| dc.contributor.advisor | Sabuncuoğlu , Suna | |
| dc.contributor.author | Mohammad Nejad, Solmaz | |
| dc.date.accessioned | 2018-09-25T06:06:29Z | |
| dc.date.issued | 2018-09-19 | |
| dc.date.submitted | 2018-09-18 | |
| dc.identifier.uri | http://hdl.handle.net/11655/4920 | |
| dc.description.abstract | Mohammadi Nejad S. Evaluation of Developmental Toxicity of Ethephon Using Embryonic Stem Cell Model. Hacettepe University, Graduate School of Health Sciences, Pharmaceutical Toxicology Program, Doctor of Philosophy Thesis, Ankara, 2018. Ethephon, a member of the organophosphorous compounds, is one of the most widely used plant growth regulators for artificial ripening. In recent years, the developmental toxicology studies have increased due to a broad interest in neurodevelopmental problems. Concerning this, new in vitro screening models such as embryonic stem cells have been established in predictive toxicology to decrease the expense and time of the experiments number of laboratory animals. The goal of this study was evaluation of the neuro-developmental toxicicity potential of “ethephon”. For this purpose, the mES cells were exposed to ethephon and the cytotoxicity, cell cycle, total antioxidant capacity, reactive oxygen species (ROS) and, expression of specific pluripotential genes including Oct-4, SOX2, NANOG, Nrf2, Nestin and neural differentiation genes such as MAP2 and β-Tubulin III were examined. Additionally, effects of ethephon on expression of stemness genes were assessed before and during neural differentiation. Our results from MTT assay with mEs cells, after 24, 48 and 96 h exposure to ethephon demonstrated that at higher doses (1280, 2560 and 5120 µM) ethephon is cytotoxic for mES cells. According to results of oxidative stress assays, it is suggested that the cytotoxic effect of ethephon is not related to the oxidative stress mechanism. In order to genetic evaluation, ethephon treated mES cells were analyzed before and after neuronal-differentiation by Real-time PCR. The expressions of several genes were evaluated. Our observations showed that ethephon is potent to induce a significant difference in expression of these genes in neuronal differentiated cellsFuture studies are necessary to understand the exact mechanism of developmental toxicity of ethephon. | en |
| dc.description.tableofcontents | ETHICAL DECLARATION V
AKNOWLEDGEMENT VI
ABSTRACT VII
ÖZET VIII
TABLE OF CONTENTS IX
SYMBOLS AND ABBREVIATIONS XII
FIGURES XIV
TABLES XV
1. INTRODUCTION 1
2. GENERAL INFORMATION 3
2.1 Organophosphorus Compounds 3
2.1.1 Physicochemical Properties of Organophosphates 4
2.1.2 Toxicity of Organophosphates 4
2.1.3 Non-Neurotoxic Effects of Organophosphates 8
2.1.4 Immunotoxic Effects of Organophosphates 10
2.2 Ethephon 10
2.2.1 Physicochemical Properties of Ethephon 11
2.2.2 Pharmacokinetics of Ethephon 11
2.2.3 Agricultural Applications of Ethephon 12
2.2.4 Toxicity of Ethephon 15
2.2.5 Acute Toxicity of Ethephon 16
2.2.6 Chronic Toxicity of Ethephon 17
2.3 Developmental Toxicity 19
2.4 Stem Cell 22
2.4.1 Classification of Stem Cells 24
2.5 Neural Development 26
2.5.1 The Pathways of Neural Differentiation 28
2.6 Stem Cell in Toxicology 35
3. MATERIAL AND METHODS 37
3.1 Chemicals 37
3.2 Instruments 39
3.3 Preparation of Solutions 39
3.3.1 Preparation of Cell Culture Medium 39
3.3.2 Preparation of Gelatin 40
3.3.3 Preparation of FBS 40
3.3.4 Preparation of Trypsin-EDTA 40
3.3.5 Preparation of MTT solution 40
3.3.6 Preparation of Neural Differentiation Culture Medium 40
3.3.7 Preparation of Retinoic Acid 40
3.3.8 Preparation of FRAP Assay Solutions 41
3.3.9 Preparation of ROS Assay Solutions 41
3.3.10 Preparation of DCFH-Diacetate 5mM 43
3.3.11 Preparation of PI solution 43
3.3.12 Preparation of RNase 43
3.4 Methods 43
3.4.1 Preparation of Gelatinized Culture Flask 43
3.4.2 Thawing Frozen Cells 43
3.4.3 Changing Culture 44
3.4.4 Cell Passage 44
3.4.5 Freezing Cells 45
3.4.6 Cell Counting by Trypan Blue Dye Exclusion 45
