Development of a Multi-Omics-Based Mechanistic Framework for Elucidating Molecular Toxicity in Aquatic Organisms = 수생생물 분자독성 기작 규명 위한 멀티오믹스 기반 분석방법론 개발

다국어 초록 (Multilingual Abstract)

Toxicity assessment has traditionally relied on targeted analyses of specific biomarkers. However, recent advances in big data analytics have expanded the application of omics-based approaches in toxicological and ecotoxicological studies. To investigate the detailed toxicological mechanisms underlying exposure to aquatic pollutants, this study applied an integrated multi-omics analysis framework. To facilitate omics-based analyses, high-quality gene and genome resources were first established for the pale chub Zacco platypus and the freshwater shrimp Neocaridina denticulata. As a common East Asian freshwater species, Z. platypus has garnered attention as a potential non-model organism for ecotoxicological assessment. Its high-quality de novo genome was assembled using combined PacBio long-read, Illumina short-read, and Hi-C–based scaffolding. Genome-wide comparisons revealed that the Z. platypus genome is the smallest among currently sequenced members of the order Cypriniformes. Notably, orthologous gene families uniquely expanded in Z. platypus were significantly enriched for functions related to detoxification and stress responses, underscoring its suitability as a genomic resource for ecotoxicological monitoring. For N. denticulata, a scaffold-level genome assembly was generated from PacBio sequencing data, yielding an estimated size of approximately 5.13 Gb— substantially larger than those reported for other species within the infraorder Caridea. To overcome the scarcity of comprehensive crustacean genomic resources, the present study complemented the genome assembly with full-length transcriptome sequencing (Iso-Seq), establishing a curated reference database of transcripts and deduced protein sequences. Based on these newly established resources, acute toxicity assessment was performed using multi-omics approaches. While toxicological pathways of specific pollutants have been partially characterized in established model organisms, comprehensive omics-based assessments of systemic toxicological responses remain limited in aquatic non-model species. I employed an integrated multi-omics framework, combining transcriptomic, proteomic, and metabolomic datasets, to elucidate the molecular mechanisms underlying pollutant-induced toxicity in Z. platypus and N. denticulata. Both species were acutely exposed to the organic pollutant triclosan and the heavy metals CdCl2 and As2O3. Integrated multi-omics analyses revealed distinct species-specific responses. In Z. platypus, exposure to all pollutants consistently induced molecular chaperones and endoplasmic reticulum (ER) stress–related markers. This was accompanied by a coordinated downregulation of energy-related metabolic pathways, reflecting a broad impairment of cellular energy metabolism. These molecular alterations were closely linked to oxidative stress, likely driven by elevated generation of reactive oxygen species (ROS). In contrast, N. denticulata showed relatively limited effects on ER stress– associated pathways. Toxicity in the shrimp was primarily manifested through mitochondrial-related responses, with glycolysis identified as a common metabolic response to aquatic pollutants. Furthermore, triclosan exposure specifically induced a compensatory metabolic response. Overall, this study successfully revealed species-specific toxicological responses to aquatic pollutants based on integrated multi-omics analyses. The multi- omics–based analytical framework established herein provides valuable insight for efficient aquatic ecosystem monitoring and risk assessment.

국문 초록 (Abstract)

