences in IGFALS levels over time might have been due to compensation of food intake by wk 9. Indeed, Forsell and coworkers (Forsell et al. 1986) demonstrated during an 8 wk study that mice fed 2 ppm only exhibited decreased food intake up to 6 wk. Furthermore, mice fed high fat diet plus 10 ppm DON exhibited decreased food intake for the first week out of a 45-day exposure (Kobayashi-Hattori et al. 2011). Another reason for the difference between 5 and 9 wk plasma IGFALS might relate to postnatal growth rate in mice which decreases over time (Lee and Dubos 1968). Since IGFALS levels are in part responsible for growth, differences in plasma IGFALS at 5 and 9 wk may reflect the decreased growth rate of mice. 132 Critical phases to employing a putative biomarker of effect are: 1) optimizing methods for acquiring and analyzing the biomarker, 2) characterizing the biomarker in human populations, which includes identifying confounding variables and 3) using the biomarker in human epidemiological studies as a predictor of long-term adverse health effects (Bonassi et al. 2001). While the method of acquiring and measuring plasma IGFALS is now reliably demonstrated in the mouse (Amuzie et al. 2011; Amuzie and Pestka 2010; Amuzie et al. 2009; Kobayashi-Hattori et al. 2011), the correlation between plasma IGFALS and DON’s weight effects in other species has yet to be established. Furthermore, treatment of various cell lines with other trichothecene mycotoxins such as T-2, nivalenol, 3-acetyl deoxynivalenol (ADON), 15-ADON and fusarenon X have resulted in increased proinflammatory cytokines such as IL-6 and TNF-α (Sugita-Konishi and Pestka 2001; Wang et al. 2012). Therefore, these other toxins could potentially decrease plasma IGFALS via increased SOCS expression resulting from aberrant proinflammatory cytokine release. Additional experiments are needed to determine the specificity of suppressed plasma IGFALS to DON as compared to other trichothecenes. Finally, an important consideration for using plasma IGFALS as a biomarker of effect in humans will be heterogeneity of plasma IGFALS levels within the human population resulting from age, sex, genetic polymorphism, food consumption patterns, socio-economic status, environmental factors and sickness behavior associated by infection and inflammation. To summarize, the data presented suggest that: 1) DON-induced depression of plasma IGFALS correlated with reduced body weight gain, 2) depression of plasma IGFALS preceded significant weight effects occurred and 3) decreased plasma IGFALS 133 might be explainable by at least two mechanisms—anorexia induction and aberrant SOCS3 regulation. Future experiments should focus on the specificity of depressed IGFALS to DON with a focus on various species and ages. If specificity is established, plasma IGFALS might be used in epidemiological studies, along with biomarkers of exposure, to predict the risk of growth suppression caused by DON in humans. 134 CHAPTER 6: DON- INDUCED WEIGHT LOSS AND ANOREXIA IS REVERSIBLE IN THE DIET-INDUCED OBESE MOUSE Abstract Obesity is a growing worldwide epidemic that results in increased risk of heart disease, cancer, diabetes, metabolic disease and the increased potential for susceptibility to environmental toxins’ adverse physiological consequences. Fusarium graminearum is a fungus that produces a toxic secondary metabolite known as deoxynivalenol (DON), which is consistently reported to impair weight gain or cause weight loss in monogastric animals. DON-induced weight changes largely occur through DON’s capacity to cause anorexia. In order to evaluate obesity as a risk factor for DON’s effects, we assessed the reversibility of DON induced weight loss in diet-induced obese in mice fed 10 ppm DON over 29 wk with the hypothesis that DON’s weight effects were reversible upon dietary DON removal. We further evaluated DON’s adverse effects on food intake upon the addition and removal of DON from very high fat diet. Results demonstrated that DON-induced weight loss was reversible in diet-induced obese mice and this effect corresponded to a robust hyperphagic response. Due to the rapid reversibility of DON-induced weight loss and anorexia, diet-induced obese mice were considered no more susceptible to DON’s weight loss effects than what has been reported in their lean counterparts. 135 Introduction As of 2011, 36% of the United States’ adult population over 20 years of age was considered obese (2012). By 2030, it was estimated that 44% of the United States’ adult population will be obese with 13 states exceeding a 60% obesity rate (Levi et al. 2012). Obesity is defined as having a Body Mass Index (BMI = weight (kg)/ height (m2)) of 30 or greater with an abnormally high percentage of body fat (Kelly et al. 2008). Obesity causes an increased risk of developing metabolic disease, high blood pressure, heart disease, particular types of cancer and leaves individuals more susceptible to some environmental toxins (Bianchini et al. 2002; Bray 2004; Brewer and Balen 2010; Lin et al. 2010; Manson et al. 1990; Meigs et al. 1997; Must et al. 1999). One potential environmental toxin that obese individuals may be more susceptible to is the mycotoxin deoxynivalenol (DON), a secondary metabolite of Fusarium graminearum. DON is a mycotoxin that often contaminates grain products at low levels worldwide. Acute DON exposure in humans is associated with gastrointestinal illness and vomiting, while sub-chronic, low- level exposure in experimental animals results in proinflammatory cytokine upregulation, anorexia, weight changes and growth hormone dysregulation (Amuzie and Pestka 2010; Flannery et al. 2011; Hughes et al. 1999; Trenholm et al. 1984). Of these adverse effects, DON’s capacity to cause weight suppression in mice over a 2 yr study period has functioned as the foundation for its regulation in grain products (Canady et al. 2001; Iverson et al. 1995). Many mechanisms are hypothesized for how DON-induced weight suppression occurs. First, anorexia serves as a primary mechanism by which DON impairs weight 136 gain (Amuzie et al. 2011; Iverson et al. 1995; Prelusky 1997; Trenholm et al. 1984). DON-induced anorexia likely results from a variety of mechanisms. First, DON’s capacity to elicit the ‘fed pattern’ of gut motility in pigs, rats and mice (Fioramonti et al. 1993; Krantis and Durst 2001a). Additionally, in mice, DON treatment has been shown to release the gut satiety hormones peptide YY (PYY) and cholecystokinin (CCK), though through the use of Y2 receptor antagonists, PYY was shown to be more efficacious at relieving DON-induced anorexia (Flannery et al. 2012). A second likely mechanism by which DON impairs weight gain is through the decrease of the important growth protein, insulin-like growth factor acid-labile subunit (IGFALS) (Amuzie and Pestka 2010; Voss 2010). Amuzie and coworkers demonstrated in growing mice fed 20 ppm DON, that DON significantly depressed IGFALS over an 8 wk time period (Amuzie and Pestka 2010). Taken together, DON-induced weight suppression is a chief concern in human susceptibility to the adverse effects of this mycotoxin. Interestingly, diet-induced obese mice were shown to be susceptible to DON’s weight-suppressive effects, an effect that corresponded to decreased food consumption. Amuzie and coworkers demonstrated that diet-induced obese (DIO) mice fed very high fat diet (VHFD; 60% kcal from fat) amended with 10 ppm DON exhibited a 23% and 40% reduction of weight and periuterine fat, respectively (Amuzie et al. 2011). Also in these studies, mice fed 10 ppm DON exhibited significant anorexia. In a separate study, DIO mice exhibited a 13% weight loss, which corresponded to decreased food intake over a 51 d period (Kobayashi-Hattori et al. 2011). Despite DON causing weight loss and anorexia in DIO mice, the reversibility of these effects are not yet known. The goal of this study was to evaluate the susceptibility of obese mice to 137 DON’s weight loss effects through the addition and removal of DON from VHFD. Furthermore, to establish the role of anorexia in weight changes, food intake was measured during these periods. Results suggested DON’s weight suppressive effects in DIO mice were reversible, an effect which corresponded to orexigenic food intake behavior. Materials and Methods Mice. Adult B6C3F1 (10 – 11 wk) mice were purchased from Charles River Breeding (Portage, MI) and acclimated to diet and environment for 1 wk before beginning experiments. Mice were housed one per cage in polycarbonate cages containing aspen bedding, nestlets and wire-top lids. Housing environment was according to a 12 h light/dark cycle and kept at a constant temperature (21 – 24° C) and humidity (40 – 55%). Mice were allowed free access to food and water for the duration of experiments. The Institutional Animal Care and Use Committee at Michigan State University approved all experiments involving mice. Diet. Very high fat pellet diet (VHFD; D12492) containing 60% kcal from fat was purchased from Research Diets Inc. (New Brunswick, NJ). Research Diets incorporated DON at 10 or 20 ppm into VHFD using a cold extrusion method. DON was obtained 138 from Fusarium graminearum cultures, extracted and purified according to a previously published method (Clifford et al. 2003). Food Intake Measurements. Mice were housed one per cage in cages containing aspen bedding sifted to uniform size for facile measurement of food spillage. Food was placed in glass jars with metal lids and weighed daily to establish intake. Experimental Design Study 1. The goal of this study was to assess weight upon the addition and removal of DON from VHFD. Four different diets were administered to 4 groups (n = 8/gp; 3/cage) of naïve mice to assess the effect of DON-removal on weight of DIO mice. One group of mice was given 10% kcal from fat diet (D12450B; Research Diets Inc., New Brunswick, NJ) and the other 3 groups given VHFD to induce obesity. At wk 9, two groups of mice given VHFD were switched to either VHFD + 10 ppm DON or VHFD + 20 ppm DON for 8 wk. Then, at wk 17 mice fed VHFD + DON were switched back to VHFD for the duration of the experiment. Weights were recorded weekly. Experimental Design Study 2. The goal of this study was to assess food intake upon the addition and subsequent removal of DON from VHFD. Mice were fed VHFD for 8 wk to induce