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

University of Mississippi eGrove

Electronic Theses and Dissertations Graduate School

2012

Mengmeng Wu

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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

MENGMENG WU

May 2012

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

(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.

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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.

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.

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

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

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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

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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.

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.

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

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

2.6.2 Cartilage staining...... 29

xii

2.6.3 Morphometric analysis…………………………...... 30

2.7 Biochemical analysis of Japanese rice fish embryos exposed to valproic acid.…....………..32

2.7.1 Treatments...... 32

2.7.2 Isolation of RNA...... 33

2.7.3 Semi-quantative RT-PCR...... 34

2.8 Statistical analysis...... 35

CHAPTER 3. RESULTS

3.1 Lethal concentration of valproic acid on Japanese rice fish embryos exposed at different developmental stages…………...... 36

3.2 Effects of valproic acid on hatching efficiency of Japanese rice fish at different developmental stages …………………………………………………………………………... 37

3.3 Morphometric analysis of Japanese rice fish exposed to valproic acid at different developmental stages...... 37

3.3.1 VPA effects on the cardiovascular system...... 38

3.3.1.1 VPA effects on heart beat……………………………………………………….………..38

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3.3.1.2 VPA effects on thrombus formation……………………………………...... 40

3.3.1.3 VPA effects on circulation……………………………………………………………….41

3.3.2 VPA effects on the hatchlings……………………………………………...... 42

3.3.3 VPA effects on the craniofacial cartilages...... 42

3.3.3.1 Neurocranial cartilages...... 43

3.3.3.1.1 Neurocranium...... 43

3.3.3.1.2 Trabeculae...... 44

3.3.3.1.3 Ephiphyseal Bar...... 44

3.3.3.1.4 Anterior Orbital Cartilage...... 44

3.3.3.1.5 Anterior Basicranial Commissure...... 45

3.3.3.1.6 Basilar Plate...... 45

3.3.3.1.7 Polar Cartilage...... 45

3.3.3.2 Splanchnocranium...... 46

3.3.3.2.1 Meckel's Cartilage...... 46

3.3.3.2.2 Quadrate...... 46

3.3.3.2.3 Basihyal...... 46

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3.3.3.2.4 Ceratohyal...... 46

3.3.3.2.5 Basibranchial 1-3...... 47

3.3.3.2.6 Basibranchial 4...... 47

3.3.3.2.7 Ceratobranchial 1...... 47

3.3.3.2.8 Ceratobranchial 2...... 47

3.3.3.2.9 Ceratobranchial 3...... 47

3.3.3.2.10 Ceratobranchial 4...... 48

3.3.3.2.11 Ceratobranchial 5...... 48

3.4 Biochemical analysis of Japanese rice fish embryos exposed to VPA prenatally and developmentally...... 48

3.4.1 Oxidative stress...... 49

3.4.1.1 Catalase...... 49

3.4.1.2. Glutathione-S-Transferase ( gst )...... 49

3.4.1.3 Glutathione Reductase ( gr )...... 50

3.4.2 Neurogenesis...... 50

3.4.2.1 Neuroligin 3b ( nlgn 3b )...... 50

3.4.2.2 Orthodenticle Homeobox 2 ( otx2 )...... 51

3.4.2.3 Wingless-typy Mouse Mammary Tumor Virus Integration Site Family, Member 1

(wnt1 )...... 51

3.4.2.4 Iroquois 3 ( iro3 )...... 51

3.4.3 Cell division...... 52

3.4.3.1 Sonic Hedgehog ( shh )...... 52

3.4.3.2 Cyclin A2 ( ccna2 )...... 52

CHAPTER 4. DISCUSSION ...... 74

CHAPTER 5. CONCLUSIONS AND FUTURE WORKS ……………………………...... 86

REFERENCES ………………………………………………………………………...... 90

VITA …………………………………………………………………………………...... 104

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LIST OF TABLES

TABLE PAGE

1. Specific features related to fetal valproate syndrome...... 22

2. Anomalies associated with valproate exposure...... 24

3. Phenotypic features of the cardiovascular system development of Japanese rice fish...... 25

4. Treatment groups classification of Japanese rice fish embryos used for determination of

effects of VPA on LC50, the cardiovascular development and the mRNA expression levels

of the related genes...... 53

5. Treatment groups classification of Japanese rice fish embryos used for measurement of

craniofacial cartilages...... 54

6. LC 50 values for Japanese rice fish embryos exposed to VPA at three different developmental

stages...... 56

7. Effect of valproate on the length of neurocranial and splanchnocranial cartilage of Japanese

rice fish...... 61

8. List of primers used in semi-quantitative RT-PCR (rRT-PCR) amplification of the mRNAs of

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Japanese rice fish embryos...... 70

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LIST OF FIGURES

FIGURE PAGE

1. Chemical structure of valproic acid...... 21

2. Child with facial dysmorphic features of FVS...... 23

3. Valproate-mediated mortality in Japanese rice fish embryos...... 55

4. Hatching efficiency of Japanese rice fish embryos developmentally exposed to

Valproate...... 57

5. Heart rates of Japanese rice fish embryos exposed to valproate during

development...... 58

6. The occurrence of thrombi and blood circulation observed on 6 dpf in embryos after

exposure to valproate at three different developmental stages...... 59

7. Representative photomicrographs displaying teratogenic effects of valproate in hatched

Japanese rice fish...... 60

8. Representative photomicrographs of neurocranial cartilages of Japanese rice fish

hatchlings...... 63

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9. Representative photomicrographs of splanchnocranial cartilages of Japanese rice fish

hatchlings...... 64

10. Effects of valproate on the development of neurocranium, trabeculae, and epiphyseal bar of

Japanese rice fish embryos...... 65

11. Effects of valproate on the development of anterior orbital cartilage, anterior basicranial

commissure, basilar plate and polar cartilage of Japanese rice fish embryos...... 66

12. Effects of valproate on the development of Meckel's cartilage, quadrate, basihyal and

ceratohyal of Japanese rice fish embryos...... 67

13. Effects of valproate on the development of basibranchials 1-3, basibranchial 4 and

ceratobranchials 1-2 of Japanese rice fish embryos...... 68

14. Effects of valproate on the development of ceratobranchials 3-5 of Japanese rice fish

embryos...... 69

15. Effects of valproate on catalase , gr and gst mRNA expression in Japanese rice fish at three

developmental stages...... 71

16. Effects of valproate on nlgn3b , otx2 , wnt1 and iro3 mRNA expression in Japanese rice fish

at three developmental stages...... 72

17. Effects of valproate on shh and ccna2 mRNA expression in Japanese rice fish at three

developmental stages...... 73

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CHAPTER 1

INTRODUCTION

1.1 Valproic acid-induced teratogenesis (fetal valproate syndrome and autistic-like behaviors)

An article in 1992 (Lindhout and Omtzigt, 1992) showed that approximately one out of every 250 pregnant mothers uses antiepileptic drugs and a large majority of these use valproate as monotherapy or in the part of polytherapy. Valproic acid (VPA, 2-propyl-pentanoic acid), a short chain branched fatty acid, was first introduced as an antiepileptic drug in 1967 in France (Perucca,

2002). In 1978, VPA was put on the market in United States to treat convulsive disorders, and has become widely prescribed worldwide since then. In the clinic, VPA is usually the preferred drug for treatment of a broad range of seizures, such as absence seizures, myoclonic seizures, generalized tonic-clonic seizures, atonic attacks and partial seizures. In addition to its antiepileptic effects, VPA is also used as a mood stabilizer in the treatment of psychiatric illnesses such as bipolar disorder (depressive-manic phases) (Gyulai et al., 2003; Keck and McElroy,

2003), and migraine (Calabresi et al., 2007). The mechanisms of action of valproate to treat convulsive disorders are suggested to be multiple. Valproate is known to deter neurons consistent

repetitive firing frequently in culture. Valproate also has an active effect on Na + currents. It is also

indicated that valproate can block the NMDA receptor-mediated excitatory currents, and increase

the levels of GABA in the brain probably through improving GABA synthesis and inhibiting its

degradation under the effects of valproate on glutamic acid decarboxylase and GABA

transaminase respectively. Valproate is also approved to inhibit the activity of GAT-1, which is a

GABA transporter, to facilitate GABA levels in the neurons. Through intensive research, valproate

is shown as a potent histone deacetylase inhibitor, however, some scientists (Gurvich et al., 2005)

believed this mechanism of action is where the teratogenic effects of valproate are derived from.

Besides its common functions, VPA is unconventionally used for the treatment of ischemia (Costa

et al., 2006), cancer (Blaheta, et al., 2005), Alzheimer’s disease (Qing et al., 2008), latent HIV

(Lehrman, et al., 2005), terminal blood loss (Alam et al., 2009), and traumatic brain injury (Dash et

al., 2010). The common adverse effects of VPA in the general population are nausea, vomiting,

gastrointestinal complaints (abdominal pain and heartburn) and a fine tremor if patients are taking

a high dose. Other toxic effects of VPA include weight gain, tremor, increased appetite,

hepatotoxicity, thrombocytopenia and alopecia (Terbach and Williams, 2009).

Compared with other antiepileptic drugs (AEDs), such as phenytoin, phenobarbital and

carbamazepine, VPA is designated to be highly toxic if used on pregnant mothers (Dravet et al.,

1992; Kaneko et al., 1999; Kini et al., 2006; Morrow et al., 2006; Diav-Citrin et al., 2008).

However, VPA has anticonvulsant effects on many different types of epilepsy and relatively absent sedative side-effects, compared with carbamazepine and phenobarbitone (Clayton-Smith and

Donnai, 1995). Therefore, VPA is classified as a Pregnancy Category D drug (Dufour-Rainfray et al., 2011) by the American Food and Drug Administration (FDA), which is defined as although detrimental effects of VPA on human fetuses are supported by available investigational and marketing evidence, potential pros of VPA may be weighed over its potential cons, and it becomes a common therapeutic drug used in pregnancies with epilepsy.

Embryogenesis is subject to the detrimental effects of teratogens (Oxendine et al., 2006).

Compared with mice and rhesus monkeys, human embryos are more susceptible to VPA

(Hendrickx et al., 1988; Ehlers et al., 1992; Ehlers et al., 1996; Al Deeb et al., 2000). Development of the human embryo can be divided into three crucial periods (Ornoy and Arnon, 1993; Arnon et al., 2000; Ornoy, 2003; Ornoy, 2006). The first important period is 0-2.5 weeks after fertilization, which is pre-organogenic. Although embryos in the pre-organogenic period are thought to be not sensitive to toxicants, highly toxic teratogens may lead pregnancies in this period into abortion or still birth. The second significant period, which starts from the 3rd week to the 8 th week post fertilization, is active organogenesis, and is theorized to be the most susceptible period (Ornoy,

2009). This theory holds that some toxicants can alter anatomical structures and functions of the organ to exhibit the damaging effects. The third critical period is the fetal period which continues

until the fetus is born. The earlier the period of the development the embryos were exposed to the teratogens, the more severe the deleterious effects were. Therefore, in general, the first trimester of pregnancy is the most sensitive period, and pregnant mothers need to take precautions to avoid contacting teratogens. Additionally, the longer the exposure continues, the more damaging the effects are. Development of the brain continues throughout pregnancy, and hence the brain can suffer from adverse effects even if exposure to VPA extends beyond the first trimester (Ornoy and Arnon, 1993; Arnon et al., 2000; Ornoy, 2003; Kultima et al., 2004; Ornoy, 2006). It is commonly accepted that VPA-induced teratogenic effects are dose and developmental stage specific (Ornoy and Arnon, 1993; Arnon et al, 2000; Ornoy, 2003; Ornoy, 2006). Therefore, three developmental stages (0-2 dpf, 1-3 dpf, 4-6 dpf) in Japanese rice fish embryos and several dosages (2, 5, 10, 20, 40 and 80 mM) were chosen for this project.

The adverse effects of VPA were first reported in 1980 (Dalens et al., 1980). In 1984, Dr.

DiLiberti (Diliberti et al., 1984) proposed a specific term "fetal valproate syndrome" to describe a well-recognized constellation of major congenital anomalies, dysmorphic features and neurodevelopmental retardations of children born to mothers taking valproic acid during pregnancy. Due to ethical constraints, VPA-induced teratogenic effects were investigated by reported cases in a prospective or retrospective methodology on children born to VPA-treated mothers during pregnancy. In addition, some subjects were not willing to participate in the

investigation, and, therefore, the results could not rule out a self-selection bias. Therefore, there is no sufficient data to draw the conclusive decisions for the phenotypes of children under the impacts of VPA-induced teratogenesis. From the available data, the adverse effects of VPA on embryonic and fetal development are categorized into three aspects (Ornoy, 2009): (1) Elevated risks of major congenital anomalies including neural tube defects, cardiovascular, orofacial and digital abnormalities, deformities of eye, skin, abdominal wall, brain, respiratory tract and genitourinary systems, etc.; (2) A specific syndrome, ‘Fetal Valproate Syndrome’, presented with facial dysmorphic features (figure 2), but also involves damage to other organs, such as the neural tube; (3) Neurodevelopmental disorders that affect cognitive, social and verbal skills, which lead to stereotypic behaviors, such as autistic-like symptoms. The neurodevelopmental disorders are probably due to neural tube defects and other abnormalities which occur in the

CNS.

Embryonic and fetal VPA exposure correlates with increased risks of neural, craniofacial, cardiovascular, and skeletal birth defects (Nau et al., 1991 and Nau, 1994), and VPA exposure induces similar symptoms in a variety of species including amphibians, fish, primates, rodents and birds (Dawson et al., 1991; Oberemm et al., 1992; Herrmann, 1993; Kratke et al., 1996;

Briner et al., 2000; Pennati et al., 2001). Neural tube defects and cardiac defects, the two most common congenital birth defects in the human population (Gurvich et al., 2005), also resulted

from VPA toxicity with high incidence. Neural tube defects, such as spina bifida, are reported to occur with 1-2% incidence in all VPA-treated pregnant mothers, and have often been seen in the lumbar and sacral positions (Dalens et al., 1980; Gomez, 1981; Bjerkedal et al., 1982; Robert et al., 1983; Lindhout et al., 1992, Omtzigt et al., 1992 and Fried et al., 2004). Lumbosacral myelomeningocele was observed at 10-20 times the rate in the general population (Dalens et al.,

1980; Gomez, 1981; Bjerkedal et al., 1982; Robert et al., 1983; Lindhout et al., 1992, Omtzigt et al., 1992 and Fried et al., 2004). Neural tube defects can be generated in the third week post fertilization. This is generally before the pregnancy can be detected or confirmed. Hence, women under treatment with VPA at childbearing age should stop usage of VPA before any planned pregnancy (Ornoy, 2009). Besides neural tube defects and cardiac defects, the major congenital anomalies also include trigonocephaly, cleft palate and lip, radial ray defects, pulmonary abnormalities, coloboma of iris/optic disc and digital defects (Kini, 2006). In general, congenital anomalies in babies born to VPA-treated mothers during pregnancy occur at a 2 to 3 times higher rate than in the general population (Yerby, 1998; Kozma, 2001). Children with FVS also show facial dysmorphic features characterized by broad forehead, thin arched eyebrows with medial deficiency, epicanthal folds with infraorbital grooves, flat nasal bridge, broad nasal root, short upturned nose, long, smooth and shallow piltrum, downturned mouth, long and thin upper lip, and thick lower lip (figure 2) (DiLiberti, et al., 1984; Winter, et al., 1987; Clayton-Smith, et al., 1995).

