Title Page
ABSTRACT
국문 초록
PREFACE
Contents
ABBREVIATION 20
CHAPTER 1. Literature review 22
1.1. General introduction to environmental pollution 22
1.1.1. Environmental pollution and toxicology 22
1.2. Pesticides 23
1.2.1. Impacts of pesticides on environmental pollution 23
1.2.2. Risks and toxic mechanisms of pesticides 25
1.2.3. Thiobencarb 26
1.2.4. Oxamyl 27
1.2.5. Flufenacet 28
1.3. Zebrafish 28
1.3.1. Characteristics of zebrafish as a test organism 28
1.3.2. Evaluation of developmental toxicity using zebrafish 30
1.3.3. Evaluation of organ toxicity using zebrafish 31
1.3.4. Evaluation of effects at the cellular levels in zebrafish 32
CHAPTER 2. Assessment of the toxic effects of pesticides using zebrafish models 34
2.1. Thiobencarb induces phenotypic abnormalities, apoptosis, and cardiovascular toxicity in zebrafish embryos through oxidative stress and inflammation 34
2.1.1. Abstract 34
2.1.2. Introduction 35
2.1.3. Materials and methods 37
2.1.4. Results 42
2.1.5. Discussion 52
2.2. Oxamyl exerts developmental toxic effects in zebrafish by disrupting the mitochondrial electron transport chain and modulating PI3K/Akt and p38 Mapk signaling 57
2.2.1. Abstract 57
2.2.2. Introduction 57
2.2.3. Materials and methods 59
2.2.4. Results 66
2.2.5. Discussion 78
2.3. Developmental toxicity of flufenacet including vascular, liver, and pancreas defects is mediated by apoptosis and alters the Mapk and PI3K/Akt signal transduction in zebrafish 83
2.3.1. Abstract 83
2.3.2. Introduction 83
2.3.3. Materials and methods 85
2.3.4. Results 92
2.3.5. Discussion 101
2.4. Fluroxypyr methylheptyl ester interferes with the normal embryogenesis of zebrafish by inducing apoptosis, inflammation, and neurovascular toxicity 105
2.4.1. Abstract 105
2.4.2. Introduction 105
2.4.3. Materials and methods 107
2.4.4. Results 112
2.4.5. Discussion 120
CHAPTER 3. Assessment of the toxic effects of a disinfectant using zebrafish embryo and human cell line 125
3.1. Embryonic exposure to chloroxylenol induces developmental defects and cardiovascular toxicity via oxidative stress, inflammation, and apoptosis in zebrafish 125
3.1.1. Abstract 125
3.1.2. Introduction 126
3.1.3. Materials and methods 127
3.1.4. Results 133
3.1.5. Discussion 141
CHAPTER 4. Adverse effects of pesticides on the bovine mammary systems 145
4.1. Fluroxypyr-1-methylheptyl ester causes apoptosis of bovine mammary gland epithelial cells by regulating PI3K and MAPK signaling pathways and endoplasmic reticulum stress 145
4.1.1. Abstract 145
4.1.2. Introduction 145
4.1.3. Materials and methods 147
4.1.4. Results 151
4.1.5. Discussion 159
4.2. Folpet promotes apoptosis of bovine mammary epithelial cells via disruption of redox homeostasis and activation of MAPK cascades 163
4.2.1. Abstract 163
4.2.2. Introduction 163
4.2.3. Materials and methods 165
4.2.4. Results 170
4.2.5. Discussion 180
CHAPTER 5. Evaluation of therapeutic effects of the phytochemical against ovarian cancer using cancer cell lines 185
5.1. 4-methylumbelliferone inhibits the proliferation of human epithelial ovarian cancer cells via G2/M phase arrest 185
5.1.1. Abstract 185
5.1.2. Introduction 185
5.1.3. Materials and methods 187
5.1.4. Results 191
5.1.5. Discussion 200
CHAPTER 6. Conclusion 204
REFERENCES 206
CHAPTER 2. 40
Table 1-1. Primer sequences for qRT-PCR analyses 40
Table 2-1. Primer sequences for qRT-PCR analyses 63
