Toxicogenomic impacts of silver nanoparticle on medaka (oryzias latipes) using cDNA microarrays

다국어 초록 (Multilingual Abstract)

This thesis has studied two main topics by using Japanese medaka (Oryzias latipes). In the first part, to strengthen the toxicogenomic study, we constructed a library of hepatic cDNA from medaka under influence of specific chemical mediated stress responses along with non-stress responded fish from the control. The functional characterization of the library was carried out for database configuration, annotation, and library analysis. Information of 1,509 high-qualities of O.latipes ESTs (531 unigenes) including 260 new ESTs was added onto GenBank and O. latipes dbEST of the US National Center of Biotechnology Information (NCBI). Continuous accession numbers are from HS104837 - HS106345. In addition, gene expression profile analyses of the cDNA microarrays followed by real time RT-PCR assays were conducted to screen particular biomarkers for 17？-estradiol (E2), nonylphenol (NP) and 2-chlorophenol (2CP). For each test chemical, two specific biomarkers were selected from the gene expression profiling of microarrays. The expression patterns of the marker genes in real time PCR analysis were consistent with the regulated gene expression patterns in microarrays. The tentative biomarkers showed unique gene expression patterns depending on chemical concentration(s) and exposure duration in real time RT-PCR analysis. The analysis accomplished of medake hepatic cDNA library and its information added to genetic and genomic resources could be sufficiently valuable specifically for aquatic toxicity studies.
The second part of the thesis is an implementation of the medaka hepatic cDNA microarrays consisting of 535 probes for discovering temporal relationships between gene expression profiles and silver nanoparticle-induced toxicological changes in liver of medaka. The results were further compared with the expression profile changes from the exposure of dissolved Ag (I) in form of AgNO3 at an equivalent mass of metallic silver to decipher how the toxic modes of the Ag-NPs differ from the toxicity of soluble Ag (I) in prolonged exposures. Six vitellogenin and chriogenin genes that dominantly induced in exposure of the nanoparticles were discovered; simultaneously those genes were specifically suppressed under AgNO3 exposure at equivalent mass of metallic silver. In addition, the chronic exposure of medaka to Ag-NPs was also studied by measuring expression levels of five stress-related biomarkers (e.g. MT, GST, p53, HSP70 and TF), two estrogenic markers (e.g. Chg-L and VTG1), and a major member of thioester-containing complement components, Orla C3-1 gene in the extended exposure period of 28 days. Using the microarrays followed by real time RT-PCR assays was valuable to evaluate toxic impacts of specific toxicants, and to screen novel biomarkers for aquatic toxicology studies. This research provides information for evaluating the risks to aquatic organisms exposed to metal nanoparticles.

다국어 초록 (Multilingual Abstract)

To strengthen the toxicogenomic study, we constructed a library of hepatic cDNA from medaka under influence of specific chemical mediated stress responses along with non-stress responded control fish. Gene expression profile analysis of the cDNA microarrays followed by real time RT-PCR assay were conducted to screen particular biomarkers for E2, NP, and 2CP. Information of 1,509 high-quality ESTs including 260 new ESTs was added onto GenBank and O. latipes dbEST. The ESTs were clustered and assembled into 159 contigs and 372 singletons. Among them, 128 contigs and 163 singletons (54.8%) were functionally characterized and 13 UniESTs (2.5%) were hypothetical proteins. Ontology analysis resulting in 282 UniESTs which involved with 2,102 GOs and 93 sequences associated with 116 enzyme codes. Gene expression analyses of the cDNA microarrays followed by real time RT-PCR assays were conducted for E2, NP, and 2CP. For each test chemical, two specific biomarkers were selected from the gene expression profiling of microarrays. The expression patterns of the marker genes in real time PCR analysis were consistent with the regulated gene expression patterns in microarrays. The tentative biomarkers showed unique gene expression patterns depending on chemical concentration(s) and exposure duration in real time RT-PCR analysis. The analysis accomplished of the hepatic cDNA library and its information added to genetic and genomic resources could be sufficiently valuable specifically for aquatic toxicity studies.