In addition, valproate treatment of pregnant mothers results in low verbal IQ and autistic-like disorders (Kini, 2006). As the epidemiological studies indicated, valproate exposure during pregnancy is associated with increased incidence of autistic-like behaviors of the offspring

(Christianson et al., 1994; Rasalam et al., 2005; Bromley et al. 2008).

Autism spectrum disorder (ASD) is an umbrella term used to define a heterogenous neurodevelopmental disorder including classic autism, Asperger syndrome, Rett syndrome, childhood disintegrative disorder and pervasive developmental disorder-not otherwise specified

(PDD-NOS) (Pardo et al, 2007; Lianeza et al., 2010). ASD is described as impairment of cognitive abilities and deficiencies in social, communicative, and verbal skills (Gaily et al., 2004). ASD is also accompanied by repetitive and sometime self-injurious actions (Landrigan, 2010). ASD correlates with attention deficit disorder, and can result in educational problems, especially related to verbal intelligence (Gaily et al., 2004). However, difficulties in social interactions or relationships are also found in numerous other neurodevelopmental disorders mentioned in the

Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) (Bishop et al.,

2007). Thus, use of only behavior criteria to diagnose ASD is not sufficient for diagnosis. Children with ASD are usually diagnosed before 3 years of age. Macrocephaly is a commonly diagnostic characteristic relevant to autism (Williams et al., 2008).

Epidemiological studies indicate increased incidence of ASD worldwide accounting for about 1 out of every 150 children in the USA with a male to female ratio of 4.3: 1 (West et al, 2009).

It is indicated that ASD is a multigenic disease, and environmental insults with deleterious effects resulting in gene mutation and epigenetic alterations, particularly when these happen at the critical developmental stages (Schanen, 2006), represent significant risk factors in development of ASD.

In 2006, resperidone was approved by the FDA to treat irritability in children and adolescents with

ASD, however, resperidone therapy can lead to significant weight gain, metabolic problems, tardive dyskinesia and neuroleptic malignant syndrome (Hong et al., 1999; Newcomer, 2005). In traditional Chinese medicine, 'Wu Chi' is a specific term to describe the symptoms that are most similar to those of ASD (Zhang, 2010). 'Wu Chi' is translated as five retardations, which are standing-up retardation, toddling retardation, hair-growth retardation, tooth-growth retardation and echolalia retardation. A prescription involving ginseng is assigned to children with the five retardations (Zhang, 2010). Ginseng contains many ingredients, such as ginsenosides which are a group of triterpenoid saponins and steroid glycosides. Ginsenosides can increase neurogenesis in vivo and in vitro under physiological and pathophysiological circumstances (Lu et al., 2010).

Additionally, ginseng can promote axonal regeneration, and it is also effective in decreasing the formation of reactive oxygen species to show its potential neuroprotective function (Rudakewich et al., 2001; Ye et al., 2009; Lu et al., 2010). Children with FVS and/or ASD need elaborative care

and particular service due to their poor verbal and coordinative skills and musculoskeletal deficiency. Children may need extra help at school and even one to one help due to the existent learning problems (Kini, 2006). Toilet training may also be highly affected, and affected children need significant training to acquire it. Physiotherapy is provided for children with motor developmental delay. Surgical intervention before the age of 6 months (Lajeunie, et al., 2001) to correct craniosynostosis can also be beneficial for the affected children.

Since valproic acid is a teratogen, its use during pregnancy is necessary to be considered

cautiously. The daily dose of VPA during pregnancy is recommended to be below 1000mg and

divided into 2-3 individual doses. The serum level of VPA needs to undergo minimal fluctuations

when taking these 2-3 doses per day (Yerby, 2003; Alsdorf and Wyszynski, 2005; Diav-Citrin et

al., 2008). Dosages above 1000mg of VPA are associated with elevated risks of teratogenic

effects (Ornoy, 2009). Vitamins of vitamin E and vitamin B family (vitamin B6, B9 and B12),

especially folic acid, are indicated to have potential protective effects for embryos and fetuses

(Elmazar et al., 1992; Al Deeb et al., 2000; Yerby, 2003). It is accepted that folic acid is aiding

rapid cell division to prevent neural tube defects, and hence it is recommended that women take

4-5 mg per day before planned conception and during the first 2-3 months of pregnancy. For

women who could not find alternative medications for VPA, it is advised that the potential

protective reagents, such as vitamin B6, B9, B12 and methionine, are prescribed at

preconception and during pregnancy. In addition, follow-up examinations are needed to detect the status of the fetus before it is born (Ornoy, 2009).

1.2 Animal Models:

A spectrum of structural and neurobehavioral anomalies arise in the development of human embryos fetuses due to the toxicity of VPA, most appropriately termed as fetal valproate syndrome.

Intensive researches have been done to determine the potential mechanisms underlying

VPA-induced teratogenesis, but none of them have provided conclusive results. Human studies of

FVS are restrained due to ethical constraints. However, several non-human animal models from mammals to non-mammals have been utilized to explore the molecular mechanisms of this disorder. Multiple mechanisms can lead to damage to embryos exposed to VPA, and certain animal models are specialized to study some specific aspects of these processes. Therefore, the choice of animal model is of vital importance in the investigation of FVS. Each animal model system has its own pros and cons, according to the addressed questions (Cudd, 2005) and comparisons between species are informative in investigating gene function and genome evolution (Wang et al., 2006).

Among the animal models used for mechanistic studies of ASD, rats and mice are the preferred models because they are mammals, phylogenetically more close to human, and detailed behavioral phenotypic data are available in many of the rodent strains (Halladay et al., 2009). However, non-mammalian vertebrates and invertebrates are valuable as high-throughput experimental

models to investigate how the nervous system performs normally and the effects of various environmental stimulants on the nervous system (Halladay et al., 2009). Although the hearts of fish and human beings possess essentially different anatomy, their heart morphogenesis, vasculogenesis and hematopoiesis are accurately controlled by conserved genetic programs in vertebrates (Lambrechts and Carmeliet, 2004). Another advantage of fish models over the mammalian models is that the embryos can survive and continue to grow even if the blood circulation is completely absent due to small sizes and low metabolism. This allows observation for many days, while mammals under the same condition will die early (Lambrechts and Carmeliet,

2004).

Japanese rice fish ( Oryzias latipes ), the Far East cousin of zebrafish, and zebrafish (Danio rerio) derived from a common ancestor about 140 million years ago (wittbrodt et al., 2002). Both of them are rapidly emerging as important fish models for research on developmental biology and toxicology (Wang et al., 2006), and interaction between biological living items and their environment. One benefit of applying the fish models rather than mammalian models includes their external (outside the body of the mother) fertilization and embryonic development (fertilized eggs and larvae), which avoids maternal/placental/fetal influences on embryo development.

Another benefit is the transparent chorions covering the embryos. Therefore, investigators can observe the developmental conditions of embryos, such as their onset of circulatory system and

normal/abnormal heart formation, directly (Wu et al., 2008). Additionally, fish models possess the characteristics of easy availability, low maintenance cost, short life cycle and their accessibility to studies of the gene function (Wittbrodt et al., 2002; Furutani-Seiki and Wittbrodt, 2004). Therefore, fish models are currently emerging as alternatives to mammalian models. Large-scale mutagenic screens in zebrafish and Japanese rice fish have revealed several orthologue genes that are highly correlated to those involved in human genetic diseases (Naruse et al., 2004) .

In zebrafish, VPA-induced teratogenic effects have been investigated by many investigators and the data were almost identical with other models. More specifically, retardation and cessation of embryonic development, yolk and cardiac edema, brain deformities, malformations of eye, otoliths and somites, cardiovascular defects, skeletal deformities including shortened and bent tail (Herrmann, 1993; Selderslaghs et al., 2009) were observed in zebrafish embryos and hatchlings after developmental VPA exposure. However, VPA analogs such as valpromide were found to be weak in producing teratogenic activity in zebrafish compared to VPA alone (Herrmann, 1993; Gurvich et al., 2005). S-valproate is associated with a significant increase in intersomitic vessel defects in zebrafish embryos (Isenberg et al., 2007). Moreover, VPA interfered with liver and exocrine pancreas formation in zebrafish during embryogenesis (Farooq et al., 2008). Furthermore, VPA suppresses seizure-like behavior and improves learning ability in adult zebrafish treated with pentylenetetrazol (Lee et al., 2010). The teratogenic effects of VPA in

zebrafish are thought to be mediated through the inhibition of HDACs (histone deacetylases)

(Gurvich et al., 2005; Menegola et al., 2006; Cao et al., 2009). Several laboratories have

established zebrafish as an applicable animal model to study mechanisms of VPA toxicity and/or

autism spectrum disorders (ASDs), and developing another fish model for comparative studies can

increase persuasive abilities of the results and allow opportunities for improvement (Wang et al.,

2006). Besides genome size strength of Japanese rice fish if compared with genome size of

zebrafish (800 Mb in Japanese rice fish and 1700 Mb in zebrafish), only the Japanese rice fish and

its nearest relative, Oryzias curvinotus , are the known non-mammalian vertebrates where the sex determining gene DMY has been identified and characterized (Matsuda, 2005; Matsuda et al.,

2002, 2003). Therefore, Japanese rice fish may be an excellent alternative non-mammalian animal model if we are looking forward to verifying what, if any, effects the gender might have in VPA toxicity. Japanese rice fish exhibit another advantage over zebrafish due to the fact that their cardiovascular development conforms mostly to the common embryonic circulatory pattern investigated in other vertebrates including the human, whereas the zebrafish has a distinctly different pattern (Fujita et al., 2006).

In Japanese rice fish, the embryonic development of the cardiovascular system has been

thoroughly studied by several investigators (Iwamatsu, 2004; Fujita, 2006). Since Japanese rice

fish grow distinct yolk veins, these may be exploited as target sites to study the effects of VPA on

the cardiovascular system (Wang et al., 2006). Therefore, the structure and development of the cardiovascular system needs to be understood if we intend to know the significance of time-dependent treatments. Table 3 outlines every developmental phase of the cardiovascular system in the embryos of Japanese rice fish. Japanese rice fish embryos after Iwamatsu stage 25 can be compared to fetuses, since these embryos (Iwamatsu stage 25 and onwards) have a well-developed cardiovascular system, several organs including the brain (organogenesis) and pectoral fin buds share corresponding features with human fetuses (Hu et al., 2006).

1.3 Metabolism of valproic acid

80-90% of consumed VPA binds to plasma proteins and is conjugated in the liver of human beings (Kini, 2006). VPA exhibits its toxicity to the fetus and becomes concentrated in the fetal compartment by diffusion across the placenta instead of placental binding (Nau H.1982;

Albani et al., 1984; Bailey and Briggs, 2005). The highest ratio of VPA serum concentration in fetuses to that in mothers can be 6, and mean ratios are 1.4-2.4 (Ornoy, 2009). This means that the fetus is exposed to a much higher concentration of VPA than the mother (Clayton-smith and

Donnai, 1995; Ornoy, 2009). In 1984, Albani (Albani et al., 1984) reported that low concentrations of VPA are excreted into human milk (less than 5% of the weight-adjusted daily dose that taken by mothers) (Von et al., 1984; The WHO Working Group, 1988). Therefore,

lactation and breast feeding by VPA-treated mothers is allowed (Albani et al., 1984; Von et al.,

1984; The WHO Working Group, 1988).

1.4 Potential Molecular Mechanisms of toxicity of VPA and autism:

The mechanism(s) by which VPA induces anticonvulsant, antimanic, and teratogenic effects are unknown, but some reports suggest that the medical and the teratogenic effects of VPA are different. Several studies indicate that teratogenic effects of VPA may be related to folic acid deficiency, induction of oxidative stress, and inhibition of HDAC (Ornoy, 2009). Furthermore,

VPA influences gamma aminobutaric acid (GABA) synthesis and breakdown (Loscher, 2002;

Calabresi et al., 2007), reduces de novo inositol synthesis (Agam et al., 2002; Shaltiel et al., 2007) and ion channel activity, causes inositol triphosphate depletion (Berridge et al., 1989; Eickholt et al., 2005; Shimshoni et al., 2007), regulates fatty acid signaling and turnover (Bazinet et al., 2006), and activates the mitogen activated protein (MAP) kinase pathway (Hao et al., 2004). VPA is also associated with carnitine depletion (Coulter, 1991), which affects mitochondrial fatty acid metabolism leading to hepatotoxicity, and hepatocellular necrosis can happen. Syndromic ASD accompanied by tuberous sclerosis, neurofibromatosis or macrocephaly presents abnormal cellular/synaptic growth rate, and this is because mTOR/PI3K pathway was activated by mutated

TSC1/TSC2, NF1, or PTEN (Pardo and Eberhart, 2007; Bourgeron, 2009). Neurexin, which is a presynaptic protein, interaction with the postsynaptic adhesion molecule neuroligin, is necessary

for synaptic maturation and synaptogenesis (Bourgeron, 2009). Neuroligin then interacts with the

scaffold protein Shank, which activates other scaffold proteins, such as PSD-95. This regulates

AMDA, NMDA and GABA receptors in the postsynaptic membranes and affects the balance

between excitatory and inhibitory electrical currents (Sudhof, 2008; Bourgeron, 2009). This

nrxn -nlgn -shank pathway is, therefore, suggested to be related to the causes or symptoms of ASD.

In addition, signaling molecules, such as neurotrophin (for example, brain-derived neurotrophic

factor (BDNF)), Reelin, hepatocyte growth factor, and neurotransmitters such as serotonin and

glutamate, are also thought to be involved in the pathogenesis of ASD (Pardo and Eberhart,

2007). VPA-induced teratogenesis probably derives from the interactions between VPA and

several genes and proteins in humans at some certain critical developmental stages. These

interactions alter the expression of several genes and result in modification of protein function.

In our study, we examined several genes to investigate their expression pattern under the

treatment of VPA. Catalase , gst and gr are oxidative stress-related genes, and were used to

investigate whether VPA has a toxic effect through an oxidative stress-related pathway. The

another five genes, nlgn3b , iro3 , wnt1 , otx2 , shh , are expressed in the brain of Japanese rice fish embryos and are involved in neurogenesis. The other two genes, cyclin a2 and shh , are associated with cell division. Most of these genes were reported to be expressed in Japanese rice fish embryos during morphogenesis (Kage et al., 2004: Wu et al., 2008, 2011), and are altered by

valproate in human and rat (Graf et al., 1998; Bambini-Junior et al., 2011; Tung and Winn, 2011).

Iro3 , otx2 , shh , wnt1 are transcription factors expressed in the neural tube of Japanese rice fish embryos at early developmental phases and are implicated importantly in brain morphogenesis

(Kage et al., 2004). The gene, i ro3 , belongs to the Iroquois family of homeoproteins and is constitutively expressed in vertebrates, flies, and nematodes (Gomez-Skarmeta and Modolell,

2002). In zebrafish, iro3 has been shown to be required for correct organizer function (Kudoh and Dawid, 2001). The gene, otx2 , is recognized as an early head-domain marker (Blitz and Cho,

1995; Pannese et al., 1995). The wnt1 gene family involves several signaling molecules showing many biological effects in the formation of the mesoderm of the gastrula (Christian and Moon,

1993; Hoppler et al., 1996; Hoppler and Moon, 1998) and the neural ectoderm (McGrew et al.,

1995, 1997) at the dorso-ventral axis during cleavage stages (Moon and Kimelman, 1998). The gene, shh , plays a critical role in cell proliferation, differentiation and patterning by activating the hedgehog signaling pathway. The shh gene is expressed in several organs, including the lung

(Bellusci et al., 1997), the fin ray (Laforest et al., 1998), the epidermis (Sire and Akimenko,

2004), somites (Fan and Tessier-Lavigne, 1994; Johnson et at., 1994; Fan et al., 1995), limb and fin buds (Krauss et al., 1993; Laufer et al.,1994), and teeth (Bitgood and McMahon, 1995; Iseki et al., 1996; Koyama et al., 1996). The cyclins, and their cyclin-dependent kinase partners the

Cdks, are the basic components of the machinery that regulates the passage of cells through cell

cycle. These selected genes were used to investigate the potential bio-signaling pathways involved in VPA-induced pathogenesis.