obesity. Then mice were split into two groups and one group was continued on VHFD while the other group was given VHFD + 10 ppm DON for 5 wk. At wk 13 mice given 139 VHFD + 10 ppm were switched back to unamended VHFD. Weights were taken weekly. Daily food intake and weights were measured from 52 – 66 d to capture the transition period from VHFD to VHFD + 10 ppm DON. Daily food intake and weights were measured 88 – 96 d to encapsulate the transition from VHFD + 10 ppm DON back to VHFD. Statistics. Statistical comparisons between two diet groups were made for each time point using a Student’s T-test unless normality failed and a Mann-Whitney Rank Sum Test was executed. Multiple diet groups at a given time point were analyzed for statistical significance using a One-Way Analysis of Variance (ANOVA) with Holm-Sidak multiple comparison procedure and when normality failed a One-Way ANOVA on Ranks with a Tukey test was utilized. Statistical significance was obtained when p < 0.05. Results Study 1. Dietary DON removal results in complete weight compensation in diet-induced obese mice At 9 wk, mice fed VHFD weighed on average between 35.4 and 37 g, while mice fed 10% kcal from fat diet weighed 24 g (Fig 6.1). Mice fed VHFD were then divided into 3 subgroups with two groups given VHFD amended with DON. By wk 17, weights of mice fed VHFD + 20 ppm DON were similar to the low-fat control. At wk 17, weights of mice fed 10% fat diet, VHFD, VHFD + 10 ppm DON and VHFD + 20 ppm DON were 28.4, 51.7, 33.3 and 29.5, respectively (Fig 6.1). DON fed mice were switched back to 140 Table 6.1 Statistics for Fig. 6.1. Different letters indicate a statistically significant difference between diet groups at that time point (p < 0.05). LFD VHFD VHFD + 10 ppm DON VHFD + 20 ppm DON LFD VHFD VHFD + 10 ppm DON VHFD + 20 ppm DON 0 b b 1 b a 2 b a 3 b a 4 b a 5 b a Week 6 7 8 b b b a a a b a a a a a a a a a a a ab ab ab b a a a a a a a a a a a ab 9 b a 10 11 12 13 14 b b b b b a a a a a b b Week 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 b b b b b b b b b b b b b b b a a a ad a a a a a a a a a a a b a ac d c c c ac a ac a ac a a a a b bc b d d d bc c c c c a a a 141 induced Figure 6.1 Weights of diet-induced obese mice upon addition and removal of dietary DON. Obesity was induced in 3 groups of mice with 2 groups being switched from VHFD to VHFD + 10 ppm DON or VHFD + 20 ppm DON at 9 wk as indicated by the first arrow. At 17 wk mice fed DON were switched back to unamended VHFD as indicated . VHFD by the second arrow. Mice fed 10% kcal from fat served as a low . low-fat control group. ntrol Weights were assessed weekly. Data (n = 8/gp) are mean ± SEM. 142 unamended VHFD at wk 17. By wk 29, mice fed DON previously weighed similar to those fed VHFD for the entire study period suggesting DON-induced weight suppression was completely reversible. Study 2. Dietary DON removal simulates appetite in diet-induced obese mice Two groups of mice were fed VHFD for 56 d resulting in average weights 34 – 35 g (Fig. 5.2). One group of mice was switched to VHFD + 10 ppm from 56 d to 92 d. By 92 d mice fed VHFD + 10 ppm exhibited a 3 g weight loss with the average weight of 31 g while mice fed VHFD weighed 43 g on average (Fig. 6.2). DON diet was removed at 92 d for mice fed VHFD to assess the effects of DON removal on food intake. Food intake dropped by 66% (57 d) relative to control values upon switching mice from VHFD to VHFD + 10 ppm and this corresponded to a rapid decrease in weight (Fig. 5.3). Interestingly, by 66 d and 90 d DON fed mice ate an average of 22% and 0% less, respectively, than control values suggesting complete food intake recovery, though weight was still depressed by 24% (10 g) relative to control values in mice fed VHFD + 10 ppm DON (Fig. 6.3). Upon DON removal (92 d) mice exhibited orexigenic behavior consuming 65% more food than controls (Fig. 6.4). By study termination, mice previously fed DON were consuming 16% more food than mice fed VHFD. Taken together, dietary DON significantly decreased food intake in mice and upon DON removal food intake was significantly increased suggesting DON could mediate hormonal signals responsible for food intake. 143 Figure 6.2 Weights of diet-induced obese mice upon addition and removal of dietary induced DON. As indicated by the first arrow, at 56 d VHFD was interchanged with VHFD + 10 ppm DON in half the mice. At 92 d, DON fed mice were switched back to non . noncontaminated VHFD as indicated by the second arrow The black lines indicate where arrow. food intake was measured (Fig 5.3 and 5.4). Weights were taken weekly. Data (n = 9 10/gp) are mean ± SEM. Different letters indicate a statistically significant difference between diet groups at that time point ( < 0.05). (p 144 Figure 6.3 Food intake and weights of mice before and after dietary DON addition. Daily A) food intake and B) weights of mice were assessed before and after a dietary addition of 10 ppm to VHFD as indicated by the black arrow. Data (n = 9 - 10/gp) are arrow. mean ± SEM. Different letters indicate a statistically significant difference between diet statistically groups at that time point (p < 0.05). 145 Figure 6.4 Food intake and weights of mice before and after dietary DON removal. Daily A) food intake and B) weights of mice were assessed before and after removal of 10 ppm dietary DON as indicated by the black arrow Data (n = 9 - 10/gp) are mean ± arrow. 146 (Figure 6.4 cont’d) SEM. Different letters indicate a statistically significant difference between diet groups at that time point (p < 0.05). Discussion In obese individuals, chronic metabolic disturbances and increased systemic inflammation may lead to increased susceptibility to environmental toxins (Emanuela et al. 2012). Here we examined the sensitivity of obese mice in response to the mycotoxin DON using weight loss reversibility and food intake as measures of toxin susceptibility. The data presented herein are the first to show that DON-induced weight loss is reversible upon DON removal and that upon DON removal, mice exhibit a hyperphagic response, an effect likely contributing to the reversibility of DON-induced weight effects. Evidence for obese individuals being more sensitive to environmental toxins was shown in humans exposed to perifluorinated chemicals via a variety of commonly used products. In obese individuals a significantly higher association between serum alanine aminotransferase, a biological marker of liver injury, and serum perfluoroctanic acid was demonstrated compared to lean individuals thus signifying obese individuals may be more susceptible to the adverse effects of this persistent environmental toxin (Lin et al. 2010). In contrast to these findings, diet- induced obese mice appeared similarly sensitive to DON-induced weight loss as non-obese mice. Evidence of this was shown in two separate studies that demonstrated the lowest observed adverse effect for DONinduced weight suppression was 5 ppm dietary DON in both obese and non-obese mice (Amuzie et al. 2011; Iverson et al. 1995). 147 DON is rapidly distributed and metabolized in mice, an effect which may contribute to weight reversibility upon DON removal. For example, in mice administered 25 mg/kg bw DON orally, DON was detected in plasma, liver, spleen and brain within 5 m of administration (Pestka et al. 2008). In the same study, only 2% of initial plasma levels were present after 24 h suggesting rapid metabolism of this toxin. Anorexia, a primary contributing factor to DON-induced weight suppression, was also shown to be rapidly reversed in mice. In mice administered 1 mg/kg bw DON via intraperitoneal injection (ip.) DON- induced anorexia was attenuated by 3 h post exposure (Flannery et al. 2011). Therefore, the rapid metabolism of DON and reversal of anorexia likely contributed to the reversibility of DON-induced weight loss in DIO mice. The physiological mechanisms controlling food intake are multifaceted and include but are not limited to complex chemical interactions between peripheral signaling molecules such as gut satiety hormones and anorexigenic and orexigenic peptides within the central nervous system (Schwartz et al. 2000). Mice administered DON orally exhibited increased hypothalamic mRNA levels of the anorexigenic peptides pro-opiomelanocortin and cocaine-amphetamine regulated transcript, an effect possibly contributing to DON’s anorectic potential (Girardet et al. 2011b). Furthermore, PYY, a gut satiety hormone that favors increased hypothalamic anorexigenic mRNA expression, was shown to be rapidly elevated in mice given an ip. injection of DON (Challis et al. 2003; Flannery et al. 2012). Interestingly, in our studies mice showed partial attenuation of DON-induced anorexia 5 d post DON exposure and a robust hyperphagic response upon dietary DON-removal. Both the attenuation of DON-induced anorexia and the hyperphagic response upon DON removal may be due to increased 148 hypothalamic expression of orexigenic peptides. Calorie restricted mice exhibited weight loss along with increase mRNA expression of the orexigenic peptides agouti-related peptide (AgRP) and neuropeptide Y (NPY) (Yu et al. 2009). Indeed, Kobayashi-Hattori and coworkers showed attenuation of DON-induced anorexia in DIO mice after 10 d exposure and this recovery corresponded to increased hypothalamic mRNA expression of AgRP (Kobayashi-Hattori et al. 2011). In our model, it is possible that increased hypothalamic mRNA expression of AgRP contributed to attenuation of DON-induced feed refusal and subsequent hyperphagic response, though this peptide was not measured in these studies. In conclusion, obese mice appeared to be equally sensitive to DON’s weight suppressive effects as was reported for non-obese counterparts. This equal sensitivity likely stems from rapid DON metabolism, attenuation of DON-induced anorexia and the robust hyperphagic response upon DON removal. Since obese individuals are reported to exhibit systemic inflammation, future studies should focus on DON’s potential to exacerbate this systemic inflammation. 