Table 2-2. List of antibodies for immunoblotting 66
Table 3-1. List of reagents 89
Table 3-2. Primer sequences for qRT-PCR analyses 89
Table 3-3. List of antibodies for immunoblotting 91
Table 4-1. Primer sequences for qRT-PCR analyses 111
CHAPTER 3. 132
Table 1-1. Primer sequences for qRT-PCR analyses 132
CHAPTER 4. 150
Table 1-1. List of antibodies and inhibitor for immunoblotting 150
Table 2-1. List of antibodies for immunoblotting 169
Table 2-2. Primer sequences for qRT-PCR analyses 169
CHAPTER 2. 43
Fig. 1-1. Thiobencarb exerts embryotoxicity on zebrafish. [A] Survival rates of thiobencarb- exposed (0, 2, 5, 10, 20, and 50 mg/L) zebrafish at 96 hpf. [B] Hatching rates of thiobencarb-... 43
Fig. 1-2. Thiobencarb induces multiple deformities in zebrafish. [A] Representative image of pathological morphologies in thiobencarb-exposed zebrafish at 96 hpf. Yellow dotted region (E):... 45
Fig. 1-3. Induction of ROS and inflammation upon thiobencarb treatment. [A and B] ROS levels in zebrafish were visualized via DCFH-DA staining. Green fluorescence indicates ROS production,... 47
Fig. 1-4. Cardiotoxicity of thiobencarb on developing zebrafish. [A] Changes in heart rhythm were evaluated by recording the number of heart beats per minute in 96 hpf zebrafish. [B] Alteration of... 49
Fig. 1-5. Impaired vasculature of zebrafish resulting from thiobencarb exposure. [A] Vascular structures on the anterior and posterior sides of thiobencarb-exposed zebrafish at 96 hpf. The... 51
Fig. 1-6. A schematic diagram demonstrating the mechanism of developmental toxicity of thiobencarb on zebrafish embryos. Zebrafish treated with thiobencarb showed growth retardation... 56
Fig. 2-1. Overall developmental toxicity of oxamyl in zebrafish. [A] Relative viability was measured after oxamyl treatment at 72 hpf. [B] Overall morphology of oxamyl-treated zebrafish at... 67
Fig. 2-2. Induction of apoptosis in response to oxamyl exposure. [A] Images of zebrafish stained with acridine orange at 72 hpf. Particles with green fluorescence that indicate apoptotic cells are... 69
Fig. 2-3. Effects of oxamyl on nervous system and notochord in developing zebrafish. [A] Images of oxamyl-treated olig2:dsRED zebrafish were captured at 72 hpf. White arrowheads denote... 71
Fig. 2-4. Effects of oxamyl on notochord, muscle, and vascular development in zebrafish. [A] Right panels are magnified images of left panels. Colored regions indicate quantified somite angle, while... 73
Fig. 2-5. Effects of oxamyl on mitochondrial respiration in the zebrafish embryo. [A] Changes in oxygen consumption rate (OCR) of zebrafish at 24 hours post-fertilization (hpf) were evaluated... 75
Fig. 2-6. Effects of oxamyl on the Akt and Mapk signal transduction pathways in the zebrafish embryo. [A] Immunoblots of phosphorylated and total forms of Akt and Mapk (Erk, Jnk, and p38).... 77
Fig. 2-7. A schematic illustration depicting mode of action of oxamyl in zebrafish. Oxamyl exposure induced morphological alterations and apoptosis. Zebrafish treated with oxamyl showed... 82
Fig. 3-1. In vivo assessment of development toxicity of flufenacet in zebrafish. [A] Mortality rate of zebrafish upon flufenacet exposure was measured at different concentrations. [B]... 93
Fig. 3-2. Impaired vascular development by flufenacet in zebrafish. [A] Images of Tg(flk1:eGFP) zebrafish strain exposed to flufenacet were captured at 96 hpf. Images in the first row show... 95