1.5 Hypothesis and objectives of the study:

It was revealed by human epidemiological studies that embryos in utero exposed to VPA during the first trimester (first 12 weeks) of pregnancy are associated with high risk of

VPA-induced teratogenesis including fetal valproate syndrome (FVS) and autism spectrum disorders (ASDs) in the offspring (Christianson et al., 1994). This indicates that VPA-induced deleterious effects are developmental stage-dependent. Three developmental stage groups (0-2 dpf,

1-3 dpf, 4-6 dpf) of Japanese rice fish embryos were chosen to investigate the effects of VPA.

Differentiation and migration of neural crest cells (NCCs) during embryogenesis determines craniofacial cartilages and bones formation. Therefore, any alteration in embryonic differentiation and migration of NCCs may affect the structure of craniofacial cartilages and bones (Wang et al.,

2006). In Japanese rice fish, the excision of the neural crest on embryos at the neurula stage will lead to defects in craniofacial cartilages and bones (Langille and Hall, 1988). During embryogenesis, the heart initiates function as the first organ prior to the advancement of other organs, and normal development and function of the cardiovascular system is very crucial for the growth of embryos of Japanese rice fish. Since embryos are supplied with nutrients that come from yolk by circulation, any disruptions occurring to circulation/circulatory system by VPA will

prohibit embryos from normal development (Hu et al., 2006). Development of the cardiovascular system and the craniofacial cartilages of fish models are subject to exogenous chemicals, such as alcohol (Wang et al., 2006). Children affected with FVS have congenital major malformations occurring in the cardiovascular system and the neurological system with characteristic facial dysmorphisms. Therefore, cardiovascular and craniofacial features were investigated and used as the phenotypic parameters to study VPA-induced teratogenesis in Japanese rice fish.

The present study had three main objectives. First, to determine the validity of VPA as a teratogen in Japanese rice fish by comparison with those effects previously observed in

VPA-treated zebrafish and mammalian models. Second, the molecular mechanisms that are suggestive to be related to VPA-induced teratogenesis (ASD and/or FVS) in humans will be investigated by studying gene expression patterns in Japanese rice fish. Finally, the applicability of the Japanese rice fish as an alternative animal model for future research on FVS and/or ASD will be studied. In this regard we have analyzed several morphological parameters including neurocraniol and splanchnocranial cartilages and mRNA expression patterns of several genes which are expressed during Japanese rice fish embryogenesis (Wu et al., 2008, 2011). Our data indicate that VPA is able to induce teratogenic effects in Japanese rice fish. These include yolk edema, malformed neurocranium and splanchnocranium, decreased hatching efficiency, reduced heart rate, increased thrombi formation, inhibited onset of blood circulation, and altered

expression of several genes, the orthologs of which have been shown to have the potential to

induce ASD in humans. Japanese rice fish ( Oryzias latipes ) embryo-larval development has been used as a unique non-mammalian model to study the molecular mechanism of FVS.

Epidemiological studies in humans indicate that VPA exposure during pregnancy induces higher incidence of autism in the offspring (Christianson et al., 1994; Rasalam et al., 2005; Bromley et al.,

2008). Therefore, teratogenic studies with VPA may throw light on understanding the mechanisms associated with ASD.

Figure 1: Chemical structure of valproic acid (Chateauvieux, et al., 2010)

Table 1: Specific features related to fetal valproate syndrome (Kini, 2006)

Neural tube defects Trigonocephaly Radial ray defects Pulmonary abnormalities Coloboma of iris/optic disc Low verbal IQ Autism and autism spectrum disorder

Figure 2: Child with facial dysmorphic features of FVS: trigonocephaly, which has been surgically repaired, broad forehead, thin arched eyebrows with medial deficiency, flat nasal bridge, infraorbital grooves, broad nasal root, short anteverted nose, long, shallow, and smooth philtrum, long and thin upper lip, and thick lower lip (Kini, 2006).

broad forehead

thin arched eyebrows with flat nasal bridge medial deficiency

infraorbital grooves broad nasal root short anteverted nose long, shallow and smooth philtrum long and thin upper lip thick lower lip

Table 2: Anomalies associated with valproate exposure (Kini, 2006)

Neural Tube Defects Congenital Heart Defects Limb Defects Spina bifida Ventricular septal defect Radial ray defect Anencephaly Atrial septal defect Polydactyly Aortic stenosis Split hand Patent ductus arteriosus Overlapping toes Anomalous right pulmonary artery Camptodactyly Ulnar or tibial hypoplasia Absent fingers Oligodactyly Genitourinary Defects Brain Anomalies Eye Anomalies Hypospadias Hydranencephaly Bilateral congenital cataract Renal hypoplasia Porencephaly Optic nerve hypoplasia Hydronephrosis Arachnoid cysts Tear duct anomalies Duplication of the calyceal system Cerebral atrophy Microphthalmia Partial agenesis of corpus callosum Bilateral iris defects Agenesis of septum pellucidum Corneal opacities Lissencephaly of medial sides of occipital lobes Dandy-Walker anomaly Abdominal Wall Defects Respiratory tract anomalies Skin abnormalities Omphalocoele Tracheomalacia Capillary haemangioma Lung hypoplasia Aplasia cutis congenita of scalp Severe laryngeal hypoplasia Abnormal lobulation of the right lung Right oligaemic lung *Rare structural abnormalities are shown in italics.

Table 3: Phenotypic features of the cardiovascular system development of Japanese rice fish (Iwamatsu, 2004)

Iwamatsu Hpf Description stage 22 ~38 The heart becomes visible. 23 ~41 The blood vessels (Cuvierian ducts and vitello-caudal vein or marginal vein) and the intermediate cell mass (ICM) or blood island become obvious. 24 ~44 The hearts begins beating with very low heart rate (32-64 beats/min), and the embryonic erythrocytes and hemangioblasts are gathered in ICM during this period. 25 ~50 The heart rate rises (70-80 beats/min) and the globular blood cells come out from the ICM (onset of circulation). 28 ~64 The blood cells display flattenly. 29 ~74 The embryonic heart starts to differentiate into atrium, ventricle, sinus venosus and bulbous aorta. 30 ~82 Arteries starts to branch to provide trunk muscles, gills and brain with blood. 34 ~121 The circulation to the pectoral fin takes place. 36 ~144 The looping of heart is accomplished.

CHAPTER 2

MATERIALS AND METHODS

The institutional animal care and use committee (IACUC) of the University of

Mississippi has approved all of the experimental animal protocols. Methods of animal maintenance, embryo collection, alcian blue staining of hatchlings, RNA extraction, purification and semi-quantitative relative (RT-PCR) techniques have been previously described

(Dasmahapatra et al., 2005; Wang et al., 2006, 2007; Hu et al., 2008, 2009; Wu et al., 2008,

2011). The research procedure used Japanese rice fish embryos and hatchlings. Each group

consisted of 8-16 embryos and each experiment was repeated 3-5 times.

2.1. Fish maintenance: Adult males and females (orange-red variety, d-rR strain) Japanese

Rice fish ( Oryzias latipes ) were maintained in a AHAB Stand-Alone recirculating system

(Aquatic Habitats), which is a Japanese Rice fish housing system, at 25 ± 1 °C, 16L:8D

photoperiod in balanced salt solution (BSS, 17 mM NaCl, 0.4mM KCl, 0.3 mM MgSO 4, 0.3

mM CaCl 2 in nano-pure water), keeping salinity at 1-1.2 ppt and pH 7.4. The Japanese rice fish

were fed with a combination of tetramin flakes (Tetra) and live brine shrimp twice a day. The

brine shrimp eggs were purchased from Ocean Star International, Inc. (10880 Wiles Road,

Coral Springs, FL 33076). Brine shrimp eggs hatched after being maintained in salty water (30

g/L NaCl) and aerated for 48 hrs at 25 ± 1 °C.

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: The clustered fertilized embryos were collected and separated manually by gently rubbing over a 20.3 cm (diameter) × 2.5 cm (depth) brass sieve (Fisher Scientific

Company) with openings that are 300 m. The status of each embryo was checked under a

phase contrast microscope (AO Scientific Instruments, Model: 1820) and the viable embryos

were kept for future research. The developmental stages of embryos were identified and

categorized as Iwamatsu described previously (2004). Three developmental stages of Japanese

rice fish embryos were tested (A group: 0-2 dpf; B group: 1-3 dpf; C group: 4-6 dpf). Fertilized

embryos with intact chorion within ~4h, 24 h (~1 day) or 96 h (~4 day) post fertilization (dpf)

were exposed to VPA (0-80mM) for 48 hours with a one time change of medium at the same

concentration of VPA at 24 h. After 48 hrs exposure, the embryos were transferred to clean

hatching solution in 48 well culture plates (one embryo/well/ml hatching solution) without VPA

and with a 50% static renewal of the medium per day until 14 dpf. The hatching solution

contained 0.0002% methylene blue (MB) to prevent fungal growth.

2.3 Lethal concentrations of valproic acid determined on embryos of Japanese rice fish

exposed at three different developmental stages: The unhatched embryos were maintained in

the 48 well culture plates until 14 dpf. The embryos without heartbeats on 14 dpf would be

considered dead. The LC 50 values (mortality of embryos) for VPA in three different

developmental stage groups were obtained by 14 dpf.

2.4 VPA effects on hatching efficiency of Japanese rich fish: Under the experimental conditions of 25±1°C, 16L: 8D light cycle mentioned above, embryos began hatching after 7 dpf. The development of the embryos treated with VPA was comparatively delayed, and therefore the alive but unhatched embryos were maintained in 48 wells culture plates until 14 dpf. The embryos that hatched earlier were recorded. The final hatching efficiency (cumulative hatching incidence of embryos at each developmental stage) was determined on 14 dpf.

2.5 Analysis of deleterious effects of VPA on the cardiovascular system of Japanese rice

fish: The cardiovascular system (circulation status and development of thrombi) of the embryos was examined on 6 dpf under the phase contrast microscope (AO Scientific Instruments) with a

50% static renewal of VPA free medium each day. The heart rates and circulation status can be investigated according to the developmental stage maps that were established by Iwamatsu

(2004). The embryos were in circulating state when there was blood flowing through any of the circulation vessels. When blood was not flowing even through a single vessel the embryos were considered as in non-circulating state. The heart rate in beats per minute of each embryo was

determined on 6, 7 and 8 dpf in ovo .

2.6. Morphometric analysis of craniofacial cartilages in Japanese rice fish hatchlings

exposed to VPA.

2.6.1. Treatments: The alive embryos were obtained and separated according to the method described above (Paragraph 2.2). Embryos at two different developmental time points (Early developmental stage: 0-2 dpf; Late developmental stage: 4-6 dpf ) were transferred to 48 wells culture plates and exposed to 1 ml hatching solution or 1ml hatching solution with 2 mM VPA

(1 embryo/ml/well). Exposure was for 48 hrs with changing of the corresponding medium at 24 hrs. After 48 hrs exposure, the embryos were washed twice with clean hatching solution and maintained in 1 ml hatching solution until hatching with a 50% static daily renewal of clean hatching solution. Ordinarily, the embryos begin to hatch after 7 dpf. Since VPA can lead to developmental delay, embryos were maintained in hatching medium until 14 dpf. To avoid post hatching growth, within 24 hrs after hatching the hatchlings were preserved in 4% paraformaldehyde.

2.6.2. Cartilage staining: The neurocranial cartilages of hatchlings were stained with 0.1 %

Alcian blue (Sigma-Aldrich, St. Louis, MO) following the protocol used in Zebrafish by Carvan

et al. (2004) with minor modifications. After hatching, the hatchlings (8 or 16 per group) were

anesthesized in MS222 (Tricaine methanesulfonate) (0.1% in hatching solution: Sigma-Aldrich,

St. Louis, MO), and then fixed in 4% paraformadehyde (Sigma-Aldrich, St. Louis, MO) in phosphate-buffered saline (PBS) with 0.1% Tween 20 (PBT) at 4 0C for 24 hrs. The fixed hatchlings were washed with nanopure water twice and then treated with 5% TCA for 10 mins.

The TCA-treated hatchlings were washed briefly with acid ethanol (0.37% HCl, 70% ethanol), and stained in 0.1% Alcian blue for 6 hrs. Excess stain was washed away with ethanol. 1% trypsin was used to digest the muscle tissues of the stained hatchlings at 37 0C for 3-4 hrs until all the muscles disappeared as determined by the absence of dark spots in the view as it appeared under the phase contrast microscope. The samples were finally preserved for long-term use in 70% glycerol at 4 0C after washing with glycerol-KOH (50% glycerol and

0.25% KOH).

2.6.3. Morphometric analysis: The neurocranial and splanchnocranial cartilages, stained with

Acian blue, were investigated under an Olympus BX 40F-3 microscope to analyze the effects of

VPA exposure during embryonic development. The entire neurocranium and splachnocranium were captured as photomicrographs using the microscope at fixed magnification (100X/40X).

Most of the photomicrographs were captured at 100X magnification, and some were at 40X magnification. The linear lengths of the cartilages of the neurocranium and the splanchnocranium were measured using imaging software (Media Cybernetics, Silver Springs,

MD). The length of the cartilages were calculated by pixel values and multiplying by the

corresponding magnification factor. After staining with Alcian blue, the ethmoid plate (EP) with lamina orbitonasalis, paired trabeculae (TB), anterior orbital cartilage (AOC), posterior orbital cartilage (POC), epiphyseal bar (EB), basilar plate (BP) with anterior (ABC) and posterior

(PBC) basicranial commissures, hypophyseal plate (HP) with paired polar cartilages (PC), and auditory capsules in the neurocranium could be visualized. In the splanchnocranium, Meckel’s cartilage (MC), pterygoid processes (PT), quadrate (QU), hyosymplectic (HYS), hyoid (HYO), basihyal (BH), ceratohyal (CH), 4 basibranchials (BB), 3 pair hypobranchials (HBR), 5 pair ceratobranchial (CBR), 4 th epibranchial (EBR) and 4 th hypobranchial (HBR) are also clearly identifiable (Figures 6-7). The nomenclature of the cartilages used in this communication was defined by Langille and Hall (1987). In the present experimental conditions, generally embryos of group E exposed to 5 mM VPA and above, were unable to hatch even after 14 dpf. In group

L, less than 25% of embryos exposed to 5 mM VPA were able to hatch. Therefore, only data for hatchlings of groups E and L exposed to 2mM VPA prenatally are presented. The individual cartilages and the entire neurocranium were used for morphometrical analysis. In the neurocranium, EP, AOC, EB and BP are linear, and therefore were measured directly. TC and

PC are not always of equal size in the left and right sides, and hence both sides were measured individually and an average was calculated. The linear length of ABC was measured from the anterior end of the ABC to the head of the notochord on both sides and then averaged. In the

splanchnocranium, QH, BH, CH, BB (1-4), CBR (1-4) are linear and were measured directly.

