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Valproic Acid-Induced Teratogenesis in Japanese Rice Fish (Oryzias Latipes) Embryogenesis

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Valproic Acid-Induced Teratogenesis in Japanese Rice Fish (Oryzias Latipes) Embryogenesis University of Mississippi eGrove Electronic Theses and Dissertations Graduate School 2012 Valproic Acid-Induced Teratogenesis in Japanese Rice Fish (Oryzias Latipes) Embryogenesis Mengmeng Wu Follow this and additional works at: https://egrove.olemiss.edu/etd Part of the Pharmacology Commons Recommended Citation Wu, Mengmeng, "Valproic Acid-Induced Teratogenesis in Japanese Rice Fish (Oryzias Latipes) Embryogenesis" (2012). Electronic Theses and Dissertations. 319. https://egrove.olemiss.edu/etd/319 This Thesis is brought to you for free and open access by the Graduate School at eGrove. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of eGrove. For more information, please contact [email protected]. VALPROIC ACID-INDUCED TERATOGENESIS IN JAPANESE RICE FISH (ORYZIAS LATIPES) EMBRYOGENESIS A Thesis presented in partial fulfillment of requirements for the degree of Master of Science In the Department of Pharmacology The University of Mississippi by MENGMENG WU May 2012 Copyright Mengmeng Wu 2012 ALL RIGHTS RESERVED ABSTRACT Valproic acid (VPA) was introduced as an antiepileptic in 1967 in France and it has become the most prescribed anticonvulsive drug therapy worldwide since then. In the clinic, valproic acid is selected to treat absence seizures, myoclonic seizures, generalized tonic-clonic seizures, atonic attacks and partial seizures. Epilepsy is the second most common neurologic disorder that affects pregnant women (0.5%-1%). Approximately 1 out of 250 pregnant women are taking antiepileptic drugs. Valproic acid is designated as a human teratogen, which induces major congenital anomalies, facial dysmorphic features, and autistic-like behaviors, which affect verbal, cognitive, communicative, and social abilities of affected children. We are developing the Japanese rice fish ( Oryzias latipes ), also known as Japanese medaka, as an animal model to study valproate-induced teratogenesis during the period of embryogenesis. Fertilized embryos of Japanese rice fish at three developmental stages (group A: 0-2 dpf; group B: 1-3 dpf; group C: 4-6 dpf) were exposed to VPA (0-80 mM) for 48 hrs. The amounts of VPA to cause 50% mortality (LC50), which were observed on 14 day post fertilization (dpf), are found to be developmental stage-specific. The LC 50 for group A (Iwamatsu developmental stage 4-10) is 1.68 ± 1.55 mM which is much lower than the LC 50 s for groups B ii (26.45 ±1.64 mM) and C (20.37 ± 3.3 mM) (Iwamatsu developmental stages 17-32). The development of the cardiovascular system was disrupted by VPA in each treatment group, displaying increased incidence of thrombus, reduced heart rates, and inhibited or delayed onset of circulation. The hatching efficiency was also reduced by VPA in each treatment group. The higher the concentration of VPA the embryos were treated with, the more severe the results were. The earlier developmental stages the treatments were targeted at, the more deleterious the effects of VPA were. VPA also caused malformation of neurocranial and splanchnocranial cartilages of hatchlings that had been exposed prenatally. The length of neurocranium, quadrate, ceratohyal and basibranchial 1-3 were all reduced in hatchlings of both groups E (0-2 pdf) and L (4-6 dpf). Trabeculae, epiphyseal bar, anterior orbital cartilage and basilar plate displayed reduction in length only in hatchlings of group E. In addition, the significant reduction in the linear length of polar cartilage and ceratobranchials 1-5 were manifested only in hatchlings of group L. Other cartilages remained unaltered in both groups E and L. It was indicated that the length of the neurocranium was reduced due to cumulative reduction of the component cartilages. iii mRNA analyses of nine genes demonstrated that the genes involved with oxidative stress remained unaltered after valproate exposure. However, the genes involved with neurogenesis (wnt1 , otx2 and nlgn3b ) and regulation of cell division ( shh and ccna2 ) showed developmental stage-specific alteration after valproate exposure. This study indicates that valproate is able to induce some phenotypic features in Japanese rice fish which are analogous to human fetal valproate syndrome (FVS), and Japanese rice fish can be used as a unique alternatively non-mammalian vertebrate model to study valproate-induced teratogenesis, including FVS. iv DEDICATION This thesis is dedicated to everyone who helped me and guided me during the past three years. In particular, I want to thank my advisor, Dr. Asok Dasmapahaptra, for research guidance, Dr. John C. Matthews, for generous help and Dr. Babu Tekwani, for your patience and kindness. I also want to thank my dear friends and my