Fig. 3-3. Impaired liver and pancreas development by flufenacet in zebrafish. [A] Images of Tg(fabp10a:dsRed; ela3l:eGFP) zebrafish strain exposed to flufenacet were captured at 96 hpf.... 97
Fig. 3-4. Apoptosis and cell cycle dysregulation induced upon flufenacet exposure in zebrafish. [A] Images of larvae undergo TUNEL staining were captured. Scale bar represents 200 μm. [B]... 99
Fig. 3-5. Flufenacet modulates Mapk and Akt signaling pathways in the developing zebrafish. Proteins extracted from total larvae upon flufenacet treatment (48 hpf) were analyzed with... 100
Fig. 3-6. Schematic diagram summarizes toxicity of flufenacet in the developing zebrafish. 104
Fig. 4-1. Overall detrimental effects of FPMH on zebrafish development. [A] Embryonic lethality was checked at the various concentrations (0, 10, 20, 25, 30, and 40 μM) every 24 h during FPMH... 113
Fig. 4-2. Analysis of apoptosis and inflammation induced by FPMH. [A] Acridine orange-stained apoptotic cells were visualized as green spots under an upright fluorescence microscope. Blue and... 115
Fig. 4-3. Malformed vascular system induced by FPMH exposure during zebrafish development. [A] Detection of fli1:eGFP zebrafish using the green fluorescent protein (GFP) channel of an... 117
Fig. 4-4. Compromised myelination induced by FPMH exposure during zebrafish development. [A] Detection of mbp:eGFP zebrafish under the green fluorescent protein (GFP) channel of an upright... 119
Fig. 4-5. Toxicity of FPMH in developing zebrafish embryos. Exposure to FPMH during the early stages of zebrafish development resulted in mortality and malformation of zebrafish larvae.... 124
CHAPTER 3. 134
Fig. 1-1. Acute developmental toxicity of chloroxylenol in zebrafish. [A] Viability of zebrafish exposed to various doses of chloroxylenol at different time points. [B] Dose-response curve... 134
Fig. 1-2. In vivo assessment of oxidative stress induced by chloroxylenol in zebrafish. [A] Representative fluorescence images of zebrafish stained with DCFH-DA at 96 hpf. Scale bar: 400... 135
Fig. 1-3. Activation of inflammatory responses and apoptosis by chloroxylenol in zebrafish. [A–D] Changes in mRNA expression levels of genes encoding mediators of inflammation—[A] cox2a,... 137
Fig. 1-4. Cardiovascular defects in zebrafish larvae exposed to chloroxylenol. [A] Representative fluorescence image of flk1:eGFP transgenic line at 96 hpf. The white arrow points to the sinus... 139
Fig. 1-5. Effects of chloroxylenol on HUVECs. [A] Viability of HUVECs exposed to 0, 0.5, 1, and 2.5 mg/L chloroxylenol for 48 h measured via MTT assay. [B] Images of tube formation by... 140
Fig. 1-6. Schematic illustration summarizing study findings regarding toxicity of chloroxylenol during zebrafish development. 144
CHAPTER 4. 152
Fig. 1-1. FPMH affects the viability of MAC-T cells and apoptosis of MAC-T cells is induced following FPMH exposure. [A] MTT assay was conducted using MAC-T cells incubated with... 152
Fig. 1-2. PI3K and MAPK signal transduction in MAC-T cells is affected by FPMH exposure. MAC-T cells were incubated with FPMH (0, 5, 7.5, and 10 μM) for 30 min. Alterations in the... 155
Fig. 1-3. Combination treatment with FPMH and signaling inhibitors to reveal pathway interactions. MAC-T cells were pretreated with LY294002, SB203580, SP600125, and U0126 (20... 156