BB 1-3 can not be identified visually, and thus the total length of BB 1-3 was considered as one parameter. BB 4 is separated from BB 1-3 visibly, and thus was measured individually. The lower jaw (MC) is curved, and therefore the length was determined from the joint points of two parts of the lower jaw (MC) to the midpoint of an imaginary line drawn linking the lower jaws ends. The 5 th CBR is also curved and its length was decided as the distance between the meeting points of the left and right 5 th CBRs and the central of an imaginary line drawn to link the anterior end of the pharyngeal teeth. The POC, auditory capsule, PBC in the neurocranium, and PT, HYS, HYO in the splanchnocranium are irregularly shaped and consequently were not used for morphometric analysis (figures 6-7).

2.7. Biochemical analysis of Japanese rice fish embryos exposed to valproic acid

RNA was extracted from the intact embryo (embryos with yolk) by Trizol reagent

(Wang et al., 2007). The primers used for rRT-PCR of mRNAs of several genes ( catalase, gr, gst, iro3, nlgn3b, otx2, wnt1, shh and ccna2 ) are mentioned in Table 8.

2.7.1. Treatments: Japanese rice fish embryos were exposed to varying concentrations of VPA at three different time points of development (A group: 0-2 dpf; B group: 1-3 dpf; C group: 4-6 dpf). Exposure was discontinued after 48 hours following a one time renewal of the medium at

24 h. In addition to controls, embryos in A group were exposed to 1 mM and 2 mM VPA, and in

B and C groups were exposed to 2 mM, 5 mM. For RNA extraction, the embryos were washed with hatching solution twice and homogenized immediately after completion of the 48 hr exposure to VPA.

2.7.2. Isolation of RNA: The total RNA was extracted from Japanese rice fish embryos by using Trizol reagent (Invitrogen, Carlsbad, CA), following the manufacturer's instruction. 0.5 ml of Trizol was added to each tube, which contained 6-8 embryos from the same treatment group. These were then homogenized and centrifuged (Labnet International, Inc., Edison, NJ; model: PRISM R) at 12,000 X g for 20 mins at 4 0C. The upper clear phase was removed to a

1.5 ml clean centrifuge tube, to which was added 0.25 l of isopropanol (Sigma-Aldrich, St.

Louis, MO, USA). The mixture was incubated at room temperature for 10 minutes, and centrifuged again at 12, 000 X g for 20 mins at 4 0C. The precipitate was cleaned with a 1 ml

75% ethanol wash. DNA from the washed RNA was removed by treating the samples with

DNase I (Promega, Madison, WI). The final 50 ul volume containing one unit of DNase I, 1 X

transcription buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl 2, 0.5 mM MnCl 2) and 40 U of

RNAsin (Promega, Madison, WI) was added to the 75% ethanol-washed RNA. After incubation at 37 0C for 20 mins, the DNase I was removed by the same methods of Trizol precipitation. The purified RNA was stored at -80 0C. The concentration of RNA was obtained with an Eppendorf Biophotometer. The purity of the RNA was determined by distinct 18S and

28S ribosomal RNA using 1% agarose gel electrophoresis with 0.1% ethidium bromide, and the

ratio of absorbance at wavelength 260 nm and at 280 nm is over 1.5.

2.7.3. Semi-quantatative RT-PCR: A first strand cDNA synthesis protocol (Invitrogen,

Carlsbad, CA) was followed to synthesize cDNA by using 1 g of DNA-free total RNA. The cDNA was synthesized in a 20 ml reaction volume that included 4 l 5 X buffer (composition),

1 l 10 mM dNTPs, 1 l 10 mM DTT, 1 l oligo dT and 200 U of superscript Ш RNA

polymerase (Invitrogen, Carlabad, CA) at 50 0C for 1 h. 1 l of synthesized cDNA in a final

volume of 20 l was used to detect the expression levels of several transcription factor mRNAs

by semi-quatitative PCR. The 20 l reaction mixture contained 10 l of 2 X buffer (composition)

(Qiagen, Valencia, CA), 1 l cDNA, 50 pM each of forward and reverse primers of the target

genes and 50 pM each of forward and reverse primers of the internal control (beta-actin). The

amplification reaction for semi-quatitative PCR was carried out in a PTC-200 Peltier Thermal

cycler (Opticon 2 MJ Research, Reno, NV). 30 cycles of denaturation at 94 0C for 30 s,

annealing at 60 0C for 1 min and extension at 72 0C for 2 mins followed initial denaturation at

94 for 2 mins. The final step was extension at 72 0C for 7 mins. 5 l of the reaction product was

separated using 2% agarose gel electrophoresis (100 voltages; room temperature) containing

0.1% ethidium bromide ( g/ml) and two bands, one of the target gene and one of the internal

control, appeared under 290-365 nm ultraviolet light. The gel electrophoresis pictures were

taken using a Versadoc Imaging System (BioRad, Hercules, CA; Model: 3000) and the band

intensities of the target gene and the internal control were determined using image analysis

software (Quantity One, BioRad). The results were expressed as the ratio of relative band

intensity of target gene to the band intensity of the β-actin gene.

2.8. Statistical analysis: LC 50 s were calculated from transformed data ( log valproate concentration) using a nonlinear regression (curve-fit) program (Graph-Pad Prism, version 5,

San Diego, CA). All other data (thrombi, vessel circulation, heart rate and hatching efficiency) were analyzed by using one way ANOVA followed by Tukey's multiple comparison post-hoc test. The rRT-PCR data were transformed (log ratio of band intensity of target gene to the band intensity of the β-actin gene) before analysis. For skeletal cartilages endpoint measurement, the

data were analyzed by unpaired Student's t test. The results were expressed as mean ± SEM of

3-4 independent experiments and P<0.05 was considered significant.

CHAPTER 3

RESULTS

3.1 Lethal concentration of valproic acid on Japanese rice fish developmentally and prenatally exposed at different developmental stages.

Three developmental stages of Japanese rice fish embryos were set as three different treatment groups and tested (A group: 0-2 dpf; B group: 1-3 dpf; C group: 4-6 dpf). Fertilized embryos of Japanese rice fish with intact chorion were exposed to valproate (0-80 mM) for 48 hrs during these three developmental stages with a one time change of the medium at 24 hr and then transferred to clean hatching solution without valproate. On day 14 post fertilization (dpf),

the calculated LC 50 values for valproate as determined from three separate independent experiments (group A has six separately independent experiments, groups B and C has three separately independent experiments) were 1.68 ± 1.55 mM with 0-2 dpf (group A) , 26.45 ± 1.64 mM with 1-3 dpf exposure (group B) and 20.37 ± 3.3 mM with 4-6 dpf exposure (group C)

(Table 3). The survival rates of embryos were severely reduced in groups B and C when the concentration of VPA was 20 mM or higher. Additionally, the embryos died completely in group

A if the VPA concentration reached 10 mM. When the concentration was 40 mM or higher, 75%

- 100% of embryos in all groups died. It is worth noting that group A is more sensitive to the toxicity of VPA since this group showed 75%-100% mortality rate occurring in all experiments at

the concentrations of 5 mM or higher. The LC 50 of group A is much lower than that of groups B and C, suggesting that the VPA toxicity is developmental-stage specific (Figure 3).

3.2 Effects of vaproic acid on hatching efficiency of Japanese rice fish at different developmental stages.

In our facility, the control embryos initiated hatching at 7+ dpf. The hatching efficiency of the embryos was altered by VPA and found to be dose-dependent. The embryos of groups A,

B and C were able to hatch (>75%) if they were exposed to VPA concentrations below or equal to 2mM. However, when embryos were exposed to 5 mM valproate, none of the group A embryos, below 50% of group B embryos and below 5% of group C embryos were able to hatch successfully (Figure 4). It is notable that group B embryos with 5 mM VPA treatment had a much higher incidence in hatching efficiency comparing with group A or C exposed to the same concentration of VPA. The reason for this will be discussed in the next chapter. None of the embryos treated with 10 mM VPA in all groups was able to hatch. Compared with controls, 5 mM or higher concentration of VPA was able to reduce the hatching efficiency significantly in all groups.

3.3 Morphometric analysis of Japanese rice fish exposed to valproic acid at different

developmental stages.

3.3.1 VPA effects on the cardiovascular system

The cardiovascular development (heartbeat, circulation status and thrombus formation) of

the embryos was examined on day 6 post fertilization (for group A 4 days after, for group B 3

days after, and for group C immediately after valproate removal from the medium).

3.3.1.1 VPA effects on heart beat

Heart rates were determined for three consecutive days (6-8 dpf) in all three groups.

Usually, the control embryos start hatching after 7 dpf. To avoid hatching effects, we have

restricted our heart rate observations in ovo from 6 to 8 dpf. Here again a dose-dependent reduction in heart rate in all groups was observed. Moreover, embryos exposed to 10 mM or higher concentration of VPA in group A, and embryos exposed to 80 mM VPA in groups B and C, displayed no heart beat in the 6-8 dpf period (Figure 5.1-5.3). In group A, the heart rate was significantly reduced in embryos exposed to 5 mM. Heart rate was not reduced in group A embryos exposed to 2 mM valproate (Figure 5.1). In groups B and C, the embryos exposed to 40 mM or lower have heart beat, however, dose-dependent reductions of the rates of heart beat in both groups were noticed (Figure 5.2-5.3). In group A, a total of 24 embryos were exposed to 5 mM VPA, and examined on three consecutive days (6-8 dpf); only 2, 1, and 1 of 24 embryos had heart beats on 6, 7, and 8 dpf respectively. Additionally, the mean rates of the heart beat on these

three consecutive days were 6.58, 5.21, and 5.40 beats per minute. These heart rates are much lower than those of groups B and C, which are above 90 beats per minute (Figure 5.1-5.3). It was shown that VPA has a more deleterious effect on the early developmental stage (group A) of

Japanese rice fish embryos. In group B, no embryo exposed to 40 mM VPA had heart beats on 6 dpf, while 8.33% and 16.67% of group B embryos had heart beats on 7 dpf and 8 dpf respectively (Figure 5.2). The heartbeat normally starts at 44 hrs post fertilization (Iwamatsu stage 24), and the embryos in group B (1-3 dpf) started VPA treatment before the onset of the heartbeats. When the embryos of group B were treated with 40 mM VPA, the heart beat of some embryos, which should initiate around 2 dpf, were delayed due to VPA toxicity and gradually started after 6 dpf. These data demonstrate that VPA can delay the onset of heart beat. All the embryos of group C started the VPA treatment after their heartbeats have already initiated. The percentage of embryos with heartbeats in group C, if they were exposed to 40 mM VPA, decreased from 66.67% on 6 dpf, to 54.17% on 7 dpf and finally down to 50% on 8 dpf (Figure

5.3). This demonstrates that high dose VPA can inhibit heartbeat after it has already initiated

(Figure 5.3). In group A, no embryo survived if the exposure concentration of VPA was above

5mM, and the heart rates of group B embryos were reduced more sharply by increasing the concentration of VPA comparing with those of group C embryos (Figure 5.1-5.3). To sum up, in groups A, B and C, the VPA-induced teratogenic effects on the onset and rates of heart beats are

dose/developmental stage-dependent.

3.3.1.2 VPA effects on thrombus formation

On 6 day post fertilization, a significant number of thrombi were noticed in the embryos that survived valproate exposure (group A 5 mM, group B 5 mM and 10 mM, and group C 20 mM, 40mM and 80 mM subgroups). Moreover, thrombi were seen only in those embryos that had developed heartbeats. When valproate treatment prevented initiation of heartbeat, no thrombi were seen. Embryos of Japanese rice fish normally start heartbeats first (44 hpf, Iwamatsu developmental stage map) and then blood circulation (50 hpf, Iwamatsu developmental stage map). Embryos of groups A and B were exposed to VPA before the initiation of heartbeat and blood circulation. Additionally, some embryos in group A (0-2 dpf) and group B (1-3 dpf) exposed to higher concentration of VPA (5 mM or higher) possessed thrombi even though they lacked blood circulation (Figure 6.1). No matter in which group, the higher the concentration of

VPA, the more thrombi appeared. In addition, with the elevated concentrations of VPA, more thrombi were shown in the embryos without circulation on 6 dpf. Some of these embryos have not developed onset of circulation, while the circulatory system of others was inhibited after its onset. An interesting phenomenon is group B (1-3 dpf) embryos treated with 20 mM valproic acid exhibited no thrombus development on 6 dpf. It was suggested that the thrombi were seen only in those embryos that have the ability to develop blood vessels. If valproate treatment (high

concentration of VPA) is able to stop blood vessel formation, no thrombus would have been seen.

Embryos in group C (4-6 dpf) were exposed to VPA after they had already started heartbeat and blood circulation. Therefore, the thrombus formation in group C was probably due to the detrimental effects of VPA toxicity which lead to injury of the endothelial cells of blood vessels

(Figure 6.1). Embryos in the late development stage (4-6 dpf) were more resistant to the

VPA-induced teratogenesis, and therefore some embryos (4-6 dpf) treated with 20mM, 40 mM or

80 mM VPA were alive and presented thrombus on 6 dpf. This is different from embryos in the early development stage (0-2 dpf and 1-3 dpf). The onset of heartbeat is a crucial factor in the thrombus formation. Onset of blood circulation also plays an important role in thrombus formation. Thrombus formation is both dose and developmental-stage dependent.

3.3.1.3 VPA effects on circulation

The onset of embryonic blood circulation was also found to be dose and developmental stage-specific (Figure 6.2). It was observed that almost all the embryos that were able to hatch successfully had well developed circulatory vessels and the blood circulated in the body and yolk through these vessels. However, of those embryos that were unable to hatch, the majority of them had either no blood vessels or difficulties in obtaining blood circulation. When observed on

6 dpf, group A embryos exposed to 2 mM, and most of group B embryos exposed to 2 and 5 mM, were able to circulate blood through vessels. Less than 5% of group A embryos exposed to

5 mM VPA, or group B embryos exposed to 10 mM VPA had vessel circulation on 6 dpf (Figure

6.2). The majority of group C embryos that were treated with 2, 5, 10 mM concentration of VPA had blood circulation on 6 dpf. However, as development proceeded in this group, a dose-dependent loss (5, 10 mM VPA ) of vessel circulation was noticed. With increasing concentrations of VPA, the incidence of thrombus was elevated and the condition of thrombus became more severe in all groups. In general, circulation could be prevented by valproate, and finally, this could prevent embryos from hatching. Embryos exposed to 10 mM VPA or higher in group A, 20 mM VPA or higher in group B were unable to start blood circulation. In group C,

46.875% of embryos exposed to 20 mM VPA exhibited blood circulation. This indicates that the late developmental stage is more resistant to VPA-induced teratogenesis. When compared to controls, the incidence of blood circulation of 20 mM VPA-treated embryos in group C was decreased significantly (53.125% ) on 6 dpf (Figure 6.2).

3.3.2 VPA effects on the hatchlings

The hatched embryos with 2 mM VPA embryonic exposure prenatally seldom exhibited bent tails, but they might manifest yolk edema. However, under the embryonic treatment of 4mM

VPA, the hatched embryos are probable to present bent tails, diminished body length and yolk edema (Figure 7).

