Proteomic analysis of daphnia magna and application for toxicity detection

20 CHAPTER 2: GENE EXPRESSIONS OF THE DHB, VTG, ARNT, CYP4, CYP314 IN DAPHNIA MAGNA INDUCED BY TOXICITY OF GLYPHOSATE AND METHIDATHION PESTICIDES ...24 2.1 A BSTRACT .... magna in respo

박사학위논문

Proteomic Analysis of Daphnia Magna and

Application for Toxicity Detection

2012 년 02 월 22 일

전 북 대 학 교 대 학 원

생물공정공학과

Le Thai-Hoang

Proteomic Analysis of Daphnia Magna and

Application for Toxicity Detection

Proteomic Analysis of Daphnia Magna and

Application for Toxicity Detection

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Table of contents

TABLE OF CONTENTS I LIST OF THE FIGURES AND TABLES V ABBREVIATION X

CHAPTER 1: LITERATURE 5

1.1 C ONTAMINATION OF AQUATIC ENVIRONMENT BY PHARMACEUTICALS AND PESTICIDES 5

1.2 P ROTEOMIC APPROACHES IN AQUATIC ECOTOXICOLOGY 8

1.2.1 Reverse Transcription Polymerase chain reaction (RT-PCR) 12

1.2.2 Proteomics analysis by two-dimensional electrophoresis 12

1.3D APHNIA MAGNA 15

1.4 T RANSGENIC ORGANISM EXPRESSING ENHANCED GREEN FLUORESCENT PROTEIN 17

1.5 A IMS OF THE THESIS 17

1.6 T HESIS ORGANIZATION 18

1.7 R EFERENCES 20

CHAPTER 2: GENE EXPRESSIONS OF THE DHB, VTG, ARNT, CYP4, CYP314 IN DAPHNIA MAGNA INDUCED BY TOXICITY OF GLYPHOSATE AND METHIDATHION PESTICIDES 24

2.1 A BSTRACT 24

2.2 I NTRODUCTION 25

2.3 E XPERIMENTAL METHODS 27

2.3.1 Daphnia magna culture 27

2.1.1 Acute toxicity test 27

2.1.1 Chronic toxicity test 28

2.1.2 Exposure experiments to analyze the gene expression 28

2.1.3 Isolation of RNA samples and Semi-quantitative reverse transcriptase-polymerase chain reaction 29

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2.1.4 Statistical analysis 30

2.4 R ESULTS AND DISCUSSION 32

2.4.1 Acute toxicity of glyphosate and methdiathion 32

2.4.2 Effects of acute toxicity of glyphosate and methidathion on gene expression 32

2.4.3 Chronic toxicity of glyphosate and methdiathion 41

2.4.4 Effects of chronic toxicity of glyphosate and methidathion on gene expression 42

2.5 C ONCLUSIONS 48

2.6 R EFERENCES 49

CHAPTER 3: TOXICITY EVALUATION OF VERAPAMIL AND TRAMADOL BASED ON TOXICITY ASSAY AND EXPRESSION PATTERNS OF DHB, VTG, ARNT, CYP4 AND CYP314 IN DAPHNIA MAGNA 53

3.1 A BSTRACT 53

3.2 I NTRODUCTION 54

3.3 M ATERIALS AND M ETHODS 56

3.3.1 Daphnia magna culture 56

3.3.2 Acute toxicity test 57

3.3.3 Chronic toxicity test 57

3.3.4 Exposure experiments to analyze the gene expression 58

3.3.5 Isolation of RNA samples and Semi-quantitative reverse transcriptase-polymerase chain reaction 58

3.3.6 Statistical analysis 59

3.4 R ESULTS 61

3.4.1 Toxicity and molecular effect after short-term exposure 61

3.4.2 Toxicity and molecular effect after long-term exposure 67

3.5 D ISCUSSION 71

3.6 R EFERENCES 73

CHAPTER 4: PROTEOMIC ANALYSIS OF DAPHNIA MAGNA EXPOSED TO GLYPHOSATE, METHIDATHION, VERAPAMIL, AND TRAMADOL 78

4.1 A BSTRACT 78

4.2 I NTRODUCTION 79

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4.3 M ATERIALS AND M ETHODS 79

4.3.1 Daphnia magna culture 79

4.3.2 Exposure experiments to analyze the protein expression 80

4.3.3 Protein isolation and concentration determination 80

4.3.4 Two-dimensional electrophoresis 80

4.3.5 Silver staining 81

4.3.6 Spots analysis 82

4.4 R ESULTS AND D ISCUSSION 83

4.4.1 Proteomic analysis of D magna exposed to glyphosate and methidathion 83

4.4.2 Proteomic analysis of D magna exposed to tramadol and verapamil 94

4.5 R EFERENCES 104

CHAPTER 5: DEVELOPMENT OF TRANSGENIC DAPHNIA MAGNA EXPRESSING GREEN FLUORESCENT PROTEINS 106

5.1 A BSTRACT 106

5.2 I NTRODUCTION 106

5.3 M ATERIAL AND M ETHODS 108

5.3.1 Daphnia magna culture and maintenance 108

5.3.2 Construction of pD18s-GFP plasmid 109

5.3.3 Microinjection 111

5.3.4 Screening transgenic Daphnia magna 111

5.4 R ESULTS AND D ISCUSSION 112

5.4.1 Construction of pD18s-GFP plasmid 112

5.4.2 Microinjection to create a pD18s-GFP transgenic daphnia 114

5.4.3 Screening the transgenic daphnia expressing green fluorescent protein 117

5.5 R EFERENCES 119

CHAPTER 6: CONCLUSIONS AND FUTURE DIRECTION OF RESEARCH 121

CHAPTER APPENDIX: PHENOL DEGRADATION ACTIVITY AND REUSABILITY OF CORYNEBACTERIUM GLUTAMICUM COATED WITH NH2-FUNCTIONALIZED SILICA-ENCAPSULATED FE 3 O 4 NANOPARTICLES 125

