Title Page
Abstract
Contents
Chapter 1. General introduction 21
1.1. Chemical contamination in the environment 21
1.2. Environmental epigenetics 22
1.3. Adverse Outcome Pathway (AOP) 30
1.4. Challenges to incorporate epigenetic modifications into chemical risk assessment 36
1.5. Aim of the thesis 38
Chapter 2. A systematic review of current state of environmental epigenetics and the applicability of epigenetic modifications in ecotoxicological assessment of chemicals 41
2.1. Introduction 41
2.2. The workflow of systematic literature review 42
2.3. Distribution of environmental epigenetics literature and categorization by toxicological terminology 43
2.4. Examination of the applicability of epigenetic modifications in an ecotoxicological context 49
2.5. Conclusion 59
Chapter 3. Caenorhabditis elegans model: Identification of repressive histone methylation-mediated reproductive toxicity of environmental chemicals using the Adverse Outcome Pathway (AOP) concept and benchmark concentration analysis 60
Abstract 60
3.1. Introduction 62
3.2. Materials and methods 67
3.2.1. C. elegans strains and cultivation 67
3.2.2. Chemical preparation 67
3.2.3. Germline GFP observations and de-silencing scoring using NL2507 strain 69
3.2.4. Reproduction assay 69
3.2.5. Enzymatic activity assay 70
3.2.6. Western blotting 71
3.2.7. Gene expression analysis 72
3.2.8. Benchmark concentration analysis 72
3.2.9. Protein sequence similarity assessment using SeqAPASS 73
3.2.10. Statistical analysis 73
3.3. Results and discussion 74
3.3.1. Effects of chemical additives on reproduction and germline silencing 74
3.3.2. Effect of chemical additives on repressive histone methylation (H3K9me3 and H3K27me3) 75
3.3.3. Histone methyltransferase-mediated reproductive defects in wild type (N2) exposed to TCS and TBBPA 78
3.3.4. Transcriptional responses of biomarker genes altered by co-treatment with HMT inhibitor, GSK343 83
3.3.5. Benchmark concentration analysis for TCS and TBBPA 85
3.3.6. Protein sequence similarity analysis using SeqAPASS 89
3.3.7. Building epigenetic AOP for TCS and TBBPA 94
3.3.8. The possible roles of repressive histone marks in environmental adaptation and transgenerational effects of chemicals 96
3.4. Conclusion 99
Chapter 4. Daphnid model: Inter- and intra-generational effects of CMIT/MIT biocide on phenotypic and epigenetic alterations 101
Abstract 101
4.1. Trans and multigenerational effects of isothiazolinone biocide CMIT/MIT on genotoxicity and epigenotoxicity in Daphnia magna 104
4.1.1. Introduction 104
4.1.2. Materials and methods 108
4.1.3. Results and discussion 115
4.1.4. Conclusion 131
4.2. Insights into the mechanisms of within-species variation in sensitivity to CMIT/MIT biocide using Daphnia sp 133
4.2.1. Introduction 133
4.2.2. Materials and methods 136
4.2.3. Results and discussion 140
4.2.4. Conclusion 158
Chapter 5. Killifish model: Differential DNA methylation and metabolite profiling of pollution adapted Fundulus heteroclitus from the New Bedford Harbor superfund site 161
Abstract 161
5.1. Introduction 163
5.2. Materials and methods 166
5.2.1. Fish sample collection 166
5.2.2. Sediment sampling and PCB analysis 166
5.2.3. Tissue dissection 166
5.2.4. Global DNA methylation 167
5.2.5. Metabolomics 167
5.2.6. Statistics and visualization 169
5.3. Results 170
5.3.1. PCB concentrations in sediments 170
5.3.2. Differential metabolites 171
5.3.3. Pathway analysis 175
5.3.4. Global DNA methylation 178
5.4. Discussion 179
5.4.1. Liver-specific DNA hypomethylation 179
5.4.2. Tissue-specific metabolic pathways 181
5.5. Conclusion 187
Chapter 6. The applicability of epigenetic markers into ecological risk assessment 188
6.1. Key findings and directions 188
6.1.1. Prospective ERA: The applicability of epigenetic changes as sensitive ecotoxicological biomarkers 189
6.1.2. Prospective ERA: Expansion of applicability domain 193
6.1.3. Retrospective ERA: The heritability of epigenetic changes and their role in adaptation 195
6.2. Testing strategies for identification of epigenotoxic chemicals in ecological risk assessment 197