3.3.3 VPA effects on the craniofacial cartilages

The craniofacial cartilages were examined within 24 hrs of hatching after staining with

Alcian blue (group E: early developmental stage without circulation (0-2 dpf treatment); group L: late developmental stage with circulation (4-6 dpf treatment)) (Table 5). The neurocranium of

Japanese rice fish embryos starts chondrification, which is to develop into cartilage or become cartilaginous in ovo on 5 dpf (Iwamatsu stage 33). This completes prior to or after hatching until hatchlings reach Iwamatsu stage 40. A typical Japanese rice fish embryo immediately after hatching possesses a chondrocranium which includes auditory and nasal capsules, a well-developed neurocranium and splanchnocranium and a minimal amount of chondral braincase elements (Figure 8-9). The detailed values for each of the treatment groups are listed in table 4. Six cartilages, POC, auditory capsule, PBC in the neurocranium and PT, HYS and HYO in the splanchnocranium were not considered for measurement due to irregular (non-linear) shapes.

3.3.3.1 Neurocranial Cartilages:

3.3.3.1.1 Neurocranium

It was observed that, in general, compared to controls the linear length of the neurocranium of hatchings in groups E and L treated with 2 mM valproate were significantly reduced (Figure 10 & Table 7). This indicates that VPA exerted a detrimental influence over the entire neurocranium.

3.3.3.1.2 Trabeculae (TC)

Compared to controls, TC was significantly reduced 8.08% in group E (0-2 dpf)

hatchlings exposed to 2 mM valproate prenatally but not in group L hatchlings (Figure 10 &

Table 7). This indicates that early developmental stages are more sensitive to the deleterious

effects resulting from VPA toxicity.

3.3.3.1.3 Epiphyseal Bar (EB)

The linear lengths of the EB in hatchlings (group E, 0-2 dpf treatment) treated with 2 mM

VPA were significantly reduced 8.17% compared with controls. EB from group L hatchlings (4-6 dpf) with 2 mM VPA treatment grow much larger than those from group E (1-3 dpf) treated with the same concentration of VPA. This indicates that VPA toxic effects are developmental-stage specific and that VPA toxic effects are more pronounced in the early developmental stage (Figure

10 & Table 7).

3.3.3.1.4 Anterior Orbital Cartilage (AOC)

AOC displayed significant reduction (10.19%) in linear length relative to controls only in group E hatchlings (0-2 dpf) treated with 2 mM VPA. The development of AOC in group L (4-6 dpf) hatchlings with 2 mM VPA exposure was not significantly affected. Compared with group E with the same concentration of VPA treatment, AOC in group L treated with 2 mM VPA grows much longer (15.28%) (Figure 11 & Table 7). These data indicate that toxicity caused by VPA is

predominant in the early developmental stage.

3.3.3.1.5 Anterior Basicranial Commissure (ABC)

ABC remained unaltered in hatchlings of 2 mM VPA-treated groups E (0-2 dpf) and L

(4-6 dpf) when compared with controls. It was determined that ABC was significantly longer in group L hatchlings with 2 mM VPA treatment than it was in group E with the same treatment

(Figure 11 & Table 7). This may be due to the fact that early developmental stage suffers more severely from VPA toxicity.

3.3.3.1.6 Basilar Plate (BP)

The linear length of BP in group E hatchlings (0-2 dpf) treated with 2 mM Valproate was reduced significantly (4.27%) when compared with controls. BP of group E hatchlings with 2 mM VPA treatment prenatally were significantly smaller (6.17%) compared with those correspondingly hatchlings in group L (4-6 dpf) (Figure 11 & Table 7). These data suggest that the teratogenic effects from VPA toxicity were more remarkable in the early developmental stage.

3.3.3.1.7 Polar Cartilage (PC)

It was shown that 2 mM VPA only decreased PC in group L hatchlings significantly

(24.47%) when exposed embryonically (Figure 11 & Table 7). VPA manifested its detrimental impacts on PC in the late developmental stage.

3.3.3.2 Splanchnocranium

3.3.3.2.1 Meckel's Cartilage (MC)

MC showed no reduction in 2 mM VPA-treated hatchlings of groups E and L compared

with controls (Figure 12 & Table 7).

3.3.3.2.2 Quadrate (QU)

Compared to controls, the hatchlings with 2 mM VPA exposure exhibit significantly

shorter QU in groups E and L respectively. QU of the hatchlings with 2 mM VPA exposure

prenatally in group E demonstrated significant reduction (6.33%) in length compared with those

treated with the same concentration of VPA in group L (Figure 12 & Table 7). It is observed that the early developmental stage is most subject to the damage derived from VPA toxicity.

3.3.3.2.3 Basihyal (BH)

Compared to controls, there were no alterations in the linear lengths of BH of the hatchlings in groups E and L respectively (Figure 12 & Table 7).

3.3.3.2.4 Ceratohyal (CH)

It was recorded that the linear lengths of CH of the hatchlings in both groups E and L treated with 2 mM VPA were decreased significantly (7.41% for group E and 8.24% for group L) compared to controls (Figure 12 & Table 7).

3.3.3.2.5 Basibranchial 1-3 (BB 1-3)

BB 1-3 revealed significant reduction in the linear lengths from the hatchlings exposed to

2 mM VPA embryonically in groups E and L, compared with controls. BB 1-3 in group E with 2

mM prenatal VPA exposure were significantly shorter (10.97%) than those in group L (Figure 13

& Table 7). It was indicated that the early developmental stage (0-2 dpf) possesses less

resistant capability to VPA toxic effects.

3.3.3.2.6 Basibranchial 4 (BB 4)

VPA produced no modification of BB 4 in any treatment groups (Figure 13 & Table 7).

3.3.3.2.7 Ceratobranchial 1 (CBR 1)

It was shown that CBR 1 of the hatchlings treated with 2 mM VPA prenatally was

reduced in length significantly only in group L when compared with controls (Figure 13 & Table

7). This indicates that CBR1 at the late developmental stage is more sensitive to VPA toxicity.

3.3.3.2.8 Ceratobranchial 2 (CBR 2)

CBR 2 displayed significant reduction in linear length only in the hatchlings of group L

treated with 2 mM VPA, when compared with controls (Figure 13 & Table 7). This indicates that

CBR2 at the late developmental stage is most subject to the toxic effects of VPA.

3.3.3.2.9 Ceratobranchial 3 (CBR 3)

Compared to controls, the linear length of CBR 3 in the hatchlings decreased significantly (11.40%) only in 2 mM VPA-treated group L. (Figure 14 & Table 7). This indicates

that the late development of CBR3 is more resistant to VPA-induced teratogenesis.

3.3.3.2.10 Ceratobranchial 4 (CBR 4)

Compared to controls, the linear length of CBR 4 was decreased significantly (17.46%) only in the hatchlings of group L embryonically treated with 2 mM VPA. The linear length of

CBR 4 in the hatchlings of group L treated with 2mM VPA displayed significant reduction when compared with those in group E (Figure 14 & Table 7). It is suggested that VPA exerts toxic effects on CBR 4 at the late developmental stage.

3.3.3.2.11 Ceratobranchial 5 (CBR 5)

It was found that, compared to controls, hatchlings displayed significantly reduced linear length of CBR 5 (25.27%) only in the hatchlings of group L that were embryonically exposed to

2 mM VPA (Figure 14 & Table 7). This indicates VPA-induced teratogenic effects on CBR 5 are more apparent at the late developmental stage.

3.4 Biochemical analysis of Japanese rice fish embryos exposed prenatally and developmentally to VPA.

To find a molecular characteristic related to VPA-induced teratogenesis in valproate-treated Japanese rice fish, we have analyzed mRNA expression patterns for nine genes that are related to oxidative stress (catalase, glutathione-s-transferase, glutathione reductase), neurogenesis (iro3, nlgn3b, wnt1, shh, otx2) and cell division (shh, cyclin a2). All of these genes

have been reported to be expressed in Japanese rice fish embryos during morphogenesis. In this experiment, Japanese rice fish embryos were exposed to two concentrations of valproate for 48 hrs in three developmental stages (group A: 0-2 dpf; group B: 1-3 dpf; group C: 4-6 dpf). Group

A embryos were exposed to 1 and 2 mM, and groups B and C embryos were exposed to 2 and 5 mM of valproate. The treatment continued for 48 hrs with a one time change of the media at 24 hrs. The embryos in each group were sacrified for mRNA analysis immediately after treatment.

The primers used for rRT-PCR are presented in Table 5.

3.4.1 Oxidative Stress

VPA did not alter the catalase, gst, or gr mRNA expression levels indicating that VPA toxicity probably was not the result of oxidative stress.

3.4.1.1 Catalase

The data obtained by rRT-PCR showed that the embryos treated with different concentrations of VPA were able to maintain equal levels of catalase mRNAs within groups A, B and C just after VPA treatment for 48 hrs (Figure 15.1). Therefore, VPA did not affect catalase mRNA levels.

3.4.1.2 Glutathione-S-Transferase ( gst )

Embryos in groups A, B and C did not show significant reduction or increase in gst mRNA expression levels when compared to their corresponding controls. In conclusion, VPA did

not have an impact on gst mRNA expression pattern. Glutathione-S-transferase mRNA

expression level significantly increased in controls on 6 dpf in contrast to those on 2 dpf. This

suggested that gst mRNA is expressed constitutively and upregulated as the development of

embryos go on (Figure 15.2).

3.4.1.3. Glutathione Reductase (gr)

The gr mRNA contents remained unaltered after valproate exposure in groups A, B and C

(Figure 15.3).

3.4.2 Neurogenesis

3.4.2.1 Neuroligin 3b ( nlgn 3b )

The neuroligin 3b mRNA expression pattern was altered in a dose and developmental stage-specific manner. When compared to controls, embryos of group B with 5 mM VPA exposure showed statistically significant reductions (84.04%) in nlgn 3b mRNA levels. It is

indicated that the nlgn mRNA expression levels tend to be reduced in VPA-treated embryos, and

the extent of reduction elevates with increasing concentration of VPA. It is believed that a larger

number (n) of experiments would make this result significant. The nlgn 3b mRNA in controls manifested an elevated expression pattern in groups B and C when compared with those in group

A, suggesting that neuroligin 3b mRNA is expressed constitutively and upregulated as the development of embryos continues (Figure 16.1).

3.4.2.2 Orthodenticle Homeobox 2 ( Otx2 )

The otx2 mRNA expression pattern was modified in a dose and developmental

stage-specific method. Compared with controls, the expression levels of otx2 mRNA were only

reduced by valproic acid in group B embryos treated with 5 mM VPA. Compared with controls

in group A, otx2 mRNA expression levels were upregulated in controls of groups B and C (on 3

dpf and 6 dpf ) (Figure 16.2). This suggested that otx2 mRNA was expressed constitutively and

its level was upregulated as the development of the embryos progressed.

3.4.2.3 Wingless-type Mouse Mammary Tumor Virus Integration Site Family, Member 1

(Wnt1 )

The wnt1 mRNA expression pattern was altered in a dose and developmental

stage-specific manner. Wnt1 mRNA expression level was only reduced in embryos of group B

treated with 5 mM valproic acid. Wnt1 mRNA content of controls on 6 dpf was less than the content on 3 dpf (P<0.0001) (Figure 16.3). It is indicated that wnt1 is constitutively expressed and its expression level was changed by the treatment of valproic acid during 1-3 dpf for 48 hrs.

3.4.2.4 Iroquois 3 ( iro3 )

The data obtained by rRT-PCR showed that the embryos from groups A, B and C with

VPA treatment maintained similar amounts of iro3 mRNA content when compared with controls

(Figure 16.4). Thus, iro3 mRNA expression levels remained unaltered after valproate exposure.

It is indicated that the toxic effects of valproic acid were not derived from modification of iro3 mRNA expression pattern.

3.4.3 Cell Division

3.4.3.1 Sonic Hedgehog ( shh )

The shh mRNA expression was modified in a dose and developmental stage-specific

manner. Only in group A were shh mRNA expression levels reduced 46.35% in embryos exposed

to 2 mM VPA (Figure 17.1). This suggests that the shh mRNA expression pattern was altered by

valproic acid in the early developmental stage of embryos, which is the cell division phase

(group A: 0-2 dpf). Therefore, VPA probably manifested its teratogenesis by inhibiting cell

division during early development of embryos.

3.4.3.2 Cyclin A2 ( ccna2 )

The mRNA expression of ccna2 was inhibited by valproic acid treatment significantly at

2 mM concentration in group A (46.33%) and 5 mM in group C (19.37%), when compared with

the corresponding controls. Since cyclin A2 is the gene that controls the progression of cell cycle

and 0-2 dpf is the critical phase for cell division, the result suggests that VPA may exert its

toxicity by reducing cell division (Figure 17.2).

Table 4: Treatment groups classification of Japanese rice fish embryos used for determination of the LC 50 s of VPA, the effects of VPA on the cardiovascular development and the mRNA expression levels of the related genes

Groups VPA treatment (hour Developmental stages at the beginning Observations after

post fertilization) of VPA treatment ( Iwamatsu, 2004) VPA removal (h)

A 0-48 4-10 ~96

B 24-72 17-20 ~72

C 96-144 31-32 ~1

Table 5: Treatment groups classifications of Japanese rice fish embryos used for measurement of craniofacial cartilages

Groups VPA treatment (hour Developmental stages at the beginning of Observations after

post fertilization) VPA treatment ( Iwamatsu, 2004) VPA removal (h)

E 0-48 4-10 ~96

L 96-144 31-32 ~1

IC50

150 Group A Group B Group C 100

50 Mortality (%)

0 -0.5 0.0 0.5 1.0 1.5 2.0 Log VPA concentration (mM)

Figure 3: Valproate-mediated mortality in Japanese rice fish embryos. Fertilized eggs of Japanese rice fish within 4 hpf (A), 24 hpf (B) and 96 hpf (C) were exposed to valproate (0-80 mM) for 48 h and then transferred to clean hatching solution. The effect on mortality was recorded on 14 dpf. The experiment was repeated 3 times and each treatment group consisted of 2 8 embryos. The calculated LC 50 values are 1.68 ± 1.55 mM for group A (r =0.83), 26.45 ± 1.64 mM for group B (r 2=0.88) and 20.37 ± 3.30 mM for group C (r 2=0.55). LC50s were calculated from transformed data (log valproate concentration) using a nonlinear regression (curve-fit) program (GraphPad Prism). Each data point represents the mean mortality precentage ± SEM (n=3). These data indicate that group A embryos were more sensitive to valproate than groups B and C.

Table 6: The LC 50 values of Japanese rice fish embryos exposed to VPA at three different developmental stages

Group LC 50

group A 1.68 ± 1.55 mM

group B 26.45 ± 1.64 mM

group C 20.37 ± 3.3 mM

Hatching efficiency 150

100 (%) 50 * Embryos Hatch Embryos * 0 * * * Control 5 mM-A 2 mM-B 5 mM-B 2 mM-C 2 mM-A C 5 mM- 10 mM-B 10 mM-C

Figure 4: Hatching efficiency of Japanese rice fish embryos developmentally exposed to valproate. Embryos at specific developmental stages (A, B, and C) were exposed to different concentrations of valproate (0-80 mM) for 48h. Then they were transferred to clean hatching solution. Parallel groups with no valproate served as controls. For group details see Table 4. Hatching was recorded from 7+ dpf. Each group consists of eight embryos and the experiment was repeated 3 times. Each bar is the mean ± SEM of 3 independent experiments. Bar head with asterisks (*) indicate that the data were significantly different (P<0.05) from the controls (no valproate).