149 CHAPTER 7: CONCLUSIONS Understanding DON-induced anorexia is critical for comprehensive risk assessment of this mycotoxin because elucidating these mechanisms will allow for 1) mitigation of anorexia and growth suppression caused by DON in livestock and potentially, humans 2) application of these mechanisms to DON-induced vomiting and 3) identification of particularly susceptible populations to DON’s adverse weight effects. Data presented herein demonstrate the sensitivity of both growing and obese mice to DON’s weight and anorectic effects. Therefore, this dissertation identified novel mechanisms by which DON caused anorexia by using a mouse model to first assess the dose and timing of DON-induced anorexia and then relating this information to elucidating mechanisms of anorexia. Data presented within this dissertation are the first to suggest both peripheral and central mechanisms contribute to DON’s anorectic effects (Figure 7.1). More specifically, DON administration resulted in release of the gut satiety peptides CCK and PYY with PYY shown to be more efficacious in preventing DON-induced anorexia though the use of peripheral injection of the Y2 inhibitor BIIE0246. However, only peripheral rather than central PYY was shown to be important for DON’s anorectic effects because centrally administered BIIE0246 did not attenuate anorexia. In the periphery, PYY acts on the vagus nerve to cause satiety (Baraboi et al. 2010). In experiments presented herein, vagotomized mice exhibited DON-induced anorexia suggesting the vagus nerve was not necessary for anorexia to occur. The discrepancy between data suggesting PYY is an important peripheral satiety factor in 150 our DON model and the data demonstrating PYY-induced satiety signaling through the vagus nerve was not necessary for DON-induced anorexia to occur is perplexing. Several alternative peripheral and central mechanisms could contribute to DON’s anorectic effects (Figure 7.2). First, PYY is expressed within the oral cavity and salivary PYY could decrease food intake through innervating cranial nerves. Salivary PYY decreases food intake as evidenced by Acosta et. al (2011) who demonstrated that 1) PYY was significantly increased in human saliva upon meal consumption, an effect independent of peripheral PYY, 2) PYY was expressed on circumvallate papillae, which contain taste buds and are innervated by the glossopharyngeal nerve, 3) Y2R receptors were also located on basal epithelial receptors on the tongue and 4) administration of aerosolized PYY to the oral cavity resulted in decreased food intake and weight gain in rodents. Thus, it is feasible that DON consumption could increase salivary PYY, which in turn decreases food intake through gustatory neuronal pathways. A second hypothesis involving central signaling would be the capacity of DON to reach the area postrema (AP), a circumventricular organ responsible for vomiting in capable species. DON reaching the AP could result in DON stimulating the chemoreceptor trigger zone, an area responsible for vomiting. Subsequently, DON exposed mice might exhibit anorexia because they are incapable of vomiting. Indeed, decreased food intake resulting from a conditioned taste aversion to saccharin caused by DON, was abolished by an AP ablation suggesting the AP likely plays a role in DON’s anorectic effects (Ossenkopp et al. 1994). 151 Figure 7.1 Suggested model of DON induced anorexia based on data collected within DON-induced collec this dissertation. 1) Systemic DON administration causes a release of peptide YY (PYY). The role of peripherally released PYY in anorexia was confirmed using the NPY2 ripherally inhibitor BIIE0246. 2) PYY has the capacity to enter the blood to reach the circumventricular organs and hypothalamus to decrease food intake; however, ; antagonizing central NPY2 receptors resulted in DON induced anorexia suggesting Y2 DON-induced PYY’s satiety effects are mediated by the periphery. 3) Vagotomized mice exhibited re DON-induced anorexia suggesting PYY’s peripheral satiety effects may be mediated by other mechanisms, potentially the glossopharyngeal nerve and 4) DON can enter the brain and hypothalamus at a dose and time consist consistent with feed refusal. Furthermore, ICV injection of DON has been reported to decrease food intake (Girardet et al. 2011b). Girardet 2011b Therefore, DON likely acts centrally as well as peripherally to reduce food intake. 152 Third, it is possible that DON could enter inside the brain inside the blood brain barrier to decrease food intake. This is suggested by the observation that intracerebroventricular injection of DON resulted in decreased food intake and increased activation of POMC neurons located in the ARC and NTS (Girardet et al. 2011b). One manner peripheral DON may enter the brain to have these effects is though the breakdown of claudin, a protein critical for maintenance of the blood brain barrier (Mullier et al. 2010). In fact, it was demonstrated that DON increased intestinal permeability by decreasing claudin expression in jejunum explants of piglets (Pinton et al. 2010). A final alternative mechanism is that DON’s anorectic effects stem from increased central nesfatin-1. Nesfatin-1 is a potent central anorexigenic peptide located within the brain, GI mucosa and adipose tissue (Oh et al. 2006; Ramanjaneya et al. 2010; Stengel et al. 2009; Zhang et al. 2010). Its anorectic effects are a result of decreased NPY/AgRP expression within the arcuate nucleus of the hypothalamus (Palasz et al. 2012). Interestingly, nesfatin-1 was shown to be elevated in central areas important for food intake in mice administered DON (Girardet et al. 2011b). In conclusion, data presented within have advanced knowledge of DON-induced anorexia and weight suppression, information applicable to risk assessment. Furthermore, this dissertation has set forth future considerations for determining mechanisms of DON-induced anorexia and identifying populations susceptible to DON’s anorectic effects. Future research should seek to identify additional susceptible populations such as the elderly, people with anorexia nervosa and people with cancer and decipher why these populations might be particularly sensitive. 153 DON-induced anorexia. 1) Oral DON results in Figure 7.2 Alternative hypotheses for DON increased PYY expression within the circumvallate papillae resulting in NPY2 receptor activation within the basal epithelium. This action might result in satiety signaling through the glossopharyngeal nerve, 2) DON within the bloodstream could reach the ough am area postrema to cause anorexia, rather than vomiting since mice are incapable of vomiting, vomiting, and 3) Both peripheral and central DON result in increased central nesfatin nesfatin-1, (Girardet et al. 2011b) which leads to decreased food intake. 154 APPENDICES 155 Appendix A: The Role of the 5-HT -1B, -2C, and -3 Receptors in DeoxynivalenolInduced Anorexia in the Mouse Abstract Deoxynivalenol is a secondary toxic metabolite elaborated from the fungus Fusarium. DON is a frequent foodborne contaminant most often found in grain products and upon consumption, has been reported to adversely affect human and animal health. DON’s capacity to cause weight suppression in growing mice, at effect consistently attributed to anorexia, serves as the foundation for its regulation. Despite the relationship between DON-induced weight suppression and anorexia, mechanisms of DON-induced anorexia are not well understood. One potential mechanism by which DON could exert its anorectic effects is through serotonergic (5-hydroxytryptamin; 5-HT) signaling. 5-HT is a neurotransmitter located in the gut and brain that serves multiple functions including the reduction of food intake. Here, we hypothesized DON administration would increase plasma levels of 5-HT which would reduce food intake through the 5-HT-3, -1B and -2C receptors. To test this hypothesis we used a standardized mouse model to compare DON’s anorectic effects in mice pre-treated with saline or pretreated with the 5-HT-3, 1B and -2C receptor antagonists granisetron, SB242289 (inverse agonist) and SB242084, respectively. Results showed DON was incapable of increasing plasma 5HT levels. Furthermore, mice pretreated with saline and the 5-HT receptor antagonists exhibited similar anorexia indicating these receptors were not important in DON-induced anorexia. Taken together, action of the 5-HT-3, -1B and -2C receptors do not appear to be critical for DON-induced anorexia to occur. 156 Introduction Each year in the United States 38.4 million people suffer from acute gastroenteritis caused by unidentified foodborne factors (Scallan et al. 2011). Deoxynivalenol (DON), a toxic secondary metabolite of Fusarium species, is one potential unidentified foodborne factor contributing to acute gastroenteritis. DON is a trichothecene mycotoxin prevalent in finished and unfinished grain products worldwide because of its heat and processing resistance (Rodrigues and Naehrer 2012). Recent studies demonstrated DON was the most prevalent mycotoxin in North America (Rodrigues and Naehrer 2012; Voss and Snook 2010), which likely results in universal human exposure. Acute DON intoxication results in symptoms of acute gastroenteritis in humans such as nausea, vomiting, diarrhea and abdominal pain (Canady et al. 2001). Adverse physiological consequences in experimental animals upon DON exposure are vomiting (susceptible species), anorexia, dysregulation of the growth hormone axis, weight loss and weight suppression (Pestka 2010b). Children are considered especially susceptible to DON’s weight effects and thus, weight suppression functions as the adverse effect by which DON was regulated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) (Pieters et al. 2002). DON’s regulation was founded on a two-year study demonstrating DON caused significant weight suppression in mice, an effect attributed in part to anorexia (Iverson et al. 1995). Numerous other investigations have also confirmed the findings that frequent DON-exposure results in weight suppression, a consequence of decreased food intake (Amuzie et al. 2011; Canady et al. 2001; Forsell et al. 1986; Young et al. 1983). Despite 157 its role in DON-induced weight suppression, the mechanisms by which DON causes anorexia are not well understood. Food intake is a complex behavior controlled by many interrelated mechanisms involving the gastrointestinal (GI) tract and central nervous system (CNS). One potential mechanism by which DON could elicit anorexia is through 5-hydroxytryptamine (5-HT; serotonin), a neurotransmitter that serves many functions including to decrease meal size (Simansky 1996). The majority of 5-HT is located in enterochromaffin (EC) cells lining the GI tract, while the rest is located in the CNS (Lam et al. 2010). Upon food consumption, 5-HT is released from EC cells into the blood where it acts on receptors within the enteric nervous system and the vagus nerve to alter gut motility and decrease food intake (Zhu et al. 2001). However, blood 5-HT can not cross the blood brain barrier meaning its action on brain structures must be via circumventricular organs (Simansky 1996). Inside the brain, 5-HT decreases food intake through the action of the 5-HT-1B and 5-HT-2C receptors which exert their action on anorexigenic expressing neurons (proopionmelanocrotin, POMC) (Zhou et al. 2005c). These neurons then signal through melanocortin – 4 receptors to decrease food intake. Both the 5-HT-1B and 5-HT-2C are G-coupled protein receptors and have been extensively linked to decreased food intake through the use of respective receptor agonists and antagonists, and through the use of genetic knock-out mouse models (Halford and Harrold 2012; Lam et al. 2010). The majority of investigations linking DON’s adverse effects to 5-HT have been performed in the pig. More specifically, the 5-HT-3 receptor antagonists ICS 205 930 and BRL 43694 A blocked DON-induced vomiting in the pig (Prelusky and Trenholm 1993), while the 5-HT-2 receptor antagonists cyproheptadine and sulpiride were only 158 slightly effective. Though 5-HT was not found in the blood of pigs administered DON (Prelusky 1994), its metabolite 5-HIAA was discovered in the cerebral spinal fluid suggesting DON may affect central serotonergic signaling (Prelusky 1993). In the mouse, the 5-HT-3 receptor antagonists ondansetron and granisetron blocked DONinduced changes in gut motility (Fioramonti et al. 1993). Currently, only one investigation into the role of 5-HT in DON-induced anorexia has been performed. Prelusky and coworkers demonstrated that the 5-HT-2 receptor antagonist cyproheptadine attenuated DON-induced anorexia suggesting DON likely acts through the 5-HT-2 receptor to reduce food intake (Prelusky et al. 1997). However, cyproheptadine is a non-specific receptor inhibitor and may have negated DON’s anorectic effects through other actions. Taken together, little is understood about the role of 5-HT and its receptors in DON-induced anorexia. Here, we sought to establish the function of serotonin and its receptors in DONinduced anorexia in the mouse. First we determined DON’s capacity to release serotonin. Then, through the use of specific 5-HT-3, -1B and -2C receptor inhibitors granisetron, SB224289 (inverse agonist) and SB242084, respectively, we clarified the function of these receptors in DON-induced anorexia. Data suggested that the 5-HT-3, -1B and -2C receptors were not critical for DON’s anorectic effects and furthermore, DON administration was not able to increase plasma 5-HT. These data suggest serotonergic signaling through these receptors may not play an important role in DONinduced anorexia. 159 Materials and Methods Mice. Naïve B6C3F1 female mice (7 – 9 wk) were purchased from Charles River Breeding (Portage, MI) and housed singly upon arrival. Mice were acclimated to the environment and 60% kcal from fat high fat diet (HFD; Research Diets Inc, New Brunswick, NJ) for 1 wk before beginning experiments. Mice were housed under ambient temperature and humidity in plastic Innovive cages (San Diego, CA). All animal experiments performed were approved by the Institutional Animal Care and Use Committee at Michigan State University. Drugs. DON was kindly provided by Dr. Anthony Krantis from the University of Ottawa and purity confirmed using HPLC. For experimental use, DON was dissolved in phosphate buffered saline. 5-HT, the 5-HT-3 receptor inhibitor granisetron, the 5-HT 2C/1B receptor agonist meta-chlorophenyl piperazine (m-CPP), the 5-HT-2C receptor antagonist SB242804 and the 5-HT-1B receptor inverse agonist SB224289 were all purchased from Tocris Bioscience (Minneapolis, MN). For use, 5-HT was dissolved in PBS as was granisetron and m-CPP. SB242084 was dissolved in 5% dimethyl sulfoxide (DMSO) and PBS. SB282289 was dissolved in distilled water for use. All drugs were administered via intraperitoneal (ip.) injection. Food intake. Food intake measurements were assessed according to a previously published protocol (Flannery et al. 2012; Flannery et al. 2011). 160 Experimental Design Assessment of DON’s capacity to increase plasma 5-HT levels. For assessing plasma serotonin release upon DON exposure, fasted mice were administered 0, 1 or 5 mg/kg bw DON in 50 µl PBS and sacrificed using 56 mg/kg bw sodium pentobarbital at 0, 15, 30, and 120 min with time of sacrifice depending on dose. Blood was removed via the inferior vena cava and placed in EDTA purple top tubes. Plasma was obtained from blood by centrifugation at 3600 g for 10 min at 4° and used for subsequent plasma 5C HT measurements. The role of the 5-HT-3 receptor in 5-HT and DON-induced feed refusal. Since the 5-HT3 receptor has been mostly implicated in 5-HT-induced vomiting, we sought to determine the role of the 5-HT-3 receptor in 5-HT-induced anorexia. Mice were administered treatments according to experiment 1, Table A.A.1. The second injection occurred 30 min after the first. Food intake was measured at 60 and 120 min. Next we sought to establish a role for the 5-HT-3 receptor in DON-induced feed refusal. Mice were administered treatments according to experiment 2, Table A.A.1. The second injection occurred 30 min after the first. Food intake was measured at 60 and 120 min. The role of 5-HT-2C and -1B receptors in DON-induced feed refusal. First we confirmed the efficacy of the 5-HT-2C receptor SB242084 in attenuating the 5-HT-2C/1B receptor agonist m-CPP induced anorexia in the mouse (experiment 3A). Next, in experiment 3B, SB242084 was administered to determine the role of the 5-HT-2C receptor in DONinduced feed refusal. Doses, volumes and groups are listed in Table A.A.1. The second 161 injection was given 30 min after the first. Food intake was measured at 10, 60 and 120 min. After experiment 3A and 3B mice were rested for 1 wk and randomized for experiment 4A and 4B. Next we confirmed the efficacy of the 5-HT1B receptor SB224289 in attenuating the 5-HT-2C/1B receptor agonist m-CPP induced anorexia (experiment 4A). Then, SB224289 was administered to determine the role of the 5-HT-1B receptor in DON-induced feed refusal (experiment 4B). Doses, volumes and groups are listed in Table A.A.1. The second injection was given 30 min after the first. Food intake was measured at 10, 60 and 120 min. 5-HT ELISA. Plasma 5-HT was measured using a Serotonin ELISA kit from Enzo Life Sciences (Farmingdale, NY) according to the manufacture’s instructions. Plate absorbance was read at 405 nm and 5-HT amounts calculated from a standard curve. Statistics. Statistics calculated using Sigma Plot 11 (Systat Software Inc., San Jose, CA). For all studies a One-way Analysis of Variance (ANOVA) was employed with a Holm-Sidak multiple comparison procedure for each time point. When normality failed a One-way ANOVA with Tukey test was utilized. Data are considered statistically significant when the comparison has a p-value < 0.05. 162 Table A.A.1 Doses and volumes for experimental (Exp.) groups during the evaluation of 5-HT -3, -1B, -2C receptor inhibitors in agonist- and DON-induced anorexia. Exp. Experiment Group Injection 1 (50 µl) Injection 2 (50 µl) 1 PBS PBS 1 Block 5-HTinduced anorexia using granisetron (GRN) 2 3 4 10 mg/kg bw GRN PBS 10 mg/kg bw GRN PBS 5 mg/kg bw 5-HT 5 mg/kg bw 5-HT 1 PBS PBS 2 Block DONinduced anorexia using GRN 2 3 4 10 mg/kg bw GRN PBS 10 mg/kg bw GRN PBS 1 mg/kg bw DON 1 mg/kg bw DON 1 5% DMSO in PBS PBS 2 3 mg/kg bw SB242084 3 5% DMSO in PBS 4 3 mg/kg bw SB242084 PBS 3 mg/kg bw mCPP 3 mg/kg bw mCPP 1 5% DMSO in PBS PBS 2 3 4 3 mg/kg bw SB242084 5% DMSO in PBS 3 mg/kg bw SB242084 PBS 1 mg/kg bw DON 1 mg/kg bw DON 1 Distilled water PBS 2 3 mg/kg bw SB242084 3 Distilled water 4 3 mg/kg bw SB242084 PBS 3 mg/kg bw mCPP 3 mg/kg bw mCPP 1 Distilled water PBS 2 3 4 3 mg/kg bw SB242084 Distilled water 3 mg/kg bw SB242084 PBS 1 mg/kg bw DON 1 mg/kg bw DON 3A 3B 4A 4B Block m-CPPinduced anorexia using SB242084 Block DONinduced anorexia using SB242084 Block m-CPPinduced anorexia using SB224289 Block DONinduced anorexia using SB224289 163 Results DON does not increase plasma 5-HT levels in the mouse. There were no remarkable differences at 15, 30 and 120 min in plasma 5-HT between mice dosed with 0, 1 or 5 mg/kg bw DON suggesting 5-HT release from the GI tract may not be important for DON-induced anorexia to occur (Fig A.A.1). The 5-HT-3 receptor does not play a role in 5-HT- or DON-induced anorexia. Mice administered only 5-HT exhibited a 56 and 28% decrease in food intake at 60 and 120 min as compared to the control, respectively (Fig A.A.2). Mice administered the 5-HT-3 receptor inhibitor granisetron 30 min before 5-HT exhibited a 46 and 19% decrease in food intake relative to control values at 60 and 120 min, respectively (Fig A.A.2). Decreases in food intake were statistically significant for mice administered 5-HT alone and granisetron plus 5-HT at 60 min, but by 120 min only mice administered 5-HT alone displayed significantly decreased food intake. These data suggest the 5-HT3 receptor may not be critical for 5-HT’s anorectic effects. Next, the role of the 5-HT-3 receptor was assessed in DON-exposed mice using granisetron (Fig A.A.3). Mice administered granisetron alone displayed similar food intake as controls except at 120 min where mice administered granisetron ate significantly less than control mice. Mice administered DON alone exhibited significantly decreased food intake relative to the control at 30, 60 and 120, with food intake depressed by 67, 31 and 22%, respectively. Mice given granisetron 30 min before DON exhibited comparable food intake as mice given DON alone indicating 5-HT-3 receptors were not needed for DON-induced anorexia to occur. 164 HT Figure A.A.1 Plasma 5-HT levels upon DON administration. Mice were administered 0, 1, or 5 mg/kg bw DON by ip. injection. Plasma 5 HT was measured at 0, 30 and 120 p. 5-HT min depending on DON dose. Data are (n = 5 6/gp). Different letters indicate statistically 5-6/gp). significant differences between treatment groups at the specified time (p < 0.05). 