6.2.1. Screening and prioritization strategies for epigenotoxic chemicals 198
6.2.2. Experimental consideration for retrospective ERA 200
6.3. Conclusion 203
References 204
Appendices 238
Appendix 1. Supplementary information: Identification of repressive histone methylation-mediated reproductive toxicity of chemical additives using the Adverse Outcome Pathway (AOP) concept and benchmark concentration analysis (Chapter 3) 238
Appendix 2. Supplementary information: Trans and multi-generational effects of isothiazolinone biocide CMIT/MIT on phenotypic and epigenetic alterations (Chapter 4.1) 241
Appendix 3. Supplementary information: Insights into the mechanisms of within-species variation in sensitivity to CMIT/MIT biocide using Daphnia sp. (Chapter 4.2) 249
Appendix 4. Supplementary information: Differential DNA methylation and metabolite profiling of pollution adapted Fundulus heteroclitus from the New Bedford Harbor superfund site (Chapter 5) 262
초록 264
Table 1.1. The history and definitions of epigenetics 23
Table 1.2. Epigenetic Adverse Outcome Pathways (AOP) from AOP Wiki database 34
Table 1.3. The major knowledge gaps and impediments that need to be addressed for the incorporation of epigenetic modifications in ecological risk assessment 38
Table 2.1. Expert decision criteria for determining the applicability of epigenetic changes as sensitive and reliable biomarkers in ecotoxicological assessment 51
Table 2.2. Literature analysis scoring sheet to determine strength of applicability 51
Table 2.3. Number of studies rated "strong", "moderate", or "weak" based on the degree of applicability of epigenetic changes as biomarkers for... 54
Table 2.4. Bisphenol A: A summary of studies showing high applicability of epigenetic modifications as ecotoxicological biomarkers 55
Table 2.5. Benzo[a]pyrene: A summary of studies showing high applicability of epigenetic modifications as ecotoxicological biomarkers 57
Table 3.1. Use category and international regulatory status of chemicals used in the study (reviewed on October 2023) 68
Table 3.2. The benchmark concentrations of triclosan (TCS) for enzymatic, transcriptional, and apical endpoints. 87
Table 3.3. The benchmark concentrations of tetrabromobisphenol A (TBBPA) for enzymatic, transcriptional, and apical endpoints. 88
Table 3.4. Summary of Level 2 SeqAPASS analysis: Ortholog detection results based on functional domain sequence similarity. 90
Table 3.5. Taxonomic groups containing model organisms with high sequence similarity to the SET domain of C. elegans histone methyltransferase. 90
Table 3.6. Taxonomic groups containing model organisms with high sequence similarity to the JmjC domain of C. elegans histone demethylase. 91
Table 4.1.1. Nominal concentration of CMIT/MIT mixture and measured concentrations of CMIT and MIT by HPLC. 116
Table 4.2.1. Acute and chronic CMIT/MIT toxicity to different daphnid species/strains. 142
Table 4.2.2. Physiological changes in three Daphnia strains exposed to CMIT/MIT. Data are presented as mean ± SE (n=10) with statistical significance. 142
Table 4.2.3. Putative critical proteins associated with difference in phenotypes and sensitivity to CMIT/MIT between DPR and DPA. 157
Table 5.1. PCB concentrations detected in sediments collected from Scorton Creek and New Bedford Harbor (ND=Not Detected) 171
Table 5.2. Differential metabolites and their fold changes in NBH fish compared to SC fish 177
Table 6.1. Tiered toxicity testing for ecotoxicity of chemical 194
Figure 1.1. Overall outline of the thesis 40
Figure 2.1. Outline of the systematic review in this study 43
Figure 2.2. Distribution of environmental epigenetics literatures categorized by research field (A) and type of epigenetic marks (B). 44
Figure 2.3. Distribution of publication on epigenetic marks in total toxicological fields (A) and ecological & ecotoxicological fields (B). 44
Figure 2.4. Distribution of publication on biological endpoints in total toxicological fields (A) and ecological & ecotoxicological fields (B). 46