Heart rate- A group 150

100

Heart rate 50 (beats/ min) ###

0 6 day (C) 7 day (C) 8 day (C) 6 day 2 mM 6 day 5 mM 7 day 2 mM 7 day 5 mM 8 day 2 mM 8 day 5 mM Treatment Groups 5.1

Heart rate- B group Heart rate- C group 150 150

# # # # # # # # # 100 100 # # # # # # #

Heart rate 50 # Heart rate (beats/ min) # # # 50 (beats/ min) # # # 0 0 6 day (C) day 6 6 day (C) day 6 (C) day 8 7 day (C) day 7 7 day 2mM day 7 6 day 2 mM 2 day 6 mM 5 day 6 mM 5 day 7 mM 2 day 8 8 day 5 mm 5 day 8 6 day 2 mM 2 day 6 mM 5 day 6 mM 2 day 7 mM 5 day 7 mM 2 day 8 mM 5 day 8 6 day 10 mM 10 day 6 mM 20 day 6 mM 40 day 6 mM 10 day 7 mM 20 day 7 mM 40 day 7 mM 10 day 8 mM 20 day 8 mM 40 day 8 7 day control day 7 control day 8 6 day 10 mM 10 day 6 mM 20 day 6 mM 40 day 6 mM 10 day 7 mM 20 day 7 mM 40 day 7 mM 10 day 8 mM 20 day 8 mM 40 day 8 5.2 Treatment Groups 5.3 Treatment Groups

Figure 5: Heart rates of Japanese rich fish embryos exposed to Valproate during development. Fertilized embryos were exposed to valproate (0-80 mM) for 48 h in groups A, B and C and then transferred to clean hatching solution. Parallel groups with no valproate served as controls. For group details see Table 4. Heart rates were examined on 6, 7, and 8 dpf. Each group contained 8 embryos per treatment. Each bar is the mean ± SEM of three independent treatment repetitions. Bar head with pound symbol (#) indicates that the data are significantly different (P<0.05) from the corresponding controls (no valproate). Figure 5.1: heart rates of group A; figure 5.2: heart rates of group B; figure 5.3: heart rates of group C. The effects of valproate on heart rates were dose- and developmental stage-specific.

Circulation Embryos with thrombus 150 150

* * * * 100 100 *

(%) * (%)

* Embryos 50 Embryos 50 with with thrombi

with vessel circulation with vessel * * 0 0 control 2mM-A 5mM-A 2mM-B 5mM-B 2mM-C 5mM-C Control 10mM-B 10mM-C 20mM-C 2 mM-A 2 mM-A 5 mM-B 2 mM-B 5 mM-C 5 2 mM-C 2 10 mM-B 10 mM-B 20 mM-C 10 mM-C 20 mM-C 40 mM-C 80 Treatment groups 6.1 Treatment Groups 6.2

Figure 6: The occurrence of thrombi and blood circulation observed on 6 dpf in embryos after exposure to valproate at three different developmental stages (groups A, B, and C). Fertilized embryos were exposed to valproate (0-80 mM) for 48 h in groups A, B and C and then transferred to clean hatching solution. Parallel groups with no valproate served as controls. For group details see Table 4. The incidences of thrombi and vessel circulation onset were examined on 6 dpf. Each group contained 8 embryos per treatment. Each bar is the mean ± SEM of three independent treatment repetitions. Bar head with asterisks (*) indicate that the data are significantly different (P<0.05) from the corresponding controls (no valproate). Figure 6.1= Incidence of thrombi in each group observed on 6 dpf. Figure 6.2= Incidence of vessel circulation onset in each group observed on 6 dpf. The effects of valproate on occurrence of thrombi and vessel circulation were dose- and developmental stage-specific.

B C A

(A) Control (B) 2mM –Group C (C) 4mM –Group C (Spine cord i s curved.)

Figure 7: Representative photomicrographs displaying teratogenic effects of valproate in hatched Japanese rice fish. Embryos (4 dpf) were exposed to 2 mM and 4 mM VPA for 48 h (4-6 dpf) and then maintained in clean hatching solution without VPA until hatching. The photographs were taken with an Olympus BX 40F-3 microscope at fixed magnification (40X). Figure 7.1: control; 7.2: embryos exposed to 2 mM VPA; 7.3: embryos exposed to 4 mM valproate. Arrow ( ) represents the area where the yolk edema was seen. In valproate-treated hatchlings, yolk edema (7.2) and bent tail (7.3) are seen.

Table 7: Effect of valproate on the length of neurocranial and splanchnocranial cartilages (um) of Japanese rice fish hatchlings

Locations Cartilag Group E Group E Group L Group L

e Name Control 2 mM VPA Control 2mM VPA

Neurocranium NRC 920±9.9 883±10.1* 945±10.0 910±9.8*

TC 198±4.6 182±2.5* 200±3.9 186±8.7

EB 306±4.7 281±4.5* 319±4.3 311±4.9

AOC 216±5.1 194±7.2* 229±4.3 229±8.2

ABC 435±6.6 424±5.6 453±7.1 444±5.3

BP 445±6.1 426±7.3* 457±11.3 454±5.6

PC 56±2.4 49±2.7 61.7±2.9 46.6±3.8*

Splanchnocranium MC 118±4.3 116±2.0 125±3.0 118±4.0

QU 335±4.8 311±7.2* 346±4.3 332±6.0*

BH 158±8.6 167±8.7 171±5.8 161±7.5

CH 270±5.8 250±5.9* 279±4.1 256±4.7*

BB 1-3 339±7.3 284±7.6* 360±5.7 319±8.7*

BB 4 43±1.8 41±2.3 46±2.6 47±3.0

CBR 1 211±6.2 199±3.9 227±6.0 189±6.1*

CBR 2 212±5.8 200±3.8 227±5.0 199±4.0*

CBR 3 213±6.5 208±3.7 228±4.7 202±4.4*

CBR 4 243±5.9 230±4.4 252±6.6 208±7.0*

CBR 5 169±5.9 163±5.3 186±7.4 139±13.8*

Japanese rice fish embryos were exposed to 2 mM VPA with or without circulation for 48 hrs at two different developmental stages (group L: treatment during 4-6 dpf; group E: treatment during 0-2 dpf). After 48 hrs treatment, they were transferred to clean hatching solution to allow the embryos to hatch. For group details see Table 5. Groups E and L hatchlings within~24 hrs after hatching were preserved in 4% PFA and used for cartilage staining with alcian blue. The cartilage length was measured with an Olympus BX 40F-3 microscope with image analysis software. NRC= Neurocranium; EP= Ethmoid plate; TC= Trabecular cartilages; EB= Epiphyseal bar; AOC = Anterior orbital cartilages; ABC= Anterior basicranial commisure; BP = Basilar plate; PC = Polar cartilages; MC = Meckel’s cartilage (lower jaw); QU = quadrate; BH= Basihyal; CH = Ceratohyal; BB1-3 = Basibranchials 1-3; BB4 = Basibranchial 4; CBR 1-5 = ceratobranchials 1-5; GWC= Groups with circulation; GWOC= Groups without circulation. The nomenclature of the cartilages used in this communication was obtained from Langille and Hall (1987). The results are expressed as mean ± SEM. The data are analyzed by unpaired t test. Asterisks (*) indicate that the value is significantly different from corresponding controls (no valproate). The deleterious impacts caused by VPA on splanchnocranial cartilages are developmental-stage dependent.

Figure 8: Representative photomicrographs of neurocranial cartilages of Japanese rice fish hatchlings (14 dpf). The hatchlings (14 dpf) were stained with alcian blue and pictures (100X) were taken with an Olympus BX 40F-3 microscope. The picture presented above displays the locations of the different cartilages in the neurocranium (dorsal view) of Japanese rice fish hatchlings.

Figure 9: Representative photomicrographs of splanchnocranial cartilages of Japanese rice fish hatchlings (14 dpf). The hatchlings (14 dpf) were stained with alcian blue and pictures (100X) were taken with an Olympus BX 40F-3 microscope. The picture presented above displays the locations of the different cartilages in the splanchnocranium (dorsal view) of Japanese rice fish hatchling

Neurocranium Trabeculae 1500 250 200 * 1000 * * 150

100 500 Length (um) Length Length (um) Length 50

0 0

M M m ntrol ntrol -2 mM o -2 m o -2 up p p-Control p-C up u up-C ro gro ou grou o E group-2 mM L gro gr E L E g L group-Control E L gr Treatment Groups Treatment Groups

Epiphyseal Bar

400 # 300 *

200

Length (um) Length 100

ol M m ontr p-2 ou roup-C gr roup-Control E L group-2 mM E g L g

Treatment Groups

Figure 10: Effects of valproate on the development of neurocranial cartilages (neurocranium, trabeculae and epiphyseal bar) of Japanese rice fish embryos. Embryos with (group L: 4-6 dpf treatment) or without (group E: 0-2 dpf treatment) circulation were exposed to 2 mM VPA for 48 hrs, with a one time change of medium at the same concentration of VPA at 24 hr. After 48 hrs treatment, the embryos were transferred to clean hatching solution, and then maintained with a 50% static renewal of medium per day until they hatched. For group details see Table 5. The hatchlings were preserved in 4% PFA within~24hrs after hatching and were stained with 0.1% Alcian blue. The cartilage length was measured with an Olympus BX 40F-3 microscope with image analysis software. The results are expressed as mean ± SEM. The data were analyzed by unpaired t test. Asterisks (*) indicate that the values are significantly different (P<0.05) from the corresponding controls and pound symbol (#) indicates the difference (P<0.05) of the data with group E at 2 mM VPA treatment. The deleterious impacts caused by VPA on neurocranial cartilages are developmental-stage dependent.

Anterior Orbital Cartilage Anterior Basicranial Commissure

250 # 500 #

200 * 400

300 150

200 100 Length (um) Length Length (um) Length 100 50

0 0 l rol M ro M t nt m on 2 m o -2 C p -Control u p up-2 mM ro ro group- roup-C g L g E L g group- E group-2 mM E L grou E L group-Control

Treatment Groups Treatment Groups

Basilar Plate Polar Cartilage 80 500 # * 400 60 * 300 40

200 Length (um) Length 20 Length (um) Length 100 0 0 l ol M tro rol nt mM m on o 2 -2 mM C C p- p- oup-2 group oup-Contr gr rou rou g E group-2 mM g L group- E gr L E L E group-Control L Treatment Groups Treatment Groups

Figure 11: Effects of valproate on the development of neurocranial cartilages (arterior orbital cartilage, anterior basicranial commissure, basilar plate and polar cartilage) of Japanese rice fish embryos. Embryos with (group L: 4-6 dpf treatment) or without (group E: 0-2 dpf treatment) circulation were exposed to 2 mM VPA for 48 hrs, with a one time change of medium at the same concentration of VPA at 24 hr. After 48 hrs treatment, the embryos were transferred to clean hatching solution, and then maintained with a 50% static renewal of medium per day until they hatched. For group details see Table 5. The hatchlings were preserved in 4% PFA within ~24hrs after hatching and were stained with 0.1% Alcian blue. The cartilage length was measured with an Olympus BX 40F-3 microscope with image analysis software. The results are expressed as mean ± SEM. The data were analyzed by unpaired t test. Asterisks (*) indicate that the values are significantly different (P<0.05) from the corresponding controls and pound symbol (#) indicates the difference (P<0.05) of the data with group E at 2 mM VPA treatment. The deleterious impacts caused by VPA on neurocranial cartilages are developmental-stage dependent

Meckel's Cartilage Quadrate 150 400 # * * 300 100

200 50 Length (um) Length Length (um) Length 100

0 0 l l l ro o mM tro tr mM ntrol 2 n o - ont o -2 p -C Con C p rou p- p- oup ou u r rou E g L group-2 mM g E group-2 mM L gr E group-C L g E L gro Treatment Groups Treatment Groups

Basihyal Ceratohyal 200 300 * * 150 200

100

100 Length (um) Length 50 (um) Length

0 0 l l M M tro m tro rol mM n nt m ntrol o 2 C Con Co p- p- u u up-Co ro ro o group- E group-2 L group-2 mM E L group-2 E g L g E gr L group- Treatment Groups Treatment Groups

Figure 12: Effects of valproate on the development of splanchnocranial cartilages (Meckel’s cartilage, quadrate, basihyal and ceratohyal) of Japanese rice fish embryos. Embryos with (group L: 4-6 dpf treatment) or without (group E: 0-2 dpf treatment) circulation were exposed to 2 mM VPA for 48 hrs, with a one time change of medium at the same concentration of VPA at 24 hr. After 48 hrs treatment, the embryos were transferred to clean hatching solution, and then maintained with a 50% static renewal of medium per day until they hatched. For group details see Table 5. The hatchlings were preserved in 4% PFA within ~24hrs after hatching and were stained with 0.1% Alcian blue. The cartilage length was measured with an Olympus BX 40F-3 microscope with image analysis software. The results are expressed as mean ± SEM. The data were analyzed by unpaired t test. Asterisks (*) indicate that the values are significantly different (P<0.05) from the corresponding controls and pound symbol (#) indicates the difference (P<0.05) of the data with group E at 2 mM VPA treatment. The deleterious impacts caused by VPA on splanchnocranial cartilages are developmental-stage dependent.

Basibranchial 1-3 Basibranchial 4 400 # 60 * 300 * 40 200

Length (um) Length 20

100 (um) Length

0 0 l M o m tr n mM mM -2 2 2 p u up-2 mM ro up-Co ro oup- oup- g ro g gr E g L E L gr E group-Control L E group-Control L group-Control Treatment Groups Treatment Groups

Ceratobranchial 1 Ceratobranchial 2 250 250 * 200 * 200

150 150

100 100 Length (um) Length Length (um) Length 50 50

0 0 l l M o ol tro mM trol m tr n n 2 2 mM -2 mM o -2 - on - p -Co p -C -Contr u -C p p up p o p rou o u r u ou gr ro g ro r g E L group E g g L E grou L E L g Treatment Groups Treatment Groups

Figure 13: Effects of valproate on the development of splanchnocranial cartilages (basibranchials 1-3, basibranchial 4, ceratobranchials 1-2) of Japanese rice fish embryos. Embryos with (group L: 4-6 dpf treatment) or without (group E: 0-2 dpf treatment) circulation were exposed to 2 mM VPA for 48 hrs, with a one time change of medium at the same concentration of VPA at 24 hr. After 48 hrs treatment, the embryos were transferred to clean hatching solution, and then maintained with a 50% static renewal of medium per day until they hatched. For group details see Table 5. The hatchlings were preserved in 4% PFA within~24hrs after hatching and were stained with 0.1% Alcian blue. The cartilage length was measured with an Olympus BX 40F-3 microscope with image analysis software. The results are expressed as mean ± SEM. The data were analyzed by unpaired t test. Asterisks (*) indicate that the values are significantly different (P<0.05) from the corresponding controls and pound symbol (#) indicates the difference (P<0.05) of the data with group E at 2 mM VPA treatment.. The deleterious impacts caused by VPA on splanchnocranial cartilages are developmental-stage dependent.