3.4.7 Neural Differentiation of mES Cells 46
3.4.8 Assessment of the Cytotoxic Effect of Ethephon on mES 47
3.4.9 MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Tetrazolium Reduction Assay 47
3.4.10 Total Antioxidant Assay 48
3.4.11 Reactive Oxygen Species Assay 48
3.4.12 Flow Cytometry Technique 49
3.4.13 Cell Cycle Analysis by Flow Cytometry 50
3.4.14 Preparation of Cells for Flow Cytometry 50
3.4.15 RNA Extraction from mES cells 51
3.4.16 RNA Qualification Assay on Gel Electrophoresis 52
3.4.17 First Strand cDNA Synthesis 54
3.4.18 Real Time-Reverse Transcription Polymerase Chain Reaction (RT-PCR) 55
3.4.19 Statistical Analysis 57
4. RESULTS 58
4.1 Results of The Cell Cycle Assay 58
4.2 Results of Cytotoxicity Assay (MTT) 60
4.3 Evaluation of Antioxidant Status by FRAP 63
4.4 Effects of Ethephon ROS Level 64
4.5 Evaluation of Morphology of mES Cells 66
4.6 Results of Gene Expression Assay in mES Cell by Real Time-PCR 68
4.7 Results of Gene Expression in Neuronal Differentiated mES Cells 71
5. DISCUSSION 77
6. CONCLUSION AND SUGGESTIONS 86
7. REFERENCES 88
8. ANNEXES
8.1 Digital Reciept
8.2 Report of Originality
9. CURRICULUM VITAE | tr_TR |
| dc.language.iso | en | tr_TR |
| dc.publisher | Sağlık Bilimleri Enstitüsü | tr_TR |
| dc.rights | info:eu-repo/semantics/openAccess | tr_TR |
| dc.subject | Ethephon | tr_TR |
| dc.subject | Cytotoxicity | |
| dc.subject | Stemness genes | |
| dc.subject | Developmental toxicity | |
| dc.subject | Neural differentiation | |
| dc.title | Evaluatıon of Developmental Toxıcıty of Ethephon Usıng Embryonıc Stem Cell Model | tr_eng |
| dc.type | info:eu-repo/semantics/doctoralThesis | en |
| dc.description.ozet | Mohammadi Nejad S. Embriyonik Kök Hücre Modeli Kullanılarak Etafonun Gelişimsel Toksisitesinin Değerlendirilmesi. Hacettepe Üniversitesi Sağlık Bilimleri Enstitüsü Farmasötik Toksikoloji Programı Doktora Tezi, Ankara, 2018. Organfosforlu bileşiklerin bir üyesi olan etafon, yapay olgunlaşma için en yaygın kullanılan bitki büyüme düzenleyicilerinden biridir. Son yıllarda, nörogelişimsel sorunlara olan yoğun ilgi nedeniyle gelişimsel toksikoloji alanındaki çalışmalar artmaktadır. Buna bağlı olarak, deneylerin maliyet ve süreleri ile kullanılan laboratuvar hayvanı sayısını azaltmak için tanımlayıcı toksikolojide embriyonik kök hücreler gibi yeni in vitro tarama modelleri geliştirilmektedir. Bu çalışmanın amacı “etafon”un nöro-gelişimsel toksisite potansiyelinin değerlendirilmesidir. Bu amaçla, mES hücreleri etafona maruz bırakılmış ve sitotoksisite, hücre döngüsü, toplam antioksidan kapasite, reaktif oksijen bileşikleri (ROB) ve Oct-4, SOX2, NANOG, Nrf2, Nestinin de dahil olduğu özel pluripotential genler ile nöral farklılaşma genleri olan MAP2 ve β-Tubulin III genlerinin ekspresyonları incelenmiştir. Nöral farklılaşma öncesi ve sonrasında, etafon uygulanmış ve etafonun kök hücre genlerinin ekspresyonu üzerindeki etkileri değerlendirilmiştir. Etfona 24, 48 ve 96 saat süreyle maruz bırakılan mEs hücrelerinde yapılan MTT analizinden elde edilen sonuçlar etefonun yüksek dozlarında (1280, 2560 ve 5120 µM) hücre proliferasyonunda belirgin bir azalma olduğunu göstermiştir. Oksidatif stres yöntemlerinden elde edilen bulgulara göre, etafonun sitotoksik etkisinin oksidatif stres mekanizmasına bağlı olmadığı düşünülmüştür. Genetik değerlendirme sonuçları, etafon uygulanan mES hücreleri, Real-time PCR ile nöronal farklılaşma öncesi ve sonrasında analiz edilmiştir. Kök hücre formu, nöral ektoderm ve dendritik dönemin çeşitli genlerinin ekspresyonu bu yöntemle değerlendirilmiştir. Sonuçlarımız, nöral farklılaşma olan hücrelerde, etafonun bu genlerin ekspresyonunu indükleyebileceğini doğrulamaktadır. Etafonun gelişimsel toksisite mekanizmasının anlaşılabilmesi için gelecekte yapılacak yeni çalışmalara ihtiyaç bulunmaktadır. | tr_TR |
| dc.contributor.department | Farmasötik Toksikoloji | tr_TR |
| dc.contributor.authorID | | tr_TR |
| dc.embargo.terms | 2 yil | tr_TR |
| dc.embargo.lift | 2020-09-26T06:06:29Z | |