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A.1 A BSTRACT 125

A.2 I NTRODUCTION 126

A.3 M ATERIAL AND M ETHODS 127

A.3.1 Preparation of Fe 3 O 4 NPs, and NH 2 -functionalized silica-encapsulated Fe 3 O 4 127 A.3.2 Bacterial strain and cultivation 127

A.3.3 Immobilization and magnetic separation of C glutamicum to NH2-functionalized silica-encapsulated Fe3O4 NPs 128

A.3.4 Field emission scanning electron microscopy (FE-SEM) 129

A.3.5 Determination of phenol 129

A.4 R ESULTS AND D ISCUSSION 130

A.4.1 Characteristics of NH 2 -functionalized silica-encapsulated Fe 3 O 4 NPs 130

A.4.2 Growth of C glutamicum in the phenol-containing medium 132

A.4.3 Immobilization of C glutamicum with NH2-functionalized silica-encapsulated Fe3O4 NPs 132

A.4.4 Biodegradation of phenol 133

A.5 C ONCLUSIONS 137

A.6 R EFERENCES 143

ACKNOWLEDGEMENTS 148

CURRICULUM VITAE 149

THESIS ABSTRACT IN KOREAN 159

adult female with parthenogenetic embryos in her brood chamber 16 Table 1 Chemical properties and description of some pesticides and

pharmaceuticals in this study 7

CHAPTER 2

Figure 1 Acute toxicity assay of D magna for glyphosate (a) and

methidathion (b) 35 Figure 2 Relative expression levels of 5 selected genes after 24h

exposures of 0, 190, 202, 214, and 234 mg/L glyphosate (a)

Dhb, (b) Vtg, (c) Arnt, (d) CYP4 and (e) CYP314 All of the data

correspond to the expression level relative to the Act gene 38 Figure 3 Relative expression levels of 5 selected genes after 24h

exposures of 0, 0.024, 0.029, 0.034, and 0.044 mg/L

methidathion (a) Dhb, (b) Vtg, (c) Arnt, (d) CYP4 and (e)

CYP314 All of the data correspond to the expression level

relative to the Act gene 39 Figure 4 Effects of 20% ethanol on the relative expression levels of 5

selected genes 45 Figure 5 Relative expression levels of 5 selected genes after 21d

exposures of 0, 2.34, 4.68 and 23.4 mg/L glyphosate (a) Dhb, (b)

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Vtg, (c) Arnt, (d) CYP4 and (e) CYP314 All of the data

correspond to the expression level relative to the Act gene 47 Figure 6 Relative expression levels of 5 selected genes after 21d

exposures of 0, 0.44, and 0.88 μg/L methidathion (a) Dhb, (b)

Vtg, (c) Arnt, (d) CYP4 and (e) CYP314 All of the data

correspond to the expression level relative to the Act gene 48 Table 1 Primer sequences of the genes used for the RT-PCR 32 Table 2 Survival and reproduction of D magna after a 21d exposure to

glyphosate 40 Table 3 Survival and reproduction of D magna after a 21d exposure to

methidathion 41

CHAPTER 3

Figure 1 Acute toxicity assay of D magna to the pharmaceuticals, i.e., (a)

verapamil HCl and (b) tramadol HCl 64 Figure 2 Relative expression levels of 5 selected genes after 24 hours of

exposure to (a) 0, 8.2, 11.0, and 15.0 mgL-1 verapamil HCl and (b) 0, 25.0, 52.0, and 90.0 mg L-1 tramadol HCl All of the data correspond to the expression levels relative to the Act gene 70 Figure 3 Relative expression levels of 5 selected genes after 21 day of

exposure to (a) 1.1 mgL-1 and 2.1 mgL-1 verapamil HCl and (b) 8.5 mgL-1 tramadol HCl All of the data correspond to the expression levels relative to the Act gene 71 Table 1 Primer sequences of the genes used for the RT-PCR 61 Table 2 Four lethal concentrations of verapamil HCl and tramadol HCl

that were used for the exposure test 65 Table 3 Survival and reproduction of D magna following a 21 day

exposure to verapamil HCl 66 Table 4 Survival and reproduction of D magna following a 21 d

exposure to tramadol HCl 67

CHAPTER 4

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Figure 1 The 2DE map shows the location of the differentiaaly expressed

proteins in response to toxicity of glyphosate The proteins were separated with pH gradient ranging from 3 to 10 and the molecular weights (MW) that are also indicated 87 Figure 2 Segment of 2D gel image showing the up-regulation of the 10

proteins induced by glyphosate 88 Figure 3 Segment of 2D gel images showing the down-regulation of the

14 proteins induced by glyphosate 89 Figure 4 The 2DE map shows the location of the differentially expressed

proteins in response to toxicity of methidathion The proteins were separated with pH gradient ranging from 3 to 10 and the molecular weights (MW) that are also indicated 91 Figure 5 Segment of 2D gel image showing the up-regulation of the 19

proteins induced by methidathion 92 Figure 6 Segment of 2D gel images showing the down-regulation of the 8

proteins induced by methidathion 93 Figure 7 Venn diagram represents the number of DEPs shared by

treatment of glyphosate and methidathion 95 Figure 8 The 2DE maps shows the location of the differentiaaly expressed

proteins in response to toxicity of tramadol The proteins were separated with pH gradient ranging from 3 to 10 and the molecular weights (MW) that are also indicated 97 Figure 9 Segment of 2D gel image showing the up-regulation of the 19

proteins induced by tramadol 98 Figure 10 Segment of 2D gel images showing the down-regulation of the 8

proteins induced by tramadol 99 Figure 11 The 2DE maps shows the location of the differentiaaly expressed

proteins in response to toxicity of verapamil The proteins were separated with pH gradient ranging from 3 to 10 and the molecular weights (MW) that are also indicated 101