Ceratobranchial 4 Ceratobranchial 3 300

250 # * * 200 200

150 100

100 (um) Length Length (um) Length 50 0 0 l M o trol tr ol M ol n m tr mM o 2 m -2 mM -2 -2 C p- p p u -Con p- o p p-Contr oup u gr rou ou gr roup-Con E L grou gr E L grou g E L g E gro L Treatment Groups Treatment Groups

Ceratobranchial 5

250

200

150 *

100 Length (um) Length 50

M m

-Control p

E group-2 mM L group-2 E grou L group-Control Treatment Groups

Figure 14: Effects of valproate on the development of splanchnocranial cartilages (ceratobranchials 3-5) of Japanese rice fish embryos. Embryos with (group L: 4-6 dpf treatment) or without (group E: 0-2 dpf treatment) circulation were exposed to 2 mM VPA for 48 hrs, with a one time change of medium at the same concentration of VPA at 24 hr. After 48 hrs treatment, the embryos were transferred to clean hatching solution, and then maintained with a 50% static renewal of medium per day until they hatched. For group details see Table 5. The hatchlings were preserved in 4% PFA within~24hrs after hatching and were stained with 0.1% Alcian blue. The cartilage length was measured with an Olympus BX 40F-3 microscope with image analysis software. The results are expressed as mean ± SEM. The data were analyzed by unpaired t test. Asterisks (*) indicate that the values are significantly different (P<0.05) from the corresponding controls and pound symbol (#) indicates the difference (P<0.05) of the data with group E at 2 mM VPA treatment. The deleterious impacts caused by VPA on splanchnocranial cartilages are developmental-stage dependent.

Table 8: List of primers used in semi-quantitative RT-PCR (rRT-PCR) amplification of the mRNAs of Japanese rice fish embryo.

mRNA Sense (5 -3) Antisense Product GenBank (5 -3) (bp) accession/ Ensembl number β-actin cctgaccctgaagtat agcgacaagatgag 542 ENSORLT00000 ccca ctggtt 017152 catalase gcggtacaacagcgc ggatggacggccttc 171 ENSORLT00000 agatgaag aagttc 002176 Glutathione reductase ggactactcctgcattc cattgactcttcctgc 165 ENSORLT00000 (gr ) ccacag gtgtgatg 000771 Glutathione-S-transfera gaacctgcagggcta ggccctcaaacatgc 241 GenBankX95200 se ( gst ) caacc gttgg Neuroligin 3b ( nlgn3b ) gatgaggtgccttatgt ccactttggtggcac 292 NM_001201508 gtttg ggtagtg Iroquois homeobox ccagacccaaaaatg ccttgtaaatcctcat 221 AB098317 protein iro3 ( iro3 ) ccacc agccg Wingless-related gtgggagatttcctca cagcaccagtggaa 338 AJ243208 MMTV integration site aggac agtgcag 1 ( wnt1 ) Sonic hedgehog ( shh ) gatgaggagaacacc cttccaccgccagtct 241 AB007129 ggagcc ggac Orthodenticle ggcctgagcttaacta ggactcgggaagatt 201 AJ000939 homeobox2 ( otx2 ) cctc gattgattttc CyclinA2 ( ccna2 ) gctacatgaagaagc gactttgagcaccag 311 ENSORLT00000 agcctg atgctc 024919

gst catalase 0.8 0.8

0.6 0.6 -actin] β β β β 0.4 0.4

0.2 0.2 [catalase: (GST: beta actin) Relative band intesity band Relative Relative band intensity band Relative 0.0 0.0 f f f f pf pf p p p p dpf dpf d d 3 6 6 6 3 d 3 d 3d l l - 2 d l- - 6 dpf m m- o M- m l- mM-2dpf M- r 2 ntro m m t tro 1 mM-2 dpf 2 mM-5 3dpf mM-o 3dpf 1 mM-2dpf n 2 m 5 m 2 mM-65 mM-dpf 6dpf contro c 2 5 2 m control- 2dpf control-2 dpf co con Treatment Groups Treatment Groups 15.1 15.2

0.25

0.20

0.15 -actin] β β β β 0.10 [GR: 0.05 Relative band intensity band Relative 0.00

pf dpf dpf dpf dpf dpf dpf dpf -2 2 -3 6 -6 rol 3 d M rol mm-6 ntrol m 5 o 1 mM-2 mM-2ont 2 mM-35 dpf 2 mm c c cont Treatment Groups 15.3

Figure 15: Effects of valproate on catalase , gst and gr mRNA expression in Japanese rice fish at three different developmental stages. Total RNA was prepared from 6-8 pooled embryos after valproate treatment at 2 dpf (group A), 3 dpf (group B) and 6 dpf (group C). This was reverse transcribed and analyzed by rRT-PCR using β-actin primers as internal control. For group details see Table 4. The results were expressed as the ratio of relative band intensity of the target gene to the band intensity of the β-actin gene. We took the log of the ratios, and these values were used for one way ANOVA followed by Tukey’s post-hoc multiple comparison test; p<0.05 was considered as significant. Each data point is the mean ± SEM of 4 separate experiments. Bar heads with an asterisk (*) indicate that the value is significantly different from the corresponding control (no valproate). Figure 15.1: catalase ; Figure 15.2: gst ; Figure 15.3: gr . The data indicate that VPA teratogenesis may not be derived from oxidative stress.

nlgn3b otx2 0.4 0.20 0.3 0.15 -actin] β β β β 0.2 * -actin] β β β β 0.10

[Otx2: 0.1 0.05 Relative band intensity band Relative [nlgn3b: 0.0

Relative band intensity band Relative * f f f f f f 0.00 pf dpf dp dp 2 dp f l M-2 pf pf M -3 dpM-3 M-6 dp d d dpf dpf m rol -3 dp mM-6 dp 2 3 dpf 3 2 mM-2dt m l- -2 dpf - 6 -5 m o ControA-1 A- on Control 6 dpf r P c A-2 A-5 m A t mM mM-2 trol- mM- 3mM P P 1 2 2 5 mM- V VP V VPA-2V on on 2 5 mM- 6 dpf VP c C control-6 dpf Treatment Groups Treatment Groups 16.1 16.2

wnt1 iro3 0.08 0.15

0.06 0.10 -actin] β β -actin] β β 0.04 β β β β

0.05

[wnt1: 0.02 * [Iro3: Relative band intensity band Relative

0.00 intensity band Relative 0.00 f f f f f f f f p p p p dp dp dp dp d pf pf pf pf pf pf pf pf pf -2 3 -3 6 d -6 d d d d l d 2 2 d d 3 d d M o M rol 2 3 6 -6 m m t mm - - ntr 5 M M ontrol-21 dpf 2 mM-2o d 2 mM-35 d 2 mm-6 trol- trol 3 trol 6 c c con mM- mM-n m mM-n m mM 1 2 o 2 5 o 2 5 Treatment Groups con c c Treatment Groups 16.3 16.4

Figure 16: Effects of valproate on nlgn3b , otx2, wnt1 and iro3 mRNA expression in Japanese rice fish at three different developmental stages. Total RNA was prepared from 6-8 pooled embryos after valproate treatment at 2 dpf (group A), 3 dpf (group B) and 6 dpf (group C). This was reverse transcribed and analyzed by rRT-PCR using β-actin primers as internal control. For group details see Table 4. The results were expressed as the ratio of relative band intensity of the target gene to the band intensity of the β-actin gene. We took the log of the ratios, and these values were used for one way ANOVA followed by Tukey’s post-hoc multiple comparison test; p<0.05 was considered as significant. Each data point is the mean ± SEM of 4 separate experiments. Bar heads with an asterisk (*) indicate that the value is significantly different from the corresponding control (no valproate). Figure 16.1: nlgn3b ; Figure 16.2: otx2 ; Figure 16.3: wnt1 ; Figure 16.4: iro3 .

ccna2 shh 0.5 0.20 0.4 * 0.15 0.3 -actin) β β β β *

-actin] 0.10 0.2 β β β β

* (ccna2: 0.1 [Shh: 0.05 Relative band intesity band Relative

Relative band intensity band Relative 0.0 0.00 f f f f f pf p p p f d d d dp pf pf 3 6 6 d dpf d dp dpf d - 6 dpf 2 2 -3dpf 3 6 6 ol-2 m-2 dpf l- l-2 dpf - - M m- l tr m tro m ro 1 2 mM- ntrol-2dpf 3 dp n mM mM m mm- mm-6 dpf o 2 mM-5 3 mM- o 2 mM-5 1 mm-2 2 5 2 5 con c c contro control-3 dpf cont Treatment Groups Treatment Groups 17.1 17.2

Figure 17: Effects of valproate on shh and ccna2 mRNA expression in Japanese rice fish at three different developmental stages. Total RNA was prepared from 6-8 pooled embryos after valproate treatment at 2 dpf (group A), 3 dpf (group B) and 6 dpf (group C). This was reverse transcribed and analyzed by rRT-PCR using β-actin primers as internal control. For group details see Table 4. The results were expressed as the ratio of relative band intensity of the target gene to the band intensity of the β-actin gene. We took the log of the ratios, and used these values for one way ANOVA followed by Tukey’s post-hoc multiple comparison test; p<0.05 was considered as significant. Each data point is the mean ± SEM of 4 separate experiments. Bar heads with an asterisk (*) indicate that the value is significantly different from the corresponding control (no valproate). Figure 17.1: shh ; Figure 17.2: ccna2.

CHAPTER 4

DISCUSSION

Valproate is preferred to treat absence seizures, generalized tonic-clonic seizures, myoclonic seizures and partial seizures. In addition, it is also very effective for treating bipolar disorder and migraine. Epilepsy is a common neurological disorder that affects 0.5-1 % of pregnant women (Fairgrieve, 2000; Holmes et al., 2001). Approximately 0.3% of all pregnant women are in need of a balanced antiepileptic drug treatment (Morrell, 2002), even though these drugs (carbamazepine, valproic acid, phenobarbital, phenytoin, lamotrigine, etc.) are known to be teratogens to children born to mothers taking them during pregnancy. These epileptic drugs are responsible for a wide spectrum of fetal malformations and severe cognitive disorders in the offspring, such as fetal valproate syndrome (FVS) and/or autistic spectrum disorders (ASDs). Since valproic acid is very specialized to treat some types of convulsive disorders and has relatively less sedative effects, it is widely used worldwide. A significant portion of pregnant women with epilepsy take valproate, either as monotherapy or as a part of polytherapy (Kini, 2006). The embryonic and fetal damages caused by antiepileptic drugs are described into three major categories (Ornoy, 2009): (1) Elevated risks of major congenital

anomalies include neural tube defects, cardiovascular, orofacial and digital abnormalities, deformities of eye, skin, abdominal wall, brain, respiratory tract and genitourinary systems, etc.;

(2) A specific syndrome, ‘Fetal Valproate Syndrome’, presented with facial dysmorphic features

(figure 2), but also involves damages to other organs, such as the neural tube;

(3)Neurodevelopmental disorders that affect cognitive, social and verbal skills, and lead to stereotypic behaviors, such as autism-like symptoms.

It is evident from the present experimental results that Japanese rice fish embryos exposed to valproate during development displayed several measurable phenotypic end points analogous with human FVS. These end points were especially apparent in the cardiovasculature, neurocranial and splanchnocranial cartilages. Human FVS which is characterized by distinctive facial appearance, a cluster of major and minor anomalies (Table 2), and dysfunctions of CNS

(Kozma, 2001). Our studies were focused on some of the phenotypic features that were altered by VPA in the Japanese rice fish, such as cardiovascular and craniofacial abnormalities that appear to correlate with the phenotypes observed in human FVS. Our data demonstrate that valproate is a teratogen in Japanese rice fish, and the teratogenic effects are specific to the developmental stages of the embryos. Usually, the earlier the exposure, the more severe the deleterious effects. It is well known that during morphogenesis, each tissue and organ have critical phases during which their development may be disrupted by various teratogens.

Therefore, to find stage-specific effects, in our model, embryos were exposed to valproate at three stages of development (groups A, B and C) (Table 4). Group A embryos were treated with

VPA during 0-2 dpf, group B embryos during 1-3 dpf, and group C embryos during 4-6 dpf. 0-2 dpf and 1-3 dpf are critical periods for cell division and neurogenesis respectively. 4-6 dpf is the

period of time after formation of the cardiovascular system. The calculated LC 50 as determined on 14 dpf suggests that the embryos, at early stages of development (group A), were more sensitive to valproate than those in late stages (groups B and C). A recent study in zebrafish

embryos by Selderslaghs et al. (2009) determined IC 50 after exposing 2-4 hpf embryos to VPA continuously for 24, 48, 72, and 144h . In an earlier study in zebrafish, Gurvich et al. (2005) reported that valproate concentrations higher than 3 mM were cytotoxic, leading to rapid developmental arrest and cell lysis (the valproate exposure period was 5.3- 24 hpf). Although the experimental conditions and the developmental stages in which the zebrafish embryos were exposed to valproate were different from our studies with Japanese rice fish (Gurvich et al., 2005;

Selderslaghs et al., 2009), it is evident that Japanese rice fish embryos were relatively more

resistant to valproate in late stages of development (groups B and C; LC 50 s are 26.45 ±1.64 mM

and 20.37 ±3.30 mM, respectively, n= 3), compared with zebrafish (LC 50 for 48 h exposure is

2.09 mM; Selderslaghs et al., 2009). In addition, we consider that the culture medium also affects

the sensitivity of the Japanese rice fish embryos to valproate. We added NaHCO 3 (∼2.38 mM) to

the hatching solution as it is required for maintaining the pH of the embryo-rearing medium.

Bicarbonates have been demonstrated to compete with valproate during cellular uptake in

Dictyostelium , zebrafish and xenopus (Terbach et al., 2011). Therefore, because of the competition, we expect that the actual concentration of valproate available to the embryo in

Japanese rice fish to induce toxicity is significantly lower than that of the calculated LC 50 values determined during the present experiments.

The deleterious effects of valproate on cardiovascular development, in our studies, were restricted to increased thrombus formation, delayed blood circulation, and lower heart rates which were found to be affected significantly and are dose and developmental-stage specific.

These effects were observed in each VPA treatment group. Embryos treated with higher concentrations of VPA displayed more severe effects. In addition, embryos treated with VPA at early developmental stages were more sensitive to VPA toxicity. Thrombi were seen only in those embryos which were able to develop heart beat. If embryos exposed to VPA before they developed blood circulation (groups A and B), some embryos showed thrombus formation on 6 dpf with heart beating but without onset of blood circulation. This may be due to the reason that blood vessels cannot develop normally to allow blood flow under VPA treatment, and thus thrombi appear. However, some embryos exposed to high concentration of VPA (20 mM) developed heart beat but did not exhibit thrombus formation. This suggests that the detrimental

impacts of high dose VPA are more severe than those of low dose VPA, and high dose VPA apparently prevents the formation of blood cells. Therefore, no thrombus appeared. When blood vessels developed before VPA treatment (C group), the VPA displayed its toxicity by acting directly on the blood vessels to cause thrombus formation. Additionally, the initiation of blood circulation was delayed in valproate exposed embryos in a dose and developmental-stage dependent manner. Moreover, group C embryos, which had established blood circulation prior to exposure to valproic acid, did not undergo any significant alterations in blood circulation during the 48 hrs of valproate exposure. If these embryos (group C) were then maintained in clean hatching solution and allowed to continue developing, thrombi became apparent, vessel circulation got slower or stopped, and the embryos were either unable to hatch or died (data not shown). The heart rate was determined on 6 th , 7 th and 8 th dpf when there was no valproate in the medium. Our data indicate that the heart rate of the embryos was reduced in a dose and developmental-stage specific manner. It was also manifested that VPA can inhibit heart beating even if it had already initiated. Several studies have indicated that valproate affects blood vessel development by inhibiting angiogenesis (Michaelis et al., 2004). In Jcl :ICR mouse, administration of valproic acid to the mother induced fetal cardiovascular abnormalities such as ventricular septal defect, endocardial cushion defect, transposition of the great arteries, double outlet right ventricle, tricuspid atresia and hypoplastic left heart syndrome(Sonoda et al., 1993).