Figure 2.5. Distribution of publication on taxa and stressors in toxicological fields (A, C) and ecological & ecotoxicological fields (B, D). 47
Figure 2.6. Distribution of environmental contaminants studied in toxicological fields (A) and ecological & ecotoxicological fields (B). 48
Figure 3.1. Concentration-response relationship between chemical exposure and reproductive toxicity. 76
Figure 3.2. Germline de-silencing status and histone methylation (H3K9me3 and H3K27me3) in worms exposed to 10 µM of five chemical additives. 77
Figure 3.3. Alterations of levels in H3K9 and H3K27-specific histone methyltransferase activity after exposure to five chemicals in wild type N2. 78
Figure 3.4. Effects of the H3K27 and H3K9-specific histone methyltransferase inhibitors on reproductive toxicity caused by TCS, TBBPA, and HBCD. 79
Figure 3.5. Effects of the H3K27-specific HMT inhibitor GSK343 on histone methylation, histone methyltransferase, and histone demethylase activities in... 82
Figure 3.6. A heatmap displaying gene expression in worms exposed to triclosan (TCS) or tetrabromobisphenol A (TBBPA) and in worms exposed to... 84
Figure 3.7. Alterations in transcriptional responses of biomarker genes under co-treatment with the HMT inhibitor GSK343. 85
Figure 3.8. Comparative analysis of the benchmark concentrations of TCS (A) and TBBPA (B) for enzymatic, transcriptional, and apical endpoints. 86
Figure 3.9. Analysis of quantitative functional SET protein domain similarity for Caenorhabditis elegans histone methyltransferase (MET-2 and MES-2)... 92
Figure 3.10. Analysis of quantitative functional JmjC protein domain similarity for Caenorhabditis elegans histone methyltransferase (JMJD-2 and... 93
Figure 3.11. Putative Adverse Outcome Pathway (AOP) for histone methyltransferase and repressive histone methylation-mediated ecotoxicity of... 95
Figure 3.12. Multigenerational and transgenerational effects of TCS and TBBPA on reproduction (A) and locomotive activity (B). 99
Figure 4.1.1. Experimental workflow for multigenerational studies: (A) Illustration of three different exposure designs (control, parental exposure,... 110
Figure 4.1.2. Effects of CMIT/MIT on mortality and reproductive capacity in D. magna 118
Figure 4.1.3. Reproductive capacity, growth, and swimming behavior of D.magna after exposure to the EC20 of CMIT/MIT (7 µg/L) 119
Figure 4.1.4. Protein-protein interaction networks and functional enrichments of differentially expressed proteins (DEPs) in D. magna after... 122
Figure 4.1.5. Phenotypic responses of daphnids to parental and multigenerational exposure of CMIT/MIT EC₂₀: Data are normalized to... 126
Figure 4.1.6. Genotoxic and epigenetic responses of daphnids to parental and multigenerational exposure of CMIT/MIT EC₂₀ 129
Figure 4.1.7. Graphical summary for Chapter 4.1. 132
Figure 4.2.1. Comparative (A) lethal concentrations (LCs) and (B) effective concentrations (ECs) of CMIT/MIT in three Daphnia strains. 143
Figure 4.2.2. Alteration of global DNA methylation levels in three Daphnia strains exposed to CMIT/MIT. 143
Figure 4.2.3. Analysis of differentially expressed proteins (DEPs) induced by exposure to CMIT/MIT LC₂₀ in the two D. pulex strains (DPR and DPA). 148
Figure 4.2.4. Graphical representation of (A) DPR and (B) DPA strain-specific KEGG pathway enrichment with differentially expressed proteins (DEPs) as... 149
Figure 4.2.5. Graphical summary for Chapter 4.2. 160
Figure 5.1. Orthogonal projection to latent structures-discriminant analysis(OPLS-DA) of metabolites detected in brain and liver tissues of Scorton Creek... 173
Figure 5.2. Comparison of fold changes in the concentrations of metabolites detected in (A) the brains and (B) livers. 174
Figure 5.3. Analysis of differential metabolites between New Bedford Harbor(NBH) and Scorton Creek (SC) fish. 176
Figure 5.4. Global DNA methylation levels in brains (A) and livers (B) of Scorton Creek (SC) and New Bedford Harbor (NBH) fish. 178
Figure 6.1. Conceptual scheme representing a testing strategy for identifying epigenotoxic chemicals combined with tiered ecotoxicity testing 201