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Figure 12 Segment of 2D gel image showing the up-regulation of the 19

proteins induced by verapamil 102 Figure 13 Segment of 2D gel images showing the down-regulation of the 8

proteins induced by verapamil 103 Figure 14 Venn diagram represents the number of DEPs shared by

treatment of tramadol and verapamil 105 Table 1 Lists of the differentially expressed proteins in D magna in

response to toxicity of glyphosate 90 Table 2 Lists of the differentially expressed proteins in D magna in

response to toxicity of methidathion 94 Table 3 Lists of the differentially expressed proteins in D magna in

response to toxicity of tramadol 100 Table 4 Lists of the differentially expressed proteins in D magna in

response to toxicity of verapamil 104

CHAPTER 5

Figure 1 Structure of recombinant plasmid pD18s-GFP (a) and nucleotide

sequence of commercial primer of pD18s constitutive promoter

in Daphnia pulex(b) 112 Figure 2 The pairwise sequence alignment between the pD18s-GFP

constructed plasmid and the 26bp DNA sequence of pD18s constitutive promoter 115 Figure 3 The optimal time for microinjection among the 15

developmental states of D magna eggs The developmental state

A is referred to state 1, 2, 3, and 4 which are optimal for microinjection The developmental state B is from the state 5th

to state 15th 118 Figure 4 Screening for transgenic D magna Under the confocal

microscope, while the green fluorescent protein can not be

observed in the control Daphnia magna (a, b, c), a strong signal

of green fluorescent is detected in the pD18s-GFP transgenic

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daphnia (d, e, f) After exposing to the methidathion (0.029mg/L), the green fluorescent was no longer observed in transgenic daphnia (g, h, k) 120 Table 1 Effect of KCL buffer, DNA type, and egg developmental state on

the transformation efficiency in microinjection 117

APPENDIX

Figure 1 TEM images of bare Fe3O4 NPs (a), silica-encapsulated Fe3O4

(b), and IR spectrum analysis for NH2 functionalization (c) 134 Figure 2 FE-SEM pictures of NH2-functionalized silica-encapsulated

Fe3O4 NPs coated C glutamicum Whole-cell scale (a), and zoom-in scale (b) 137 Figure 3 Effect of the ratio of NH2-functionalized silica-encapsulated

Fe3O4 NPs and the DCW of C glutamicum (w/w) on the immobilization efficiency 138 Figure 4 Time course of phenol biodegradation using the C glutamicum

cells coated with NH2-functionalized silica-encapsulated Fe3O4 NPs 141 Figure 5 Reusability of C glutamicum coated with NH2-functionalized

silica-encapsulated Fe3O4 NPs for phenol degradation activity 142 Figure 6 FE-SEM pictures of C glutamicum coated with NH2-

functionalized silica-encapsulated Fe3O4 NPs after fourth reuse batch of phenol degradation There were 3 random sites selected for taking images with the whole cell scale on the left side (a, c and e), and zoom-in scale on the right side (b, d and f) where the adsorption of NPs on the cell surface can be observed 143 Figure S1 Relationship between the DCW and OD600 value of C

glutamicum 144 Figure S2 Time course of cell growth of C glutamicum in MSMY medium

without phenol (black symbol), and with 50 ppm phenol (white symbol) 145

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Abbreviation

LC50 Lethal concentration of 50%

RTPCR Reverse Transcription Polymerase chain reaction

2DE Two-dimensional electrophoresis

DEPs Differentially expressed proteins

pD18 Constitutive promoter of Daphnia pulex 18s ribosomal RNA EGFP Enhanced green fluorescent protein vector

Dhb Daphnia hemoglobin

Vtg Vitellogenin

Arnt Aryl receptor nuclear translocator

CYP4 Cytochrome P450 family 4

CYP314 Cytochrome P450 family 314

NPs Nanoparticles

FE-SEM Field emission scan electron microscopy

TEM Transmission electron microscopy

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Proteomic Analysis of Daphnia Magna and

Application for Toxicity Detection

Thai-Hoang Le Department of Bioprocess Engineering

The graduate school Chonbuk National University

Abstract

While the pressure on our environmental health has increased by the introduction of many new classes of pollutants (e.g., pesticides and pharmaceuticals), the traditional toxicity assessment methods (e.g., acute, chronic test), which based on only the simple endpoints such as growth rate, survival, and reproduction, are not capable of detecting them The advance approaches based on the molecular responses such as gene expression and proteomic analysis were focused in this thesis in attempt to predict the toxicity to aquatic organisms; to understand the action mode of the toxicity; and to discover novel biomarkers for detection of these contaminants in the aquatic environment In addition, an invivo system for a rapid detection of toxic chemicals in the aquatic environment was developed by creating a transgenic water

flea (D magna) able to express green fluorescent protein

Firstly, the expression of 5 selected genes including hemoglobin (Dhb), vitellogenin (Vtg), aryl hydrocarbon receptor nuclear translocator (Arnt), cytochrome P450 4 family (CYP4) and cytochrome P450 314 family (CYP314) was studied on D

magna in response to the toxicity of some typical pesticides (i.e., glyphosate,

methidathion), and pharmaceuticals (i.e., verapamil and tramadol) These 5 selected genes were well known for environmental stress Through the method of reversed transcriptase polymerase chain reaction (RTPCR), the alternation in expression