In murine pluripotent embryonic stem cells, differentiation of cardiomyocytes was inhibited by valproate in a dose-dependent manner (Na et al., 2003). Moreover, valproate inhibited the differentiation of embryonic heart cells by reducing the contractile activity in chick heart micromass and D3 mouse embryonic stem cell-derived cardiomyocytes in vitro (Ahir and Pratten,

2011). Although the parameters we were able to measure in cardiovascular disruption were limited (formation of thrombi, delay in vessel circulation and reduced heart rate), we expect that these defects in Japanese rice fish are the result of inhibition of angiogenesis (Michaelis et al.,

2004).

Healthy cardiovascular development plays an important role in the successful hatch.

Therefore, Japanese rice fish embryos that were able to hatch needed to have well-developed circulatory vessels and normally developed blood circulation. Hatching efficiency is also dose and developmental-stage specific. Embryos treated with higher concentrations of VPA had less possibility to hatch. Embryos treated with VPA at early developmental stages were asssociated with decreased incidence of hatching. The Japanese rice fish hatchlings developed yolk edema, bent tails, and reduced body length after valproate exposure. These changes are believed to be analogous to the spinal anomalies observed in human children with FVS.

Our analyses of craniofacial cartilages indicate that valproate induced a phenomenon of microcephaly in Japanese rice fish (Table 7). We noticed that although B and C group embryos

have higher LC 50 values of valproate compared to group A, the successful hatching of these embryos (B and C) was not seen in those treated with concentration of VPA greater than 10 mM.

This indicates that the morphogenesis of the embryos treated with concentrations of VPA greater than 10 mM was significantly affected by sublethal concentrations of valproate. Therefore we have analyzed the cartilages of the neurocranium and splanchnocranium of those embryos that hatched successfully (Table 7). The embryos of two treatment groups (Table 5), group E: 0-2 dpf

VPA (without circulation) or group L: 4-6 dpf VPA (with circulation), were preserved in 4% PFA within 24 h of hatching and stained with Alcian Blue (Hu et al., 2009). Previously, the preservation of hatchlings was done at 10 dpf, and if an embryo hatched earlier (in our culture conditions the embryos initiate hatching after 7 dpf), they were maintained in hatching solution without food until 10 dpf (Hu et al., 2009). In the present experiment, to avoid post hatch growth, we preserved the embryos within 24 hrs of hatching. We observed that the measured length of the neurocranium and other cartilages in control hatchlings (untreated embryos) were significantly shorter than those we reported in our previous studies (Hu et al., 2009), since our previous studies measured cartilages in Japanese rice fish on 10 dpf. Our current data indicate that valproate significantly reduced the linear length of the neurocranium in both groups E and L embryos when compared with controls. Epidemiological studies also showed that children exposed to valproate prenatally have microcephaly (Almgren et al., 2009). However,

macrocephaly not microcephaly is considered as the most consistent phenotypic feature of autism spectrum disorder (ASD). To find VPA-sensitive neurocranial and/or splanchnocranial features, we have extended our investigation to 6 individual cartilages in the neurocraniuam (TC,

ABC, EB, PC, AOC, BP) and 11 individual cartilages in the splanchnocranium (MC, QU, BH,

CH, BB 1-3, BB4 and 5 CBRs). We found that compared to controls the linear lengths of TC, EB,

AOC and BP in the neurocranium and QU, CH, and BB1-3 in the Splanchnocranium were significantly reduced in group E hatchlings exposed to 2 mM valproic acid during embryonic development (Table 7). It was also shown that, compared to controls, the linear lengths of PC in the neurocranium and QU, CH, BB 1-3, and CBR 1-5 in the splanchnocranium were reduced in group L hatchlings exposed to 2 mM valproic acid during embryonic development. Therefore, it appears that the reduction in the linear length of the neurocranium is the result of cumulative reductions of the component cartilages.

The third aim of this study was to find an affected molecular marker of FVS and/or ASD.

For that, we have analyzed mRNA expression levels of nine genes ( catalase , gst , gr , nlgn3b , otx2 , wnt1 , iro3 , shh and ccna2 ) in Japanese rice fish embryos at three developmental stages (Table 4) exposed for 48 hrs to two different concentrations of valproic acid (for group A, 1 and 2 mM, for groups B and C, 2 and 5 mM) (Wu et al., 2012). The selected genes belong to three categories; oxidative stress ( catalase , gr and gst ), neurogenesis ( nlgn3b , Iro3 , wnt1 and otx2 ) and regulation

of cell division ( cyclin a2 and shh ). The expression of many of the selected genes has been

shown to be altered by valproate in human and rat (Graf et al., 1998; Bambini-Junior et al., 2011:

Tung and Winn, 2011). Analysis of mRNA was done by rRT-PCR (Hu et al., 2008) using β-actin as internal control (Wang et al., 2006; Wu et al., 2008; 2011). The oxidative stress-related genes

(gst , gr , and catalase ) have been shown to be constitutively expressed in Japanese rice fish during development (Wu et al., 2011). Although oxidative stress is a potential cause of teratogenesis, surprisingly, compared to controls, we did not find any significant alteration in the mRNA expression pattern of catalase , gr and gst mRNA in Japanese rice fish embryos due to exposure to valproate at any stage of development (Figure 15). Epileptic children exposed to

VPA also displayed no significant alterations in serum levels of catalase (Peker et al., 2009).

Therefore, it appears that valproate-induced teratogenesis in Japanese rice fish is mediated through genes other than those that participate in mediating the process of oxidative stress.

As valproate is able to induce neural tube defects in human embryos, we have analyzed the mRNA expression patterns of five genes ( nlgn3b, iro3, wnt1, otx2, shh ) that are expressed in the brain of Japanese rice fish embryos. Neuroligins (nlgn) are a family of postsynaptic cell-adhesion molecules that play roles in synaptic maturation through association of their presynaptic partners the neuroxins (nrxn) (Ichtchenko et al., 1995; 1996). Our data indicate that expression of nlgn3b mRNA is developmentally regulated in Japanese rice fish and tended to be

reduced in all valproate-treated groups compared to the corresponding controls. Valproate modulates the gene expression patterns of nlgn and nrxn in mice (Kolozsi et al., 2009). Iro3 belongs to Iroquois family of homeoproteins and is constitutively expressed in vertebrates, flies, and nematodes (Gomez-Skarmeta and Modolell, 2002). In zebrafish, Iro3 has been indicated to inhibit formation of motoneurons and to facilitate formation of interneurons (Lewis et al., 2005).

However, our analysis by rRT-PCR indicated that the expression of iro3 mRNA in Japanese rice fish is constitutive and remained unaltered either during development or after valproate exposure

(Figure 16.4). This suggests that iro3 is not a potential target of valproate in Japanese rice fish embryogenesis.

Our analysis of mRNA expression of shh, wnt1 and otx2 showed that the mRNA expression levels of wnt1 and otx2 genes were significantly reduced only in B group (1-3 dpf exposure) embryos treated with 5 mM VPA. In the other two treatment groups (A and C), these mRNAs remained unaltered. Shh was significantly reduced in group A (2 mM), and in the other groups (groups B and C) it remained unaltered. The developmental stage specific response of these genes cannot be explained properly from this study. However, available literature indicates that the teratogenic effects of valproate are also mediated by wnt1 signaling molecules

(Shimshoni et al., 2007). Shh played a central role in cell proliferation, differentiation and patterning by activating the hedgehog signaling pathway. Polydactyly/arhinencephaly (Pdn/Pdn)

mice exposed to valproate during gestation have exencephaly, but shh mRNA content remained unaltered (Maekawa et al., 2005). Otx2 is recognized as an early head-domain marker (Bliz and

Cho, 1995; Pannese et al., 1995). Microarray analysis in mouse embryos exposed to valproate prenatally identified several genes including otx2 as potential target genes of valproate-induced teratogenesis (Kultima et al., 2010). In Japanese rice fish, further studies are needed to understand the mechanisms of wnt1 , shh and otx2 regulation by valproate.

Our data further indicate that ccna2 mRNA expression during embryogenesis of Japanese rice fish was reduced by valproic acid in a developmental stage and dose specific manner. The cyclins and their cyclin-dependent kinase partners (CDKs), are the basic components of the machinery that regulates the passage of cells through cell cycle. Cyclin A2 deficiency is marked by early embryonic lethality. Valproate is able to downregulate cyclin A expression in gastric carcinoma cell lines (Zhao et al. 2011). Therefore, abnormality in cell division due to disruption in ccna 2 expression probably is the partial reason to explain the developmental delay and reduced hatching efficiency by valproate as we observed in our model.

Although the molecular endpoints are not established from this study, the present investigation indicates that some of the consequences of damages induced by valproate such as organ-specific effects in heart and blood vessels, dysmorphogenesis in neurocranial cartilages, and modification in neuronal gene expression, in Japanese rice fish are parallel with other

organisms such as zebrafish and have potential to provide an opportunity to use the Japanese rice fish as a model to study FVS and ASD.

CHAPTER 5

CONCLUSIONS AND FUTURE WORKS

In this study, the Japanese rice fish was considered as a unique non-mammalian vertebrate

model to study VPA teratogenesis. The higher the concentration of VPA in each group, the more

incidence of death happened. The calculated LC 50 value of group A (treatment during 0-2 dpf)

was 1.68 ± 1.55 mM, which was much lower than that of group B (treatment during 1-3 dpf; LC 50

for group B was 26.45 ± 1.64 mM ) or group C (treatment during 4-6 dpf; LC 50 for group C was

20.37 ± 3.3 mM). VPA has deleterious effects on cardiovascular development. The experiments manifested that VPA reduced heart rates, increased incidence of thrombus and inhibited onset of circulation in each group. VPA also decreased hatching efficiency, and resulted in axial malformations. Yolk edema and curved spinal cord were viewed on hatchlings treated with VPA prenatally. The higher the concentration of VPA the embryos were treated with, the more severe the effects were. The earlier the exposure, the more dangerous the VPA treatments were. In addition, VPA caused deformed craniofacial cartilages. The length of the neurocranium, quadrate, ceratohyal and basibrancials 1-3 was reduced in both groups E (0-2 dpf) and L (4-6 dpf).

Trabeculae, epiphyseal bar, anterior orbital cartilage and basilar plate displayed reduction of

length only in group E (0-2 dpf). Polar cartilage and ceratobranchials 1-5 become shorter only in group L (4-6 dpf). However, anterior Basicranial commissure, meckel's cartilage, basihyal and basibrancial 4 remained unaltered. While there are no conclusive outcomes of craniofacial cartilages drawn from this study, most of the neurocranial and splanchnocranial cartilages were modified by VPA. If the neurocranial or splanchnocranial cartilage became malformed through

VPA treatment, the length of it would be reduced. The experiments revealed that VPA induced the abnormal morphological features (yolk edema, bent tails and microcephaly) in the hatchlings, and the embryos at early developmental stages are more sensitive to VPA toxicity. Some genes ( gst, gr and catalase ) that were indicated to be involved in oxidative stress remained unaltered in VPA treated-embryos, however, the expression levels of some genes ( nlgn3b, otx2, wnt1 ) correlated with neurodevelopment were modified only in 5 mM VPA-treated embryos of group B (1-3 dpf).

Shh and cyclin A2 are associated with cell division, and their expression levels were all reduced in embryos treated with 2 mM VPA at the early developmental stage (group A, 0-2 dpf). The early developmental stage (0-2 dpf) is known to be a very crucial period for cell division. Hence, shh and cyclin A2 were indicated to be implicated in the VPA-induced teratogenesis, e.g. neural tube defects, by interrupting continuation of cell cycles. The teratogenic features of Japanese rice fish induced by VPA were analogous to those of human FVS. In addition, all of these available characteristics which could be regarded as parameters of FVS evaluation have potential to validate

this Japanese rice fish as a certified and beneficial model to study VPA-induced teratogenesis,

even though more intensive investigation is needed to complete this validation.

Several problems remain unsolved from this study. These provide some additional

directions for future investigation. Most Children affected with FVS have neurodevelopmental

disorders and show autistic-like behaviors, affecting the cognitive, verbal and communicative

abilities. Autism spectrum disorder (ASD) is behaviorally defined, and therefore any

neurodevelopmental disorders displaying autistic-like behaviors will be attributed as ASD. The

Japanese rice fish can further be used to detect their behaviors induced by VPA to evaluate their

potential as a model to study ASD. Epidemiological studies report the ratio of males to females

diagnosed with autism spectrum disorder (ASD) is 4.3: 1.0 (Fombonne, 2003) and the molecular

mechanism of this phenomenon is as yet unknown. Thus, scientists can concentrate on the

epigenetic modification of genes, such as Dmy, to study the gender specific induction of ASD.

Dmy (dmrt1bY; DM-domain gene on the Y chromosome), the male determining gene, is only

found in Japanese rice fish ( Oryzias latipes ) (Matsuda et al., 2002, 2003) and its nearest relative

Oryzias curvinotus (Kondo et al., 2003).

In our study, we did not establish detailed and specific molecular endpoints. However several genes were suggested to be involved in the pathogenesis of FVS induced by VPA. In the future, it will be desirable to do real-time reverse transcription PCR to test the expression patterns

of these genes by quantifying their mRNAs. In addition, it will be desirable to do in situ hybridization to detect the locations of these genes and to measure their expression levels.

Although we hypothesize that oxidative stress induced by VPA results in the pathogenesis of FVS, there were no alterations in the oxidative stress related genes ( gst, gr, catalase ) that we evaluated in this study. In future, it will be desirable to determine whether other pathways related to oxidative stress are affected. Consequently, lipid peroxidation and the ratio of NAD+: NADH which are indicators of the redox state of a cell, are proposed as potential test parameters.

From this study we have established that most neurocranial and splanchnocranium cartilages were affected by VPA. Neural crest (NC) cells are the precursors of these cartilages.

Many signal molecules, such as sonic hedgehog (shh), fibroblast growth factor (fgf), Wingless

(wnt) and Notch, are associated with the migration of NC cells from the CNS. Therefore, in the future, investigations on these signaling molecules and their alterations by VPA are necessary.

Until now only one medication, risperidone, has been approved by the Food and Drug

Administration (FDA) of the USA for the pharmacologic management of autistic-like behaviors in

Children. After establishing this animal model, we can further our research to test the ability of some natural products, such as Ginseng and Ginkgo biloba, as potential preventive agents for the

FVS and/or ASD (autism spectrum disorder). It is promising that some natural products are more effective with less side effects.

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VITA

Mengmeng Wu grew up in Nanjing, Jiangsu province, China. She attended local schools for primary and secondary studies. She was accepted as an undergraduate student in Nanjing

University of Chinese Medicine, China in September 2005 and graduated in June 2009 with a

Bachelor Degree in Pharmaceutical Engineering of Traditional Chinese Medicine. After graduation, Mengmeng went to America to further her education in Pharmaceutical Sciences.

From August 2009, Mengmeng joined University of Mississippi as a graduate student in the department of Pharmacology in School of Pharmacy. During the recent three years, she tried all her best to accomplish the tasks required, and is currently a candidate for the degree of Master in

Pharmaceutical Sciences with emphasis in Pharmacology.

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