165 Figure A.A.2 Evaluation of the 5 5-HT-3 receptor in 5-HT induced anorexia. To confirm 5HT 5 HT causes anorexia and that the 5 5-HT-3 receptor was involved, mice were administered 3 treatments according to Experiment 1, Table A.A.1. Food intake was measured at 60 ording A.A.1. and 120 min. Data are (n = 6 - 7/gp). Different letters indicate statistically significant differences between treatment groups at the specified time (p < 0.05). 166 5-HT-3 receptor in DON-induced anorexia. Mice were induced Figure A.A.3 Evaluation of the 5 administered treatments according to Experiment 2, Table A1.1. Food intake was ts measured at 60 and 120 min. Data are (n = 9 - 10/gp). Different letters indicate statistically significant differences between treatment groups at the specified time (p < 0.05). 167 The 5-HT-1B receptor inverse agonist SB224289 attenuates m-CPP- but not DONinduced anorexia. First, the ability of the 5-HT-1B receptor inverse agonist SB224289 to attenuate agonist-induced anorexia was assessed (Fig A.A.4A). Mice administered SB224289 alone ate similar amounts of food as the control. Mice administered only m-CPP, a 5HT-1B/2C receptor agonist, exhibited significant food intake inhibition with decreases of 88, 81, 71 and 81% relative to the control at 15, 30, 60 and 120 min, respectively. Mice pretreated with SB224289 before m-CPP displayed significantly greater food intake than those administered m-CPP alone demonstrating the efficacy of SB224289 on agonistinduced anorexia. Additionally, the function of the 5-HT-1B receptor in DON-induced feed refusal was evaluated (Fig A.A.4B). Control mice and mice administered SB224289 alone exposed ate similar amounts for the total 120 min measurement time. Mice administered DON alone ate 68, 60, 52 and 45 % less than the controls at 15, 30, 60 and 120 min, respectively. Food intake of mice pretreated with SB224289 before DON mirrored that of DON exposed mice indicating the 5-HT-1B receptor was unnecessary for DON-induced anorexia to occur. The 5-HT-2C receptor antagonist, SB242084 attenuated m-CPP- but not DON-induced anorexia. First the efficacy of the 5-HT-2C receptor inhibitor SB242084 in attenuating agonist-induced anorexia was evaluated (Fig A.A.5A). The 5-HT-1B/2C receptor agonist 168 m-CPP decreased food intake significantly by 76, 73, 76 and 82% at 15, 30, 60 and 120 min compared to control values, respectively. Interestingly, SB242084 administered alone also significantly depressed food intake at 60 and 120 minutes relative to control values. When SB242084 was administered 30 min before m-CPP, SB242084 significantly inhibited m-CPP’s negative effects on food intake. Subsequently, the capacity for SB242084 to attenuate DON-induced anorexia was assessed (Fig A.A.5B). No statistically significant differences in food intake between any treatment groups at any time points were discovered; however, DON decreased food intake by 32, 29, 70 and 56% at 15, 30, 60 and 120 min relative to the control, respectively. Food intakes of both DON-exposed groups were similar suggesting that the 5-HT-2C receptor was not necessary for DON-induced anorexia. Discussion Foodborne DON contamination is of public health concern because of its adverse gastrointestinal effects in humans, and anorectic and negative growth effects in experimental animals. Here we assessed the capacity of DON to elevate plasma 5-HT levels at a time course consistent with DON-induced anorexia. Additionally, the function of the 5-HT-3, -1B, and -2C receptors in DON-induced anorexia was assessed using respective receptor inhibitors. DON exposure did not increase plasma 5-HT nor did the 5-HT-3, -1B or -2C receptor inhibitors attenuate DON-induced anorexia suggesting these receptors may not be necessary for DON’s anorectic effects. 169 Figure A.A.4 Evaluation of the 5HT 5HT-1B receptor in m-CPP and DON-induced anorexia. induced ano The capacity of SB224289 to attenuate A) m m-CPP- and B) DON-induced anorexia was induced evaluated at 30, 60 and 120 min. Treatments are listed in Table A.A.1, experiments 3A and 3B. Data are (n = 6/gp). Different letters indicate statistically significant d differences between treatment groups at the specified time (p < 0.05). 170 Figure A.A.5 Evaluation of the 5HT 5HT-2C receptor in m-CPP and DON-induced anorexia. induced The capacity of SB242084 to attenuate A) m m-CPP- and B) DON-induced anorexia was induced evaluated at 30, 60 and 120 min. Treatments are listed in Table A.A.1, experiments 4A and 4B. Data are (n = 6/gp). Different letters indicate statistically significant differen differences between treatment groups at the specified time (p < 0.05). 171 In these experiments, the results demonstrating the inability of DON to increase plasma serotonin levels in mice in were similar to those of Prelusky and coworkers showing DON could not raise plasma 5-HT levels in pigs upon intragastric and intravenous administration (Prelusky 1994). Conversely, our result indicating the 5-HT2C receptor SB242084 did not attenuate DON-induced anorexia was opposite that of Prelusky and coworkers who demonstrated the 5-HT-2C receptor inhibitor, cyproheptadine abolished DON-induced anorexia in the mice (Prelusky et al. 1997). Cyproheptadine serves as both an antihistamine and antiserotonergic agent that also exhibits affinity for the dopamine -3 and muscarinic acetylcholine receptors -1 – 5 (Eltze et al. 1989; Kennett et al. 1997; Moguilevsky et al. 1994; Toll et al. 1998). Unlike cyproheptadine, SB242084 is a potent brain penetrable 5-HT-2C agonist that displays 100-fold selectivity over other 5-HT receptors (Kennett et al. 1997). Since cyproheptadine is non-specific, it likely blocked DON’s anorectic effects through mechanisms not specific to the 5-HT-2C receptor. Taken together, both peripheral and brain 5-HT2C receptors may not play an essential function in DON-induced anorexia. The 5-HT-1B receptor is also known to mediate food intake as evidenced by 1) results demonstrating the 5-HT-1B agonists RU24969, TFMPP and mCPP dose dependently reduced food intake in rats (Kennett and Curzon 1988), an effect attributed to satiety rather than nausea or decreased locomotor activity (Kitchener and Dourish 1994) and 2) results presented herein demonstrating the inverse agonist of the 5-HT-1B receptor SB224289 partially attenuated m-CPP induced anorexia in the mouse. No data currently exist of the capacity of SB224289 to cross the blood brain barrier; however, serotonergic signaling through the 5-HT-1B receptor occurs within the brain (Bruinvels 172 et al. 1994; Varnas et al. 2005), and m-CPP-induced anorexia was attenuated by peripheral administration of SB224289 suggesting this inverse agonist affected central signaling through the 5-HT-1B receptor. Mice pretreated with SB224289 still exhibited DON-induced anorexia indicating central 5-HT-1B receptors are likely not involved in DON’s anorectic effects. Previous studies evaluating the function of the 5-HT-3 receptor in food intake have shown mixed results. For example, the 5-HT-3 receptor agonist 2-methyl-5-HT failed to alter food intake in food deprived rats as did the 5-HT-3 antagonist ondansetron (Mazzola-Pomietto et al. 1995; Sugimoto et al. 1996). Concurrent with this idea, granisetron failed to attenuate 5-HT induced anorexia in our experiments suggesting the 5-HT-3 receptor was not necessary for 5-HT’s food intake effects. These results differ from Hayes and Covasa who demonstrated the efficacy of ondansetron in attenuating anorexia caused by serotonin creatinine sulfate complex (Hayes and Covasa 2005). Contradictory results between the aforementioned study and the one presented herein are likely due to differences in the 5-HT-3 receptor antagonists and doses utilized. Although mice are incapable of vomiting, the 5-HT3 receptor is most recognized for its role in 5-HT-induced emesis in vomiting species (Endo et al. 2000). Emesis caused by 5-HT often results from consumption of noxious substances and chemotherapeutic or radiation treatment. Interestingly, the 5-HT-3 was found to mediate DON-induced vomiting in pigs (Prelusky and Trenholm 1993). Though experimental results presented herein indicated the 5-HT-3 receptor was not important for 5-HTinduced anorexia, this receptor may nevertheless be important in DON-induced anorexia, if anorexia served as a substitute for DON-induced vomiting in the mouse. 173 However, DON-exposed mice pretreated with granisetron exhibited similar decreases in food intake as mice administered DON alone indicating 1) the 5-HT-3 receptor was not critical for DON-induced anorexia and 2) that anorexia may not be a surrogate for DONinduced emesis. Mechanisms involved in DON-induced anorexia are not well elucidated. Mouse studies suggested that DON targets the central nervous system resulting in increased hypothalamic anorexigenic peptides, an effect which could lead to anorexia (Girardet et al. 2011b). These studies also confirmed the role of the central nervous system in DONinduced anorexia by demonstrating that DON caused anorexia when injected into the lateral ventricle of the brain at a dose lower than would cause anorexia when injected peripherally (Girardet et al. 2011b). Another potential mechanism of DON-induced anorexia is the capacity of DON to cause a release of gut satiety hormones other than 5-HT. Recently, it was demonstrated that ip. DON administration caused increased plasma levels of the gut satiety hormones peptide YY and cholecystokinin and this corresponded to the dose and timing of DON-induced anorexia (Flannery et al. 2012). Since these peptides can alter orexigenic and anorexigenic signaling within the hypothalamus, gut hormone release may be one way by which DON functions to cause signaling changes within the brain. In conclusion, food intake is a very complex, redundant process involving many mechanisms. Data presented herein showed that DON did not cause an increase in plasma 5-HT nor did it have its anorectic actions through the 5-HT-3, -1B or -2C receptors. Therefore, anorexia resulting from DON’s effects on serotonergic signaling likely involves mechanisms other than the 5-HT-3, -1B or -2C receptors. 