독성 평가는 전통적으로 특정 표적 대상의 바이오마커 기반 분석에 의존해 왔으나, 최근 빅데이터 분석 기술의 발전으로 오믹스 기반 접근법이 주목받고 있다. 본 연구에서는 수생 오염물질 노출에 따른 독성 기작을 규명하기 위해 멀티오믹스 분석을 적용하였다. 이를 위해 먼저 멀티오믹스 분석의 기반이 되는 유전자 및 유전체 자원을 담수 어류 피라미(Zacco platypus)와 담수 새우 새뱅이(Neocaridina denticulata)를 대상으로 구축하였다.
피라미는 동아시아 전역의 담수 생태계에 널리 분포하며, 생태독성 및 환경 모니터링을 위한 잠재적 모델 어종으로 인식되고 있다. 본 연구에서는 long-read, short-read, Hi-C 시퀀싱을 통해 염색체 수준의 고품질 유전체를 구축하였다. 피라미는 잉어목(order Cypriniformes)에 속한 종들 중 가장 작은 유전체 크기를 보였으며, gene family 분석을 통해 해독 및 스트레스 반응 관련 유전자군이 특이적으로 확장된 것을 확인했다. 이러한 결과는 피라미가 생태독성 및 환경 모니터링을 위한 유망한 모델 생물임을 시사한다.
새뱅이의 경우, long-read 시퀀싱을 이용하여 scaffold 수준의 유전체를 구축하였다. 새뱅이는 십각목(infraorder Caridea)에 속한 다른 종들에 비해 상대적으로 큰 유전체 크기(약 5.13 Gb)를 가지며, 갑각류는 일반적으로 유전체 구조가 크고 복잡하여 고품질 유전체 분석이 제한적이다. 이에 본 연구에서는 전사체 분석(Iso-Seq)을 통해 유전자 데이터베이스를 구축하였다.
이와 같이 구축된 유전자 자원을 기반으로 다차원 오믹스를 활용한 수생오염원에 대한 급성 독성 반응을 연구하였다. 제브라피시나 마우스와 같은 모델 생물에서는 이러한 오믹스 분석을 통한 수생 오염원의 독성 기작이 일부 보고되었으나, 비모델 수생 생물에서 전반적인 독성 반응을 통합적으로 이해하기 위한 멀티 오믹스 연구는 여전히 부족하다. 본 연구에서는 전사체, 단백체, 대사체를 통합한 멀티 오믹스 플랫폼을 활용하여 수생오염원 노출에 따른 피라미와 새뱅이에서의 분자 수준 독성 기작을 규명하고자 하였다.
두 수생 생물은 대표적인 유기 오염원인 트리클로산과 무기 오염원인 중금속 CdCl2 및 As2O3에 급성 노출되었으며, 통합 멀티오믹스 분석을 통해 분자 반응을 평가하였다. 피라미에서는 모든 오염물질 노출 조건에서 분자 샤페론 및 소포체(endoplasmic reticulum, ER) 스트레스 관련 지표의 활성화가 공통적으로 관찰되었다. 동시에 아미노산 및 탄수화물 대사의 억제로 인한 에너지 대사 교란이 확인되었으며, 이는 활성산소종(reactive oxygen species, ROS) 증가에 의해 매개되는 산화 스트레스와 연관된 것으로 판단된다.
반면, 갑각류인 새뱅이에서는 ER 스트레스 관련 경로의 영향이 상대적으로 제한적인 반면, 미토콘드리아 관련 반응을 중심으로 독성이 관찰되었다. 특히 새뱅이는 해당과정(glycolysis)이 수생 오염원에 대한 공통적인 반응으로 나타났으며, 트리클로산 노출 시에는 보상적 대사 반응이 추가적으로 유도되었다.
종합적으로 본 연구는 멀티오믹스 분석을 통해 수생 오염물질에 대한 독성 반응이 종 특이적으로 상이함을 규명하였으며, 이는 수서생태독성 평가에서 비모델 생물 기반 멀티오믹스 접근의 중요성을 제시한다. 본 연구를 통해 구축한 멀티오믹스 기반 분석 방법이 향후 오믹스를 이용한 수생태계 독성 모니터링에 대한 인사이트를 제공할 수 있을 것으로 기대한다.

목차 (Table of Contents)