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levels of the 5 selected genes were examined in D magna exposed to several

sublethal concentrations of pesticides and pharmaceuticals Our results indicated that

pesticides and pharmaceuticals gave different impacts on the gene expression in D

magna The 24h exposure to pesticides caused a significant reduction of expression

level of the 5 selected genes Among these 5 genes, CYP4 and CYP314 genes which

expressed differently in exposure to glyphosate and methidathion, respectively; could be considered as the potential biomarkers of these pesticides However, the 21d exposure to some low concentrations of glyphosate and methidathion (i.e., 1%, 2% and 10%LC50) showed slightly up-regulation of the 5 genes which indicated the chronic toxicity of these chemicals may stimulate some acclimation mechanism to help organisms to overcome the adverse impacts from the toxic environments In case of pharmaceuticals, while the 24h exposure to verapamil and tramadol gave slight impacts on the expression level of the 5 genes, the 21d exposure to these

chemicals considerably reduced the expression of Vtg gene which is the biomarker

for the reproduction ability of oviparous animals

In the proteomic study, the expression of the entire set of proteome in Daphnia

magna was studied in response to the pesticides and pharmaceuticals using the

technique of two-dimensional electrophoresis (2DE) Particularly, the total protein samples were isolated from the daphnia which were exposed to LC50 and LC75 concentrations of pesticides and pharmaceuticals for 24h Next, through 2DE method, the total protein samples were separated by the first dimension of pH range from 3 to 10 and the second dimension of molecular weight After the silver staining step, the protein profile in the gel images were analyzed by Progenesis solfware for determination of the differentially expressed proteins (DEPs) in comparison with control samples Our results showed that a large number of DEPs were found in

Daphnia magna in response to the toxicity of glyphosate, methidathion, verapamil

and tramadol Particularly, the found up-regulated proteins by glyphosate, methidathion, verapamil and tramadol were 10, 19, 10, and 7, respectively; and the down-regulated proteins by these chemicals were 12, 8, 8, and 8, respectively In the

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further work, a MALDI-TOF analysis could be performed in order to identify the specific proteins

One of the most concerned studies in this thesis is how to create a transgenic water

flea, D magna, able to express a green fluorescent protein to establish a novel

system for a rapid detection of environmental stress A 32bp DNA fragment

containing the 18s-ribosome constitute promoter of daphnia was generated by invitro

annealing the commercial primers at 80oC This daphnia promoter of 18s-ribosome (pD18s) was inserted and replaced the CMV promoter fragment that was located

ahead to the gfp gene in the EGFP vector (Enhanced Green Flourescent Protein)

The successful recombinant plasmid confirmed by the DNA sequencing was introduced into the earlier developing-state daphnia eggs (round shape) using microinjection technique After 48h incubation in the 20oC chamber, the injected eggs able to develop to juveniles were observed at the 488nm wavelength by the confocal fluorescent microscope to confirm the presence of green fluorescent protein Our results show that the green fluorescent protein was substantially found in the upper part of the transgenic organisms (e.g., head, back) while no fluorescent signal

was detected in the control organism It demonstrated that a transgenic D magna

expressing green fluorescent protein was firstly generated and would be promising

to build a novel testing system for easily monitoring environmental stress

Keywords: Daphnia magna, toxicity, transcriptomics, proteomics, transgenic

daphnia, nanoparticles,

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Chapter 1

Literature

through spray drift, volatilization, drainage and leaching (Cerejeira et al., 2003; Pereira et al., 2009), pharmaceuticals may enter the aquatic environments via biomedical, veterinary medicine, agricultural, and industrial routes (Flaherty et al.,

2005) Consequently, a large number of the bioactive different compound used as ingredients for the pharmaceutical and pesticides are entering waste water and receiving water bodies (rivers, lakes, etc.) without being examined for special

environmental effects (Richardson et al., 2005)

In the scope of this thesis, several typical pesticides and pharmaceuticals were selected for study based on their high toxicity and popularity

Glyphosate, N-Phosphonomethylglycine (fig 1a), is a nonselective and

broad-spectrum herbicide used to kill weeds competing with crops Glyphosate, an active ingredient in many commercial weed-killing formulations (e.g., Roundup, Rodeo), is

popularly used in agriculture, silvicultural, and urban environment (Borggaard et al.,

2008) Due to its high water solubility and highly usage, exposure of non-target organisms in environment to glyphosate becomes an emerging concern to ecotoxicologists (Tsui, 2003)

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Methidathion, an organophosphate insecticide (fig 1b), is used to control a

broad spectrum of agricultural insects and mite pests in terrestrial food crops Methidathion, an active ingredient in many commercial products (e.g., Somonic, Somonil, Supracide, Suprathion, Ultracide, and etc.), is used to protect plants from insects with sucking, chewing mouthparts such as scale, moths, and aphids According to Environmental Protection Agency (USEPA), methidathion, highly toxic and classified as EPA toxicity class I (Danger), kill insects by disrupting the nervous system and causing paralysis(Washburn, 2003)

Verapamil HCl , an L-type calcium channel blocker of the phenylalkylamine

class (Fig 1c), is used to control ventricular rate in the treatment of some cardiovascular diseases such as hypertension, angina pectoris, cardiac arrhythmia Recently, verapamil, among top five risk-based of pharmaceutical compounds in the aquatic environment, was found to significantly increase the risk for many types of

cancer (Beiderbeck-Noll et al., 2003; Fent et al., 2006)

Tramadol HCl, (1R,2R)-rel-2-(dimethylamino)methyl- methoxyphenyl)cyclohexanol (fig.1d), a narcotic-like pain reliever, is used similarly

1-(3-to codein in treating moderate 1-(3-to severe pain (Flick et al., 1972) It is a synthetic agent as a 4-phenyl-piperidine analog of codeine and appears to affect the GABAergic, noradrenergic, and serotonergic systems (Keith Budd, 1999) Not only verapamil, tramadol is also among top five pharmaceutical compounds dangerous to our aquatic environment Although these two medicines have commonly been used worldwide, few discussions about their adverse effects on the aquatic environment have been carried out so far

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Table 1: Chemical properties and description of some pesticides and pharmaceuticals in this study

Chemicals Molecular structure Descriptions

Glyphosate

(169.02 g/mol)

A nonselective and broad-spectrum herbicide

An active ingredient in many commercial weed-killing formulations (e.g., Roundup, Rodeo)