174 Appendix B: Model Considerations for Toxicological Endpoint Measurements in the Mouse Abstract The Food and Drug Administration mandates safety assessment for implementation of a new drug or food additive in humans. These safety assessments often include evaluation of toxicological endpoints assessed in rodents, particularly mice. Though mice are considered robust toxicological models due to their physiological similarity to humans, many internal and external factors can influence measurement variability. Here we assessed the contribution of two external factors, diet consistency and animal handling, on selected toxicological endpoints in mice. In the first study, mice were fed either pellet or powder diet spiked with the foodborne mycotoxin, deoxynivalenol at doses 0, 0.5, 1, 2.5, 5 and 10 ppm for 9 wk. Upon experimental termination the no observed adverse effect level (NOAEL) for weight, plasma insulin-like growth factor 1 (IGF1), plasma insulin-like growth factor acid-labile subunit (IGFALS) and hepatic mRNA expression for IGF1, IGFALS and suppressor of cytokine signaling 3 (SOCS3) were assed with the hypothesis that the NOAELS for various endpoints would differ based on diet consistency. In study 2, we assessed the effect of animal handler on food intake with hypothesis that person administering an intraperitoneal injection would influence food intake in mice. Results demonstrated that NOAELS for various toxicological endpoints depended on diet consistency. Furthermore, animal handler significantly affected food intake in mice with mice paired with the less familiar handler exhibiting significant decreases in food intake. Taken together, both diet consistency 175 and animal handler need to be considered as variables when assessing important toxicological endpoints. Introduction In the United States, safety and efficacy testing is required by the Food and Drug Administration for use of a new food additive or drug in humans (FDA 2012). When assessing an adverse chemical, specific endpoints relevant to potential target tissues are often evaluated. Endpoint results often depend on preparation of chemical stock solutions, duration of study, species utilized along with chemical dose, duration and route of administration. Rodents are frequently used in toxicological studies due to their ease of handling and availability. With the exception of the ability to vomit, the physiological systems of rodents mirror those in humans making them valuable tools for studying potential toxicities of xenobiotics in humans (Gad 2007). Of rodents, the mouse is the most frequently used model for toxicity testing due to their low cost and the availability of genetically mutated breeds (Gad 2007). One undesirable aspect of using mouse models is the inherent discrepancies among various mouse models which may affect the outcome of toxicological endpoints. These discrepancies include age, strain and sex of the mouse. For example, three different breeds of Swiss mice, ICR, NMRI and CFW-1 were administered yessotoxin, a poisonous toxin produced by shellfish, and the lethal dose 50 (LD 50) determined (Aune et al. 2008). Researchers found the LD 50 of yessotoxin depended on mouse strain with sensitivity CFW-1 > NMRI > ICR. Furthermore, female mice exhibited a 1.3 fold lower 176 LD 50 than males (Aune et al. 2008). In another murine model evaluating the toxicity of the foodborne mycotoxin deoxynivalenol (DON), male mice exhibited significantly more weight suppression and anorexia than female mice over the two-year study period (Iverson et al. 1995). Taken together, differences among mouse models need to be carefully assessed before choosing the right model for the toxicological endpoints of interest. Here we assessed the effect of diet consistency and animal handler on various physiological endpoints using two different mouse models. In study 1, we assessed the no observed adverse effect level of DON using weight, plasma insulin-like growth factor 1 (IGF1), plasma insulin-like growth factor acid-labile subunit (IGFALS), and hepatic mRNA expression of IGF1, IGFALS and suppressor of cytokine signaling 3 in weanling mice fed identical diets with the exception of diet consistency. These endpoints were chosen because recently our laboratory linked the adverse weight effects of the mycotoxin DON with depressions in the potent growth regulator IGFALS, an effect that occurred through increased SOCS3 (Amuzie and Pestka 2010; Amuzie et al. 2009) (FIG.1.). Furthermore, the Joint FAO/WHO Committee on Food Additives based its regulatory limits of DON on the no observed adverse effect level for growth suppression in mice (Canady et al. 2001). In study 2, we used a previously published mouse model to assess the effects of compound administration by two different animal handler on food intake. Results demonstrated that both diet consistency and animal handler significantly affected toxicological endpoints in these studies suggesting the importance of these two factors in establishing validated mouse models used for regulatory decisions regarding xenobiotics. 177 Materials and Methods Mice. In study 1, weanling (3 wk) female B6C3F1 mice were purchased from Charles River and housed 4 per cage. For study 2, adult (8 – 10 wks) B6C3F1 female mice were purchased from Charles River, housed singly and allowed to acclimate to diet and environment for 1 wk before beginning experiments. Approval for all animal experiments was obtained from Michigan State University’s Institutional Animal and Care and Use Committee as overseen by the National Institutes of Health. Diet. DON was purified from Fusarium graminearum rice cultures as previously described and added to a pelleted AIN-93G diet by Research Diets (D10012G; New Brunswick, NJ) (Clifford et al. 2003) for study 1. According to Research Diets, DON was solubilized in ethanol and added in appropriate concentrations to a powder mix, mixed homogenously, and pellets are cold extruded. To obtain a powder diet, pellets were ground to uniform size using the Magic Bullet Blender (Homeland Housewares, Sherman Oaks, CA) and sifted using a flour sifter to maintain uniform particle size. Mice were fed ad-libitum in glass containers with metal assistors and cages kept in ventilated Type II cage racks. Mice in Study 2 received high fat diet (HFD) purchased from Research Diets Inc. (45% kcal from fat; D12451). Mice were fed in accordance with a previously designed mouse model by Flannery and researchers (Flannery et al. 2012; Flannery et al. 2011). Briefly, mice were fasted for 8 h and given two intraperitoneal (ip.) injections of 50 µl phosphor-buffered saline (PBS) 30 min apart upon the dark light cycle. Mice were provided with two pellets of HFD and food intake measured at indicated intervals. 178 Experimental Design Study 1. Effect of diet consistency on selected toxicological outcomes. Mice (n = 8 per group) were exposed to DON in either AIN-93G pellet or powder diet (Research Diets, New Brunswick, NJ) at concentrations of 0, 0.5, 1, 2.5, 5, and 10 ppm DON. Mice were weighed weekly and euthanized after 9 weeks of DON exposure via an ip injection of 56 mg/kg bw sodium pentobarbital. Blood was collected using heparin syringes (50 IU, BD, Franklin Lakes, NJ ) from the caudal vena cava and immediately put on ice. Blood was centrifuged at 3000 x g for 10 minutes, plasma collected and aliquoted, and frozen at - 80° The large lobe of the liver was e xcised, put in 1 mL of RNAlater and C. frozen at – 80° Plasma was used to determine DON concentration, IGF-1 and C. IGFALS. Liver tissue was used to determine relative mRNA expression of SOCS3, IGF1 and IGFALS. Study 2. Effect of animal handler on food intake. Fasted mice (n = 5/ gp) received two ip. injections of 50 µl phosphate-buffered saline (PBS) 30 m apart and were immediately given food. Two different laboratory personnel referred to as ‘animal handlers’ performed injections. Food intake was measured at 30, 60 and 120 m. ELISAS. Plasma IGF1 was tested using Quantikine ELISA (MG-100) Kit (R & D systems, Minneapolis, MN) according to the manufacturer’s instructions. IGFALS was assayed using a previously described method with an alteration in sample dilution (Amuzie and Pestka). For both IGF1 and IGFALS, plates were analyzed at 450 nm on 179 an ELISA Plate Reader (Molecular Devices, Menlo Park, CA) and individual samples were quantified from a standard curve using Softmax software (Molecular Devices). Quantitative Real-Time PCR. RNA was extracted from hepatic tissue by homogenization in 1mL Tri-Reagent (Molecular Research Center Inc., Cincinnati, OH) and extracted according to the manufacturer’s protocol. SOCS3, IGF1 and IGFALS mRNA reactions were prepared by using Taqman One-Step Real-time PCR Master Mix and Assays-on-Demand primer/probe according to the manufacturer’s protocol (Applied Biosystems, Foster City, NY). Relative mRNA expression was determined using an ABI PRISM 7900HT Sequence Detection System mRNA was amplified and mRNA quantified relative to B2 microglobulin. Fold changes in mRNA expression were determined using the 2 (-∆∆Ct) method as previously described (Livak and Schmittgen 2001). Statistics. A One-Way Analysis of Variance (ANOVA) was used to determine statistical significance relative to the control at a given time point. A One-Way ANOVA was also utilized to determine statistical differences in plasma levels of hormones and hepatic mRNA expression. A Holm-Sidak multiple comparison procedure was used with the One-Way ANOVA unless normality failed and then a Dunnett’s test was employed. To compare two groups a t-test was applied. For all comparisons statistical significance was reached with p < 0.05. 