다국어 초록 (Multilingual Abstract)

A hepatic cDNA microarray consisting of 513 unique genes has been conducted to evaluate toxic impacts of silver nanoparticles (Ag-NPs) on the liver of medaka. The number of significantly altered genes was time and dose dependent. The larger number and the most over lapping of changed genes came from the higher dose (25 ？g/l) compared to the lower dose (1 ？g/l) of Ag-NP exposures. The comparison with the gene expression profile of exposure to 25 ？g/l dissolved Ag (I) evidenced that there existed distinguishable toxic fingerprints between the two silver forms. Nonetheless, molecular function classifications of transcription profiles of exposure both silver types were somehow similar. The conspicuous induction of estrogenic-related genes of medaka exposed to Ag-NPs is an interesting phenomenon. The specifically and dominantly altered genes by each exposure silver form were listed. Additionally, the chronic toxicity test has been conducted for twenty eight days to characterize the hepatic expression levels of eight marker genes after exposure medaka to dissolved Ag (I) or Ag-NP by using real time RT-PCR analyses. This extends my previously published work to include three additional biomarkers and three later time points. In comparing with the control, the significantly induction of MT and GST genes in livers of the fish exposed to 1 ？g/l Ag-NPs was observed at various time points during the test period. The Orla C3-1 (medaka) gene was slightly induced only with 1 ？g/l Ag-NPs at 7-day exposure while the suppression of p53 and HSP70 was recorded in all exposures at the end of the test. The gene encoding transferrin was repressed at 21-day by both silver types and at every exposure dosage. These results revealed the Ag-NPs increase metal detoxification, oxidative and inflammatory stress, lastly, stimulate immune responses in medaka. The conspicuous induction of choriogenin L and vitellogenin 1 in male fish exposed to Ag-NPs, especially at 7- and 21-day, compared with the exposures of AgNO3 or control was the first attempt to examine estrogenic effects of Ag-NPs.

목차 (Table of Contents)

• CONTENTS
• CHAPTER 1. INTRODUCTION 1
• 1.1. Overview 2
• 1.2. Literature Survey 4
• 1.2.1. Research History and Oryzias Latipes Database Update 4
• 1.2.2. Nanomaterials and Silver Nanoparticles 9
• 1.2.3. Econanotoxicology and Fish 12
• 1.3. Scope and Organization of the Dissertation 15
• CHAPTER 2. CONSTRUCTION AND CHARACTERIZATION OF JAPANESE MEDAKA (ORYZIAS LATIPES) HEPATIC CDNA LIBRARY AND ITS IMPLEMENTATION TO BIOMARKER SCREENING IN AQUATIC TOXICOLOGY 18
• 2.1. Abstract 19
• 2.2. Introduction 20
• 2.3. Materials and Methods 23
• 2.3.1. Fish Housing 23
• 2.3.2. Chemicals, Exposure and Sampling Methods 24
• 2.3.3. RNA Isolation and cDNA Library Construction 25
• 2.3.4. Contig Assembly and Sequence Analysis 27
• 2.3.5. Reverse Transcription and Real time RT-PCR 28
• 2.4. Results and Discussion 34
• 2.4.1. Generation of Hepatic cDNA Library 34
• 2.4.2. Sequence Assembly and Annotation 36
• 2.4.3. Gene Ontology Annotation 38
• 2.4.4. Pathway Analysis 39
• 2.4.5. Microarray Chip Validation and Biomarker Screening 40
• 2.5. Conclusions 45
• CHAPTER 3. TOXICOGENOMIC ANALYSIS OF EXPOSURE MEDAKA (O. LATIPES) TO SILVER NANOPARTICLES 57
• 3.1. Abstract 58
• 3.2. Introduction 60
• 3.3. Materials and Methods 67
• 3.3.1. Chemicals and Particle Characterization 67
• 3.3.2. Fish Housing, Exposure and Sampling Methods 68
• 3.3.3. Plasmid Isolation and cDNA Chip Fabrication 70
• 3.3.4. RNA Extraction and Amplification 71
• 3.3.5. cDNA Microarray Hybridization 72
• 3.3.6. Scanning and Data Mining 73
• 3.3.7. Reverse Transcription and Real time RT-PCR 75
• 3.4. Results and Discussion 82
• 3.4.1. Characterization of the Silver Nanoparticles 82
• 3.4.2. Determination of Lethal Concentration in Acute Toxicity Test 82
• 3.4.3. Construction of Medaka Hepatic cDNA Microarray Chips 83
• 3.4.4. Gene Expression Profile in Medaka Exposed to Ag-NPs and Dissolved Ag (I) 85
• 3.4.5. Differential Transcription Profiles between Exposures of Medaka to Ag-NPs and Dissolved Ag(I) 88
• 3.4.6. The Chronic Effects of Ag-NPs and Dissolved Ag (I) on Gene Expression in Liver of Male Medaka 91
• 3.5. Conclusions 100
• CHAPTER 4. CONCLUDING REMARKS 131
• 4.1. Concluding Remarks 132
• 4.2. Suggestions for Further Studies 136