Methidathion

(302.34 g/mol)

Organophosphate insecticide Used to control a broad spectrum of agricultural insects and mite pests in terrestrial food crops

An active ingredient in many commercial products (e.g., Somonic, Somonil, Supracide, Suprathion, Ultracide, and etc.),

P O

OH OH

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1.2 Proteomic approaches in aquatic ecotoxicology

While these emerging contaminants are complex and pressing a serious concern

on the environmental health, current risk assessment models based on the conventional toxicity test (i.e., acute and chronic toxicity test) are not sufficient to examine totally risks of pollutants on the non-target organisms in the aquatic environment, especially on predicting their toxicity to aquatic organism and environmental fate Basically, gene expression is changed by toxicants as either a direct or indirect result of a toxicity exposure (Nuwaysir et al., 1999) The changing

at molecular level (e.g., gene and protein expression) in the organism exposing to toxic environment should firstly occur and result in a consequent alternation at body level (e.g., survival, growth, reproduction, etc…) Therefore, the research approach

on the gene and protein expression should be a more sensitive indicator to detect the environmental contaminants than the conventional toxicity methods

Proteomics is the large-scale study on the proteome, which is the entire set of proteins produced by a cell, tissue, or organism Like transcriptomics, the proteomics study is considered as a “bottom-up” approach from molecular to population level (Fig 1) which will enhance opportunity to study the phenotypic and genotypic basis of fitness (Snape, 2004) Comparing to the traditional single endpoint methods, the proteomic technique is helpful to maximize the information obtained from limited testing organisms and hence to reduce the future level of routine ecotoxicity testing; to identify the mechanism of toxicity of pollutants that may assist to develop predictive simulation model of toxic effects; to link molecular and cellular biomarkers with higher level population and ecosystem responses; to predict potential ecological risk assessment issues for new chemicals and emerging technologies (Snape, 2004)

Particularly, analyzing the gene and protein expression can assess reactions of

an organism in response to an environmental stressor Figure 1 illustrates how a pollutant can lead to a change in expression level of specific gene and protein in an exposed organisms (Poynton et al., 2009) First, in the toxic environment, the

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organism is exposed to a chemical pollutant which enters and distributes throughout its body The pollutant interacts with cells and cellular components in a manner dependent on its chemical properties which results in a specific cellular damage To protect itself from the toxicant of mitigate adverse effect of the stressor, the organism reacts to the pollutant at multilevel through changing the expression level

of genes and proteins The particular group of those genes and proteins is dependent

on the specific action mode of the pollutant Therefore, particular pattern of genes and proteins can act as a fingerprint for a specific mode of action of the pollutant

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Figure 1: Conceptual framework of ecotoxicogenomics (Snape, 2004)

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Figure 2: Gene and protein expression profiling as a tool to study organism responses to pollutants ((Poynton et al., 2009)

1.2.1 Reverse Transcription Polymerase chain reaction (RT-PCR)

In cells, many cellular decisions related to survival, growth, and differentiation, significant affected by surrounding environment, are reflected in changed pattern of gene expression and quantitation of the transcription level of specific genes has been always central to any research into gene function (Zamorano et al 1996) In general, four methods have been employed popularly for the quantification of transcription including northern blotting and in situ hybridisation, RNAse protection assays and the reverse transcription polymerase chain reaction (RT-PCR) Among these methods, the RT-PCR is the most sensitive method for the detection of low-concentration mRNA, often isolated from limited tissue samples (Bustin, 2000) Additionally, RT-PCR is a simple and flexible quantification method to compare levels of mRNAs in different sample population to characterise patterns of mRNA expression, and to discriminate between closely related mRNAs (Stephen et al., 2008) This method includes two major steps: reverse transcription and polymerase chain reaction In the first step, reverse transcription, total mRNA molecules in the sample are reversely transcribed into total cDNA molecules using the reverse transcriptase enzyme In the second step, polymerase chain reaction, the total cDNA molecules are used as DNA template for a usual PCR reaction to amplify the target gene using specific primers The quantity of the target PCR product represents for the amount of specific mRNA in the primary RNA sample By quantitating the mRNA level, we can analyze the expression level of a specific gene in the organism

1.2.2 Proteomics analysis by two-dimensional electrophoresis

While RTPCR technique permits to measure mRNA amount for a study of gene expression, two-dimensional electrophoresis technique (2-DE) is capable to analyse proteome for the comprehensive structural and quantitative information on all proteins in the organisms (Hebestreit, 2001) To analyse the changes in protein expression between two distinct samples, several techniques are used including 2DE, mass spectrometry, or protein array These approaches can be combined with DNA microarray to provide a better insight into the impact mechanism of a stressor in the exposed organism Recently, proteomics analysis is obtaining importance for biomarker and drug discovery (Celis, 2004) and starting produce results in the field

of ecotoxicogenomics (Shrader et al., 2003) At present, 2DE is considered as a core

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technique in proteomics for protein efficient separation and semiquantitative analysis A typical procedure to perform a 2DE experiment is schematized in the

figure 2 (Rabilloud et al., 2011) Basically, it includes 5 main steps such as sample

preparation (step 1), the first separation, isoelectric focusing (step 2), strip equilibration (step 3), the second separation, sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE (step 4), and protein detection (step 5) Firstly, the raw biological material must go to an extraction and purification process for a final soluble protein sample (step1) Then, the sample is loaded onto a pH gradient gel strip oriented with the acidic side at the anode and the basic side at the cathode After the IEF step, the proteins have reached their pI and thus have no remaining electrical charge (step 2) To negatively charge the proteins, the strip is equilibrated

in a SDS-containing buffer (step 3) Next, the gel strip containing negative-charge proteins is loaded on top of a SDS-PAGE gel for a second-dimension separation based on the molecular mass (step 4) Finally, the protein spots in the gel are detected by staining gel with dye solution (e.g., coomassie brilliant blue, silver nitrate, and etc.) (step 5)