180 Results Study 1. No observed adverse effect level for toxicological endpoints is different based on diet consistency DON-induced weight suppression in weanling animals was measured over a period of 9 wks. Mice were fed AIN-93G pellet or ground pellet diet (powder) containing 0, 0.5, 1, 2.5, 5 or 10 ppm DON ad-lib and weights obtained weekly. Though not significant, mice fed pellet diet with 10 ppm DON exhibited an average weight decrease of 8% relative to the control mice at wk 9 (Fig. A.B.1). At no time point were any average weights for any dose statistically significant for mice fed pellet diet. In contrast, mice fed powder diet show statistically significant weight suppression at wk 6 for mice fed 5 ppm DON and at wks 2 through 8 in mice fed 10 ppm DON, respectively (Fig. AB.1). Mice fed pellet and powder diet weighed 15 g at the beginning of the study (wk 0), but by wk 9 pellet fed control mice weighed about 22 g, while control mice fed powder diet weighed about 28 g. This growth discrepancy may have contributed to significant weight suppression detected in powder fed mice and not pellet fed mice. For mice fed both pellet and powder diets, no statistically significant difference was observed at any DON dose for plasma IGF1 (Fig. A.B.2 and Fig. A.B.3). Mice fed pellet diet exhibited a 18% decrease in plasma IGF1 at 1 ppm DON relative to the control, while mice fed powder diet showed a 12% increase relative to the control. Therefore, mice fed pellet diet were slightly more sensitive to IGF1 depression than mice fed pellet diet. A similar pattern was observed for plasma IGFALS. Plasma IGFALS was significantly depressed at 5 ppm DON in mice fed pellet diet, but no significant depressions were observed in mice fed powder diet (Fig. A.B.2 and Fig. A.B.3). 181 In contrast to the effects of diet consistency on plasma hormone parameters, hepatic mRNA expression for IGF1 and IGFALS depression was more sensitive endpoints in powder fed mice than those fed pellet (Fig. A.B.4). IGF1, IGALS and SOCS 3 mRNA expression were not statistically significant relative to the control in mice fed pellet diet; however, in powder fed mice, mRNA expression of IGF1 and IGFALS was statistically significant at 2.5 and 10 ppm, and 10 ppm, respectively. Overall, SOCS3 increased as DON-concentration increased though no statistical differences were observed for either diet consistency. Study 2. Animal handler significantly influences food Mice were given two ip. injections of PBS by two different people and food intake measured (Fig. AB.5). Food consumption of mice injected by handler 2 was significantly lower than those injected by handler 1 at 2 h. This trend was also observed for 30 and 60 m. These data suggest the person handling the mouse is an important factor when measuring food intake. 182 Figure A.B.1 Relationship of diet consistency to DON-induced weight suppression. Mice induced were fed A) pellet or B) powder diet of AIN 93G amended with 0, 0.5, 1, 2.5, 5 or 10 AIN-93G ppm DON and weights taken weekly. Data (n = 8/gp) are mean ± SEM. An asterisk indicates a statistically significant difference relative to the control (p < 0.05). 183 Figure A.B.2 Effect of diet consistency on plasma IGF1 and IGFALS upon DON exposure in mice fed pellet diet Mice were fed pellet diet of AIN-93G amended with 0, diet. 93G wi 0.5, 1, 2.5, 5 or 10 ppm DON and plasma removed upon experimental termination (9 wk). Data (n = 6 - 8/gp) are mean ± SEM. An asterisk indicates a statistically significant difference relative to the control (p < 0.05). 184 Figure A.B.3 Effect of diet consistency on plasma IGF1 and IGFALS upon DON exposure in mice fed powder diet. Mice were fed powder diet of AIN 93G amended with AIN-93G 0, 0.5, 1, 2.5, 5 or 10 ppm DON and plasma removed upon experimental termination (9 wk). Data (n = 6 - 8/gp) are mean ± SEM. An asterisk indicates a statistically significant difference relative to the control (p < 0.05). 185 Figure A.B.4 Effect of diet consistency on hepatic mRNA expression of IGF1 and IGFALS upon DON exposure. Mice were fed A) pellet or B) powder diet of AIN-93G A amended with 0, 0.5, 1, 2.5, 5 or 10 ppm DON and hepatic tissue removed upon experimental termination (9 wks). Data (n = 6 - 8/gp) are mean ± SEM. An asterisk indicates a statistically significant difference relative to the control (p < 0.05). 186 Figure A.B.5 Food consumption relative to animal handler. Mice were give two ip. injections of PBS 30 min apart by two different handlers. Data (n = 5/gp) are mean ± SEM. An asterisk indicates a statistically significant difference (p < 0.05). 187 Discussion The data presented herein confirm diet consistency and animal handler as two other variables that need to be considered when developing a mouse model for toxicity testing. When evaluating DON’s adverse endpoints, data suggested mouse weight, plasma hormone levels and hepatic mRNA expression depended on diet consistency. Furthermore, the person administrating the experimental treatment played a significant role in the outcome of food intake. When designing experiments, not accounting for these two parameters may yield misleading experimental results. As previously reported in mice, diet amended with DON caused weight suppression (Amuzie et al. 2011; Amuzie and Pestka 2010); however, the no observed adverse effect level for DON-induced weight suppression was different depending on the diet type utilized (Table A.B.1). In both powder and pellet diet, mice fed 10 ppm DON exhibited similar end weights (19 – 20 g). In contrast, mice fed powder diet displayed an end weight 4.7 g more than mice fed pellet diet. This weight discrepancy between pellet and powder fed mice likely contributed to the difference in NOAELS for weight suppression between these two diets. Table A.B.1 The no observed adverse effect level using plasma IGF1, IGFALS and hepatic mRNA expression of SOCS3, IGF1 and IGFALS as biological endpoints. Plasma Levels Hepatic mRNA Expression Diet Type Weight IGF1 IGFALS SOCS3 IGF1 IGFALS Pellet 10 ppm 10 ppm 10 ppm 10 ppm 10 ppm 10 ppm Powder 2.5 ppm 10 ppm 2.5 ppm 10 ppm 1 ppm 5 ppm 188 Diet consistency and hardness has been reported to affect growth of mice fed diets with identical nutrient composition. Ford and researchers demonstrated that mice fed hard pellet diets gained significantly less weight than mice fed softer pellets, an effect attributed to decreased food intake resulting from problematic pellet consumption (Ford 1977). In this same study, researchers discovered that mice fed a diet of a finer particle size exhibited greater food to weight conversion. Researchers suggested the reason for this was more efficient digestion of the food and greater food consumption (Ford 1977). Therefore, in our studies, mice fed pellet diet likely exhibited decreased weight gain compared to powder consuming mice because pellet size and hardness made the diet difficult to digest and consume. Differences among plasma IGF1 and IGFALS and hepatic IGF1, IGFALS and SOCS 3 mRNA expression were also demonstrated between pellet and powder fed mice. Reduced plasma IGF1 and IGFALS were more apparent in mice fed pellet diet than in powder diet. This disparity may be due to powder fed mice exhibiting 22% more growth than pellet fed mice, which could have resulted in higher basal hormone levels masking the growth hormone effects of DON. In contrast, mice fed powder diet showed greater sensitivity to DON-induced depressions of hepatic IGF1 and IGFALS mRNA. It is possible that mice fed powder diet were exposed to DON both nasally and orally with increased bioavailability of DON orally due to diet particle size. Indeed, DON tissue concentrations in mice exposed nasally were up to three times higher than mice exposed to DON orally (Amuzie et al. 2008). In this same study, nasally exposed mice exhibited up to 10 greater induction of pro-inflammatory cytokine mRNA expression as compared to orally exposed mice, though this effect was dependent also on tissue type 189 and cytokine of interest. Therefore, it was likely in this study that DON exposed mice fed powder diet exhibited greater DON- induced suppression of hepatic IGF1 and IGFALS mRNA due to nasal DON exposure, though DON’s mRNA effects were not yet reflected in corresponding plasma hormone levels. Food intake in mice is a complex behavior that is dependent upon but not limited to environmental factors including temperature, light cycle and season, and physiological factors such as body composition, sex, age and social interaction (Schwartz et al. 2000; Woods et al. 2000). Habituation, noise, smell and handling techniques are innate characteristics of investigators that may also affect food intake of mice. For example, a continuous debate about anorexigenic potential of the hormone peptide YY continued among scientists until it was discovered that PYY was in fact highly anorexigenic but this effect was masked due to improper habituation of mice by investigators (Batterham et al. 2002; Hagan 2002). In our model, animal handler 1 consistently handled mice, while animal handler 2 only helped during injections making lack of habituation, differences in smell and handling techniques likely reasons for food intake differences between mice injected by two different people. In conclusion, data presented herein demonstrate diet consistency and animal handler as two factors that potentially affect outcomes in toxicological studies. Since regulatory decisions are often based on physiological endpoints that may be affected by these two confounding factors, it is of utmost importance to identify and minimize these factors when designing experimental models. 190 Appendix C: Protein Kinase R is Not Critical for Deoxynivalenol-Induced Weight Suppression Abstract Deoxynivalenol (DON) is a mycotoxin that is frequently found in unprocessed and processed grain products throughout the world. DON’s adverse biological consequences in experimental animals include proinflammatory cytokine induction, anorexia, weight loss and weight suppression, with the latter serving as its basis for regulation. DON’s capacity to elicit robust proinflammatory cytokine induction originates from its ability to rapidly activate the double stranded protein kinase R (PKR) and subsequent mitogen activated protein kinase signaling cascade. Using a PKR-knock out (KO) mouse model, our goal was to assess the role of PKR in DON-induced weight suppression. Both PKR-KO and WT mice were fed very high fat diet (VHFD) or VHFD containing 10 ppm DON for 9 wk with weights taken weekly and percent peri-uterine fat determined upon experimental termination. Data demonstrated that PKR-KO mice fed DON exhibited slight attenuation of DON-induced weight suppression, though this effect was not significant. Additionally, PKR-KO mice fed VHFD or VHFD + 10 ppm DON exhibited increased fat percentage relative to the respective wild type controls. In all, the data suggested PKR is not necessary for DON-induced weight suppression. 