Fig. 1-4. Stimulation of the ER stress response in MAC-T cells following FPMH treatment. MAC- T cells were incubated with 0, 5, 7.5, and 10 μM FPMH for 4 h. [A] Immunoblots showing the... 157
Fig. 1-5. Effect of FPMH on calcium dysregulation in MAC-T cells. Following a 24 h incubation with 0, 5, 7.5, and 10 μM FPMH, MAC-T cells were stained with rhod-2 [A and B] and fluo-4 [C... 158
Fig. 1-6. Schematic illustration presenting the mechanisms of FPMH cytotoxicity in MAC-T cells. 162
Fig. 2-1. Effects of folpet on the viability of MAC-T cells. [A] The number of viable MAC-T cells was measured after folpet treatment (0, 0.1, 0.5, 1, 2, 5, 10, and 50 μM, 48 h) using the 3-(4, 5-... 171
Fig. 2-2. Folpet mediated the apoptosis of MAC-T cells via disruption of the mitochondrial membrane potential and calcium homeostasis. [A and B] Annexin V/propidium iodide staining and... 173
Fig. 2-3. Effects of folpet on redox homeostasis in MAC-T cells. [A and B] Results of 2′,7′- dichlorofluorescein diacetate (DCFH-DA) staining and flow cytometry analyses of MAC-T cells... 175
Fig. 2-4. Modulation of MAPK cascades in MAC-T cells upon folpet exposure. [A] Immunoblot of each MAPK signaling molecule (phosphorylated form and total form of ERK1/2, JNK, and p38)... 176
Fig. 2-5. Activation of the MAPK cascade was mediated via ROS induction by folpet in MAC-T cells. [A and B] Results of 2′,7′-dichlorofluorescein diacetate (DCFH-DA) staining and flow... 177
Fig. 2-6. Cytotoxicity of folpet was mitigated through free radical scavenging with N- acetylcysteine. [A] Cytotoxicity of folpet on MAC-T cells was measured after sole treatment of 8... 178
Fig. 2-7. Effects of folpet on transcription of casein-encoding genes. [A–D] Results of quantitative real-time PCR showed alteration in mRNA levels of [A] CSN1S1, [B] CSN1S2, [C] CSN3, and [D]... 179
Fig. 2-8. A putative mechanism of action of folpet in MAC-T cells. In MAC-T cells, folpet exposure generates excessive ROS, enhancing MAPK signal transduction activation and lipid... 184
CHAPTER 5. 192
Fig. 1-1. Effects of 4-MU on ES2 and OV90 cell proliferation. [A] A BrdU cell proliferation assay was performed to measure the anti-proliferative effects of 4-MU (0, 0.25, 0.5, 1, 2, 4 mM) on ES2... 192
Fig. 1-2. Effects of 4-MU on cytoplasmic calcium concentration in [A] ES2 and [B] OV90 cells. Cytoplasmic calcium concentration was measured by flow cytometry using fluo-4 AM and data... 193
Fig. 1-3. 4-MU induced ER stress and increased mitochondrial calcium levels in ES2 and OV90 cells. [A] Protein expression levels of cleaved ATF6α, GRP78, and GADD153 were determined... 195
Fig. 1-4. Regulation of PI3K/AKT and MAPK signaling by 4-MU. Phosphorylation levels of [A] AKT, [B] S6, [C] ERK1/2, [D] P38, and [E] JNK were determined by western blotting analyses... 197
Fig. 1-5. Pretreatment with pharmacological inhibitors before 4-MU treatment alters the phosphorylation of signaling proteins. Phosphorylation levels of [A] AKT, [B] S6, [C] ERK1/2,... 197
Fig. 1-6. Effects of the co-treatment with 4-MU and pharmacological inhibitors on human ovarian cancer cells. Cells were incubated with 20 μM LY294002, SB203580, or SP600125 or 10 μM... 199
Fig. 1-7. Illustration of the hypothetical action of 4-MU on intracellular calcium levels, the endoplasmic reticulum (ER), and signal transduction in human epithelial ovarian cancer cells.... 203