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Figure 3: Scheme of principle of 2D gel electrophoresis (Rabilloud et al., 2011)

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1.3 Daphnia magna

Daphnia, planktonic crustaceans that belong to the Phyllopoda, use the leaf-like legs to produce a water current for the filtering apparatus Whole bodies of daphnia are enclosed by a double-wall chitin carapace Daphnia have up to 10 pairs of appendages including antennules, antennae, maxillae, mandibles, 5 or 6 limbs, and a pairs of claws at the end of the abdomen The body length of daphnia ranges from less than 0.5mm to more than 6mm (Fig 4)(Elbert, 2005)

Daphnia is already an established model species in toxicology This water flea

is not only used to evaluate the toxic effects of chemical on aquatic system but also play an important role in building up regulatory criteria by environmental agencies (e.g., Environmental Protection Agency EPA, Organization for Economic Cooperation and Development OECD)(Shaw et al., 2008) This is probably because daphnia have many interesting characteristics that make it become useful in biological and toxicological research Particularly, they are numerical abundance, wide distribution, important role in aquatic food web, high sensitivity to a wide range of chemicals, a short lifecycle, ease of manipulation in the laboratory (Soetaert

et al., 2006) As a results, Daphnia are considered as a sentinel species of freshwater water bodies (e.g., lakes and ponds), where their decline serves as an indicator of

environmental problems (Hanazato et al., 1995)

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Figure 4: The functional anatomy of Daphnia This draw illustrates an adult female with parthenogenetic embryos in her brood chamber (Elbert, 2005)

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1.4 Transgenic organism expressing enhanced green fluorescent protein

Based on the alternation of gene expression level by the toxicant exposure, the transcriptomic and proteomic approach are able to identify the novel biomarkers

specific to the toxic impacts (Kato et al., 2010) Once the specific biomarker gene is

identified, it can be introduced together with a reporter gene into a host organism to create a transgenic organism which is able to monitor easily the presence of the

corresponding toxic factor in the environment Among the reporter genes, gfp gene,

generated green fluorescent protein which can be easily detected in the dark room, has been employed to create a transgenic organism for various applications For instance, a transgenic zebrafish was generated to establish a novel in vivo test

system for a rapid detection of environmental estrogens (Chen et al., 2010) To create the transgenic zebrafish, a pzVtg sequence, a promoter of zebrafish

vittelogenin gene which is considered as a biomarker for endogenous estrogens, was

cloned to upstream of gfp gene in the pEGFP vector, which can produce green fluorescent protein, to construct a recombinant plasmid of pzVtg-EGFP which was

then introduce into zebrafish embryo In purpose of creating a bioreactor to produce pharmaceutical proteins, a transgenic chickens able to expressing the enhanced

green fluorescent protein was generated (Kwon et al., 2004) In another research, a

transgenic mice expressing green fluorescent protein was generated by injecting

directly the foreign DNA into the ovaries of fertile mice(Yang et al., 2007) Taking together, it is promising to develop a transgenic D magna using gfp gene as a

reporter system to establish a novel in vivo system to detect toxic factors in environment

1.5 Aims of the thesis

In this thesis, I studied on the toxicity assessment of pesticides and

pharmaceuticals in the aquatic environment using D magna I was especially

interested in alternations at the molecular level in organisms such as gene and

protein expression in D magna in response to the toxic environments

Understanding the molecular responses of exposed organisms to toxic chemical in

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the environment will help to interpret the mode of toxicity as well as uncover the potential biomarkers of toxic factors Furthermore, this thesis was an attempt to develop an in vivo system for a rapid and sensitive detection of environmental stress

To achieve those goals, the experiments in this thesis were designed into 4 main parts as following:

1 Characterize toxicity of two typical pesticides (i.e., glyphosate and methidathion), and two typical pharmaceuticals (i.e., verapamil and tramadol) using the conventional acute and chronic toxicity test

2 Investigate the changing in gene expression of the 5 genes including Dhb,

Vtg, Arnt, CYP4, and CYP314 in Daphnia magna in response to both short-term

and long-term exposure to glyphosate, methidathion, verapamil and tramadol

3 Analyze the protein expression in entire set of proteins in Daphnia magna

and identification of the differentially expressed proteins (DEPs) caused by the toxicity of glyphosate, methidathion, verapamil and tramadol

4 Develop a transgenic Daphnia magna able to expressing the green

fluorescent protein in response to the toxic chemicals and apply as a novel system to easily and sensitive detect the environmental stress

1.6 Thesis organization

The thesis was divided into 6 main chapters and 1 appendix chapter:

Chapter 1 is “Literature” providing some basic information which is necessary

to understand the importance and contribution of the other chapters in this thesis It includes knowledge about research background, the current problems in the aquatic environment, the solving approaches, and the purposes of the studies in this thesis Chapter 2 is “Gene expression in D magna induced by toxicity of glyphosate and methidathion pesticides” in which the expression level of 5 selected genes

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including Dhb, Vtg, CYP4, and CYP314 in response to both short-term and

long-term exposure to those chemicals were examined using the RTPCR method to quantitate the mRNA amount of each genes generated through the transcription process Thereby, the effects of these toxic chemicals on the gene expression in organism were elucidated

Chapter 3 is “Toxicity evaluation of verapamil and tramadol based on toxicity

assay and gene expression patterns in D magna” Toxicity of two typical

pharmaceuticals was studied with the focus on their influence on the expression of the 5 selected genes in compared to the physiological responses such as growth, survival, and reproduction in D magna

Chapter 4 is “Proteomic analysis of D magna exposed to glyphosate, methidathion, tramadol, and verapamil” The alternation of the proteomic expression

in D magna after exposing to those toxic chemicals was investigated using dimensional electrophoresis method The differentially expressed proteins (DEPs)

two-by each chemical were determined and identified in order to screening the potential biomarkers