191 Introduction Deoxynivalenol is trichothecene mycotoxin that upon consumption causes symptoms of gastrointestinal illness in humans and pro-inflammatory cytokine induction, weight suppression, anorexia and growth hormone dysregulation in experimental animals (Pestka 2010b). Since DON is resistant to processing, it is frequently found in grain products resulting in recurrent exposure in humans and animals (Canady et al. 2001). As a result, understanding the mechanisms by which DON causes its adverse effects are of utmost importance for comprehensive risk assessment of this mycotoxin. In experimental animals, DON targets the immune system, more specifically leukocytes, resulting in systemic induction of pro-inflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor –α (TNF-α) within 1 hr of DON exposure (Amuzie et al. 2009). Though in-vivo studies are more biologically relevant than in-vitro models for human exposures to DON, cell models are valuable for discerning specific signaling cascades involved in DON toxicity. The RAW 264.7 cell line serves as cardinal model for elucidating signaling cascades involved in DON’s immune toxicity (Zhou et al. 2005a; Zhou et al. 2005b; Zhou et al. 2003). DON is considered a translational inhibitor despite this toxin’s ability to increase pro-inflammatory cytokine mRNA and protein expression, a contradiction that occurs because DON increases mRNA stability of IL-6 and TNF-α (Chung et al. 2003; Jia et al. 2006; Pestka et al. 2004). DON causes translational inhibition through a mechanism termed ‘ribotoxic stress response.’ Ribotoxic Stress Response refers to DON’s ability to activate the mitogen activated protein kinase signaling cascade through protein kinase R (PKR) and hemopoeitic kinase (Pestka 2010a). PKR is a double-stranded protein 192 kinase that serves to defend against viruses and may facilitate IL-6 induction (Pestka 2008). Evidence that PKR is a key regulator of DON-induced cytokine increases is demonstrated by experiments showing that PKR activation occurs within 5 min post DON administration in the macrophage and that PKR inhibitors suppression DON’s initiation of the MAPK signaling cascade and stimulation of TNF-α (Zhou et al. 2005b; Zhou et al. 2003). Though the immune system is a main target of DON, DON is regulated by the Joint WHO/FAO Expert Committee on Food Additives (JECFA) based on the no observed adverse effect level for growth suppression in mice (Canady et al. 2001). Growth suppression serves as the basis for DON regulation because children are considered particularly vulnerable to DON’s growth effects due to their rapid rate of growth and their high consumption of grains per kilogram body weight (Pieters et al. 2002). Recent evidence from mouse models suggests a mechanistic link between DONinduced proinflammatory cytokine stimulation and its negative growth effects through the action of suppressor of cytokine signaling 3 (SOCS3) on growth hormone signaling (Amuzie and Pestka 2010). More specifically, oral DON administration systemically induced IL-6 and TNF-α with in 1 hr post exposure and the decrease of these proteins coincided with robust increase in hepatic SOCS3 expression at 3 hr (Amuzie et al. 2009). Robust SOCS3 increases were associated with significant decreases in hepatic insulin-like growth factor acid-labile subunit (IGFALS) mRNA expression, a protein critical for postnatal growth (Amuzie et al. 2009). While this model relates DON’s growth 193 effects with its early immune effects, how DON’s immediate adverse intracellular effects, such as PKR activation, are linked to DON’s weight effects is not yet known. Using a PKR-knock out (KO) mouse model, we sought to determine PKR’s effects on DON-induced weight suppression with the hypothesis that PKR-KO mice would exhibit less weight suppression than wild type (WT) mice upon dietary DON exposure. Data suggested that PKR-KO mice exhibited slightly attenuated DONinduced weight and body fat suppression than the WT controls, though this effect was not significant. Materials and Methods Mice. C57BL6 adult female wild-type and PKR-knockout were a gift from Dr. Kauffman (University of Michigan) and bred by Van Andel Institute (Grand Rapids, MI). The PKR null genotype were verified using polymerase chain reaction (PCR) of tail snips from PKR-KO and WT mice. All experiments received prior approval by the Institutional Animal Care and Use Committee at Michigan State University as directed by the National Institutes of Health Animal Care “Guidelines for Animal Research.” Diet. Mice were given either very high fat pellet diet (VHFD; 60% kilocalories from fat; D12492, Research Diets Inc., New Brunswick, NJ) or VHFD amended with 10 ppm DON by Research Diets Inc. according to their standardized procedure. DON was obtained and purified from Fusarium graminearum cultures as previously described (Clifford et al. 2003). Experimental Design. Upon arrival mice were randomized according to genetic make-up into 4 groups (n = 5 - 6/gp): wild-type mice fed VHFD, wild-type mice fed VHFD + 10 194 ppm DON, PKR-KO mice fed VHFD and PKR-KO mice fed VHFD + 10 ppm DON. Mice were housed 3 per cage and allowed unrestricted access to food and water. Cages contained aspen bedding with wire top lids and filter bonnets to avoid DON food from spilling outside the cage. Mice were weighed weekly for 15 wks and then sacrificed using 50 mg/kg bw sodium pentobarbital. Peri-uterine fat was excised and weighed for percent fat determination. Statistics. Differences between groups at any given time point were determined by a One-Way Analysis of Variance (ANOVA) with Holm-Sidak comparison procedure or a Kruskal-Wallis One-Way ANOVA with Tukey Test when equal variance failed. Results PKR-KO mice fed DON exhibit similar DON-induced weight suppression as WT Despite total weight being similar between DON fed WT and PKR-KO groups, PKR-KO mice fed DON exhibited more cumulative weight gain (Fig. A.C.1 and A.C.2). At experimental termination WT and PKR-KO mice gained a total of 14 g and 15 g, respectively, while WT and PKR-KO fed 10 ppm DON gained a total of 3 g and 7 g, respectively (Fig. A.C.2). Though DON exposed WT mice gained 57% less weight than DON exposed PKR-KO mice, the differences in cumulative weight gain were not statistically significant. 195 Figure A.C.1 Weights of WT and PKR KO mice fed 10 ppm DON. Mice were fed VHFD PKR-KO or VHFD + 10 ppm DON for 15 wks with weights taken weekly. Data (n = 5 - 6/gp) are mean ± SEM. Different letters indicate statistically significant difference between groups at that time point (p < 0.05). 196 Figure A.C.2 Cumulative weight gain of WT and PKR-KO mice fed 10 ppm DON. Mice KO were fed VHFD or VHFD + 10 ppm DON for 15 wks with weights taken weekly and cumulative weight gain determined by subtracting each weeks weight from weight at wk weight 0. Data (n = 5 - 6/gp) are mean ± SEM. Different letters indicate statistically significant difference between groups at that time point (p < 0.05). 197 Figure A.C.3 Body fat percentage of WT and PKRKO mice fed 0 or 10 ppm. Mice were fed according to Fig. 1 and sacrificed at 9 wk and peri-uterine fat excised. Data (n = 5 uterine excised 6/gp) are mean ± SEM. Different letters indicate statistically significant difference among statistically groups (p < 0.05). 198 PKR-KO mice exhibit greater percentage of peri-uterine fat as compared to WT Upon experimental termination, peri-uterine fat made up 7.5% body weight in WT mice fed HFD while peri-uterine fat accounted for 9.8% in PKR-KO mice (Fig. A.C.3). As expected, groups of mice fed 10 ppm DON contained a lower percent body fat that mice fed VHFD though it was not statistically significant. DON exposed WT mice had 3.1% body fat whereas PKR-KO mice fed 10 ppm DON showed 6.5% body fat (Fig. A.C.3). Though not statistically significant, percent body fat was higher in PKR-KO mice than WT irrespective of diet. Discussion Growth suppression functions as the harmful physiological effect by which regulatory limits of DON are based, however, the manner by which DON-induced growth suppression is linked to DON’s immediate intracellular toxicity is not yet understood. The data presented herein suggest PKR is likely not important in DON’s adverse weight effects, despite PKR-KO animals fed DON exhibiting a slight attenuation of cumulative weight gain. Both WT and PKR-KO mice fed VHFD + 10 ppm DON exhibited significant weight suppression relative to the VHFD controls. These results agreed with data from Amuzie and researchers showing that mice fed VHFD + 10 ppm DON exhibited a 24% weight suppression and 83% reduction in peri-uterine fat relative to controls over 10 weeks (Amuzie et al. 2011). Furthermore, data from Kobayashi-Hattori and coworkers demonstrated that diet-induced obese mice fed VHFD + 10 ppm DON exhibited a 13% weight loss and 12% fat loss over 51 d, an effect attributed to decreased food 199 consumption (Kobayashi-Hattori et al. 2011). Taken together, DON suppresses weight gain and promotes weight loss in mice fed VHFD. Starting at wk 8, PKR-KO mice fed DON gained slightly more weight overall than DON-fed WT mice, though this effect was not statistically significant. Since PKR activation is upstream of proinflammatory cytokine induction, the difference in weight gain between genetic groups may be due to the attenuation of DON- induced proinflammatory cytokine stimulation and subsequent anorexia and growth hormone dysregulation potentially caused by these cytokines. However, tissue cytokines, IGFALS and food intake were not measured in these studies. PKR is thought to be one signaling molecule linking the systemic inflammation and metabolic disease seen in obesity. For example, using mouse embryonic fibroblasts from PKR-KO mice, it was shown that PKR interacts directly with the insulin receptor substrate – 1 (IRS1), a receptor critical for insulin sensitivity and growth hormone signaling (Nakamura et al. 2010). As compared to WT controls, PKR-KO mice fed VHFD also exhibited decreased blood glucose levels, depressed weight gain and reduced body fat mass, effects that were not attributable to decreased food intake (Nakamura et al. 2010). Contrary to these findings, PKR-KO mice in our studies did not exhibit depressed weight gain compared to WT controls and though not statistically significant, PKR-KO mice actually showed increased peri-uterine fat relative to wild type controls. The discrepancies between previously published data and our results may be due to difference in mouse species, sex and fat distribution. 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