parents, Ms. Sun, Wanying and Mr. Wu, Zhenggen. v LIST OF ABBREVIATIONS ABC Anterior basicranial commissure ANOVA One-way analysis of variance AOC Anterior orbital cartilage ASD Autism spectrum disorder BB Basibranchials BH Basihyal BP Basilar plate CBR Ceratobranchial ccna2 CyclinA2 CH Ceratohyal dpf Days post fertilization EBR Epibranchial EB Epiphyseal bar EP Ethmoid plate vi FVS Fetal valproate syndrome gr Glutathione reductase gst Glutathione-S-transferase hpf Hours post fertilization HYO Hyoid HYS Hyosymplectic HBR Hypobranchial HP Hypophyseal plate iro3 Iroquois homeobox protein MC Meckel’s cartilage NC Neural crest nlgn3b Neuroligin 3b nrxn Neurexin Otx2 Orthodenticle homeobox 2 PC Polar cartilage PBC Posterior basicranial commissure vii POC Posterior orbital cartilage QU Quadrate qRTPCR Quantitative real-time polymerase chain reaction ROS Reactive oxygen species rRTPCR Relative reverse transcription polymerase chain reaction shh Sonic hedgehog TB Trabeculae VPA Valproic Acid wnt1 Wingless-related MMTV integration site 1 viii ACKNOWLEDGEMENTS I would like to express my gratitude to all the people who made my graduate work and this thesis possible. First of all, I would like to thank my adviser, Dr. Asok Dasmahapatra, who allowed me to join his lab and provided me with his precious guidance. During the past three years, he supported me with the experimental materials and environment and extensive scientific training. He was always very patient to answer my questions. Secondly, I would like to thank Dr. John C. Matthews for his constant encouragement, generous help and great advises through my own times of stress, difficulty and anxiety. He is a life mentor and a great educator, like a beam of sunshine in the winter. I also want to thank one of my committee members, Dr. Babu Tekwani for his patience, kindness, excellent advises and critical reading of this thesis. I also feel grateful to Dr. Kristie Willett, for offering equipments in her lab, and Dr. Ikhlas A. Khan for funding. I also want to thank all the help, friendship, a variety of support and sponsorship from all the faculty, staffs, fellow graduate students, technicians, all the fish feeders and my dear friends. ix I greatly appreciate Department of Pharmacology, The University of Mississippi to offer me this golden opportunity to allow me to further my education here. During the past three years, I can feel my progress. I know this period of life journey in Oxford will be a critical time for my entire life. All the memories here will be treasured at the bottom of my heart. Finally, I want to give my big thank to my parents, Ms. Sun, Wanying and Mr. Wu, Zhenggen, for your endless love. I love you. x TABLE OF CONTENTS PAGE ABSTRACT.....................................................................................................................................ii DEDICATION………………………………………………………………….…………………v LIST OF ABBREVIATIONS………………………………………………………………..…...vi ACKNOWLEDGEMENTS............................................................................................................ix TABLE OF CONTENTS………………………………………………………………………....xi LIST OF TABLES…………………………………………………………………………...…xvii LIST OF FIGURES……………………………………………………………………………..xix CHAPTER 1. INTRODUCTION 1.1 Valproic acid-induced teratogenesis (fetal valproate syndrome and autistic-like behaviors)….………………………………....................................................................................1 1.2 Animal model..………………………………………………………………………….........10 1.3 Metabolism of valproic acid………...……………………………………………….............14 xi 1.4 Potential molecular mechanisms of VPA toxicity and autism………….................................15 1.5 Hypothesis and objectives of the study....................................................................................18 CHAPTER 2. MATERIALS AND METHODS 2.1 Fish maintenance ………………………………………………....….........……………...…26 2.2 Treatments used to investigate the lethal concentrations of valproic acid and the effects of valproic acid on hatching efficiency and on the cardiovascular system at three different time points ...…………………………………………………………………………………..............27 2.3 Lethal concentrations of valproic acid determined on embryos of Japanese rice fish exposed at three different developmental stages ……………………………………........….....................27 2.4 VPA effects on hatching efficiency of Japanese rice fish........................................................28 2.5 Analysis of deleterious effects of VPA on the cardiovascular system of Japanese rice fish ...28 2.6 Morphometric analysis of craniofacial cartilages in Japanese rice fish hatchlings exposed to VPA................................................................................................................................................29 2.6.1 Treatments…………………......................………………...................................................29
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