Chapter 5 is “Development of transgenic Daphnia magna expressing the green fluorescent protein” The transgenic Daphnia magna was created by introduction of

a recombinant plasmid pD18s-GFP into the daphnia eggs using microinjection method The inserted promoter pD18s in this plasmid is a constitutive promoter of daphnia pulex 18s ribosomal RNA, a strong promoter controlling the high expression of the associated genes without any required inducers

Chapter 6 is “Conclusion and future direction of research” which outlines some main interesting results achieved from this thesis research Furthermore, some directions for the future research are suggested in order to develop these findings in thesis for the contribution in understanding the toxicity mechanism in daphnia and in the progress of risk assessment

The appendix chapter is “Phenol Degradation Activity and Reusability of

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Corynebacterium glutamicum Coated with NH2-functionalized Silica-Encapsulated Fe3O4 Nanoparticles” This study ia an attempt to develop a novel method to

immobilize and separate Corynebacterium glutamicum for phenol degradation was

developed using Fe3O4 nanoparticles (NPs) The Fe3O4 NPs were encapsulated with silica and functionalized with NH2 groups to enhance their capacity to adsorb on the cell surface

Beiderbeck-Noll, A B., Sturkenboom, M C J M., Linden, P D v d., Herings, R

M C., Hofman, A., Coebergh, J W W., Leufkens, H G M and Stricker, B H C., 2003 Verapamil is associated with an increased risk of cancer in the elderly: the Rotterdam study European Journal of Cancer 39, 8

Borggaard, O K and Gimsing, A L., 2008 Fate of glyphosate in soil and the possibility of leaching to ground and surface waters: a review Pest Management Science 64, 441-456

Bustin, S A., 2000 Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays Journal of Molecular Endocrinology 25, 24

Celis, J E., 2004 Proteomic Characterization of the Interstitial Fluid Perfusing the Breast Tumor Microenvironment: A Novel Resource for Biomarker and Therapeutic Target Discovery Molecular & Cellular Proteomics 3, 327-344 Cerejeira, M J., Vianab, P., Batistaa, S., Pereiraa, T., Silvaa, E., Val!erioa, M J., Silvaa, A., Ferreirab, M and Silva-Fernandesa, A M., 2003 Pesticides in Portuguese surface and ground waters Water Research 37, 9

Chen, H., Hu, J., Yang, J., Wang, Y., Xu, H., Jiang, Q., Gong, Y., Gu, Y and Song, H., 2010 Generation of a fluorescent transgenic zebrafish for detection of environmental estrogens Aquatic Toxicology 96, 53-61

Elbert, D., Ed (2005) Ecology, Epidemiology and Evolution of Parasitism in Daphnia Basel, Universität Basel

Fent, K., Weston, A and Caminada, D., 2006 Ecotoxicology of human pharmaceuticals Aquatic Toxicology 76, 122-159

Flaherty, C and Dodson, S., 2005 Effects of pharmaceuticals on survival, growth, and reproduction Chemosphere 61, 200-207

Hal, N L W v., Vorst, O., Houwelingen, A l M M L v., Kok, E J., Peijnenburg,

Hebestreit, H F., 2001 Proteomics: an holistic analysis of nature 's protein Current Opinion in Pharmacology 1, 8

Kato, Y., Kobayashi, K., Watanabe, H and Iguchi, T., 2010 Introduction of foreign

DNA into the water flea, Daphnia magna, by electroporation Ecotoxicology 19,

Miracle, A L and Ankley, G T., 2005 Ecotoxicogenomics: linkages between exposure and effects in assessing risks of aquatic contaminants to fish Reproductive Toxicology 19, 321-326

Nuwaysir, E F., Bittner, M., Trent, J., Barrett, J C and Afshari, C A., 1999 Microarrays and Toxicology: The Advent of Toxicogenomics MOLECULAR CARCINOGENESIS 24, 7

Pereira, J L., Antunes, S C., Castro, B B., Marques, C R., Gonçalves, A M M., Gonçalves, F and Pereira, R., 2009 Toxicity evaluation of three pesticides on non-target aquatic and soil organisms: commercial formulation versus active ingredient Ecotoxicology 18, 455-463

Poynton, H C and Vulpe, C D., 2009 Ecotoxicogenomics Emerging technologies for emerging contaminants JOURNAL OF THE AMERICAN WATER RESOURCES ASSOCIATION 45, 12

Rabilloud, T and Lelong, C., 2011 Two-dimensional gel electrophoresis in proteomics: A tutorial Journal of Proteomics

Richardson, S D and Ternes, T A., 2005 Water analysis Emerging contaminants and current issues Analytical Chemistry 77, 32

Shaw, J R., Pfrender, M E., Eads, B D., Klaper, R., Callaghan, A., Colson, I., Gilbert, D and Colbourne, J K., 2008 Daphnia as an Emerging Model for Toxicological Genomics Advances in Experimental Biology 2, 55

Shrader, E A., Henry, T R., Greeley, M S., Jr and Bradley, B P., 2003 Proteomics

in zebrafish exposed to endocrine disrupting chemicals Ecotoxicology 12, 8 Snape, J., 2004 Ecotoxicogenomics: the challenge of integrating genomics into aquatic and terrestrial ecotoxicology Aquatic Toxicology 67, 143-154

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Soetaert, A., Moens, L., Vanderven, K., Vanleemput, K., Naudts, B., Blust, R and

Decoen, W., 2006 Molecular impact of propiconazole on Daphnia magna

using a reproduction-related cDNA array Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 142, 66-76

Tsui, M., 2003 Aquatic toxicity of glyphosate-based formulations: comparison between different organisms and the effects of environmental factors Chemosphere 52, 1189-1197

Washburn, A D., 2003 The Environmental Fate of Methidathion 2003

Watson, A., Mazumder, A., Stewart, M and Balasubramanian, S., 1998 Technology for microarray analysis of gene expression Current Opinion in Biotechnology

9, 6

Yang, S., Wang, J., Cui, H., Sun, S., Li, Q., Gu, L., Hong, Y., Liu, P and Liu, W.,

2007 Efficient generation of transgenic mice by direct intraovarian injection of plasmid DNA Biochemical and Biophysical Research Communications 358, 266-271

Zamorano PL, Mahesh VB & Brann DW 1996 Quantitative RT-PCR for neuroendocrine studies A minireview Neuroendocrinology 63 397–407 Flick, Kurt; Frankus, Ernst, (1972) “1-(m-Substituted Phenyl)-2-Aminomethyl Cyclohexanols", US patent 3652589, issued 28 March 1972

Stephen, J W., Travis, J W., Kent, E V., 2008 Semiquantitative Real-Time PCR for Analysis 30 of mRNA Levels Methods in Molecular Medicine 79, 1940-6037 Lothar Eggeling, Michael Bott (2005) Handbook of corynebacterium glutamicum CRC Press

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Chapter 2

Gene Expressions of the Dhb, Vtg, Arnt, CYP4, CYP314

in Daphnia magna Induced by Toxicity of Glyphosate

and Methidathion Pesticides

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Chapter 2: Gene Expressions of the Dhb, Vtg, Arnt, CYP4,

CYP314 in Daphnia magna Induced by Toxicity of Glyphosate

and Methidathion Pesticides

caused physiological effects with different patterns in D magna, especially

metabolisms related to CYPs On the other hand, only vitellogenin (Vtg), which was

responsive to the estrogenic potency, did not show any differences in D magna after exposure to glyphosate or methidathion

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2.2 Introduction

Pesticides, the chemicals used commonly in agriculture to control pests, pathogens and weeds, have been contributing to the serious contamination of the aquatic environment through spray drift, volatilization, drainage and leaching (Cerejeira et al., 2003; Pereira et al., 2009) Among various pesticides, methidathion

is a highly toxic insecticide used to control a wide spectrum of agricultural insect and mite pests In the other hand, glyphosate, the active ingredient in many commercial weed-killing formulation (e.g., Roundup), is widely used in agricultural, silvicultural and urban environment (Borggaard, K.O., and Gimsing, L.A., 2008) The results are an increasing detection of these pesticides in the environment, especially in aquatic system may have some ecotoxicological impacts on non-target aquatic organisms (Tsui et al., 2003; Vorkamp et al., 2002)

D magna, a freshwater crustacean, has been used extensively to evaluate the

toxic effects of chemical on aquatic system (USEPA, 2002) because of their high sensitivity to a wide range of chemicals, a short lifecycle, and ease of manipulation

in the laboratory In addition, the daphnia are ubiquitous and play a key role in aquatic food web (Soetaert et al., 2006) Conventionally, the toxicity assays (e.g., acute or chronic toxicity tests) have widely been used to evaluate the aquatic toxicity

as well as the adverse impacts of the toxic chemicals on aquatic organisms based on the phynotic endpoints such as the survival, growth, and reproduction (Colleen et al., 2005; Heckmanna et al., 2007) These body responses result from some molecular responses (e.g., gene expression) in organisms that expose to a toxic environment Therefore, changing in gene expression in the organisms should happen first, and be

a more sensitive indicator to the toxic chemicals than the body responses (Jo et al., 2008; Le et al., 2010; Le et al., 2011)

Notably, when an organism is exposed to a toxic environment, the metabolic activity in the organism will change to overcome the adverse effects (Ankley and Villeneuve, 2006) Certain genes, possessing the particular functions, express differently in organisms that are exposed to toxicants For instance, hemoglobins

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(Dhb), the protein are distributed widely in all organisms, are composed of multiple

two-domain chains with a relatively normal oxygen binding activity when found in

the hemolymph of D magna (Tokishita et al., 1997; Anderson et al., 2008) Vitellogenin (Vtg) is a major lipoprotein in many oviparous animals and has been

used as a useful biomarker to examine the hazardous effects of endocrine disrupting compounds (EDCs) (Jones et al., 2000; Kato et al., 2004) Aryl hydrocarbon

receptor nuclear translocator (Arnt) activates the transcription of the genes that

encode the enzymes involved in metabolizing aryl hydrocarbons, such as dioxin and endocrine disruptors in mammalian cells and marine, freshwater and avian species

(Tokishita et al, 2006) Hence, the Arnt gene probably responds to the altered

metabolic effect of exposure to aryl hydrocarbons Cytochrome P450s (CYPs) are a large and ubiquitous super-family of heme proteins that are encoded by receptor-dependent transcriptional activation genes, and are a class of proteins that respond to the hazardous effect of toxic chemicals (Snyder, 2000) CYPs are categorized into 4

different families, i.e., mitochondrial, CYP2, CYP3-like, and CYP4 family

(Bradfield et al., 1991; Baldwin et al., 2009) In the present study, the hazardous

effects of two selected pesticides (i.e., glyphosate, and methidathion) on D magna

were examined by studying the changes in the gene expressions of five stress

responsive genes, including Dhb, Arnt, Vtg, CYP4, and CYP314 using the method of

reverse transcription polymerase chain reaction (RT-PCR) This technique is a simple and effective tool to study the gene expression (Stephen et al., 2008) The expression level of a gene is measured by determining the mRNA amount generated

by that gene via the transcription Through the gene expression analysis, the

molecular responses of D magna exposed to pesticides can be determined to

provide an insight into the action mode of chemicals

The aim of this study was to evaluate toxicity of the two pesticides and to

analyze their adverse effects on the expression patterns of different genes in D

magna Thereby, the mechanisms controlling the gene expressions were indirectly

studied in response to the addition of pesticides, i.e., glyphosate and methidathion

Five different genes including hemoglobin (Dhb) (Ha and Choi, 2009), vitellogenin