• Abstact i
• Preface iv
• Table of Contents v
• List of Tables x
• List of Figures xiv
• Chapter 1. Introduction 1
• 1.1. Aquatic pollutants 1
• 1.1.1. Organic pollutant-Triclosan 1
• 1.1.2. Inorganic pollutant-Heavy metal 6
• 1.2. Multi-omics approach 12
• 1.3. Limitation in aquatic ecotoxicological assessment 12
• 1.4. Zacco platypus as a vertebrate model for ecotoxicological studies 13
• 1.5. Neocaridina denticulata as an invertebrate model for ecotoxicological studies 14
• 1.6. Objectives 15
• Chapter 2. Materials and Methods 16
• 2.1. Animals 16
• 2.1.1. Experimental organisms 16
• 2.1.2. Ethics statement 16
• 2.2. Construction of gene and genome resources 17
• 2.2.1. Genomic DNA extraction 17
• 2.2.2. Library construction, sequencing, and assembly 17
• 2.2.3. Hi-C sequencing and chromosome-scale scaffolding 18
• 2.2.4. Assessment of genome completeness 18
• 2.2.5. Genomic repeat analysis and RNA profiling 19
• 2.2.6. Gene prediction and annotation 19
• 2.2.7. Unigene set construction using full-length transcriptome sequencing 20
• 2.2.8. Functional gene annotation 21
• 2.2.9. Gene family identification and phylogenetic analysis 21
• 2.3. Acute toxicity tests 22
• 2.3.1. Animal exposure 22
• 2.3.2. Measurement for actual exposure concentration and bioaccumulation 23
• 2.4. Transcriptome analysis 24
• 2.4.1. RNA extraction, sequencing, and data pre-processing 24
• 2.4.2. Unigene set construction using full-length transcriptome sequencing 24
• 2.4.3. Functional annotation and mRNA expression profiling 25
• 2.4.4. Validation using quantitative real-time polymerase chain reaction 25
• 2.5. Proteome analysis 28
• 2.5.1. Protein extraction and digestion 28
• 2.5.2. TMT labeling and offline high-pH reversed-phase fractionation 28
• 2.5.3. DDA and DIA analysis by nano-LC-MS/MS 30
• 2.6. Metabolome analysis 32
• 2.6.1. Metabolite extraction 32
• 2.6.2. Quantitative metabolomics analysis 33
• 2.6.3. Peak annotation and bioinformatics analysis 33
• 2.7. Multi-omics integration 34
• 2.8. Validation 35
• 2.8.1. Measurement of physiological parameters 35
• 2.8.2. Biochemical analyses 35
• Chapter 3. Construction of gene resource from non-model aquatic organisms 38
• 3.1. Whole-genome analysis of Zacco platypus 38
• 3.1.1. Genome sequencing and assembly 38
• 3.1.2. Comparative analysis of transposable elements and non-coding RNA profiling47
• 3.1.3. Gene prediction and functional annotation based on genome data 55
• 3.1.4. Gene family identification and phylogenetic analysis 59
• 3.1.5. Genome-wide identification of expanded gene families relevant to ecotoxicological responses in Z. platypus 62
• 3.2. Construction of a gene information database for Neocaridina denticulata using transcriptomic data 63
• 3.2.1. Genome sequencing and scaffold-level assembly 64
• 3.2.2. Iso-Seq analysis and transcript clustering 64
• 3.2.3. Functional annotation of genes 70
• Chapter 4. Multi-omics–based toxicity assessment in aquatic organisms 73
• 4.1. Triclosan 73
• 4.1.1. Toxicological mechanisms of TCS in Zacco platypus 73
• 4.1.1.1. Multi‑omics profiling in response to acute exposure 73
• 4.1.1.2. Integrated transcriptomic and proteomic response to TCS 82
• 4.1.1.3. Integrated genes and metabolomic response to TCS 92
• 4.1.1.4. Biochemical analyses 98
• 4.1.2. Species-specific molecular pathway in Zacco platypus 100
• 4.1.2.1. Unfolded protein response identified at transcript and protein levels 101
• 4.1.2.2. ROS-associated pathways identified through gene-metabolite interaction analysis 103
• 4.1.2.3. Non-monotonic alterations in lipid metabolism in response to TCS 106
• 4.1.3. TCS-targeted pathways in Neocaridina denticulata 113
• 4.1.3.1. Multi-omics responses to acute exposure 113
• 4.1.3.2. Oxidative stress and antioxidant defense responses 121
• 4.1.3.3. Integrated transcriptomic and proteomic responses to TCS 123
• 4.1.3.4. Integrated genes and metabolites in TCS-specific responses 131
• 4.1.4. Species-specific molecular pathway in Neocaridina denticulata 135
• 4.1.4.1. Unconventional antioxidant defense and ROS generation 135
• 4.1.4.2. Compensatory mechanisms in central metabolic pathways 136
• 4.1.4.3. Ca2+-dependent mechanisms under TCS exposure 137
• 4.2. Cadmium chloride 144
• 4.2.1. Toxicological mechanisms of CdCl2 in Zacco platypus 144
• 4.2.1.1. Multi‑omics profiling in response to acute exposure 144
• 4.2.1.2. Antioxidant defense and apoptosis responses 148
• 4.2.1.3. Integrated transcriptomic and proteomic response to CdCl2 150
• 4.2.1.4. Integrated genes and metabolomic responses to CdCl2 158
• 4.2.2. CdCl2-targeted pathways in Neocaridina denticulata 162
• 4.2.2.1. Multi-omics responses to acute exposure 162
• 4.2.2.2. Antioxidant defense and apoptosis responses 166
• 4.2.2.3. Integrated transcriptomic and proteomic responses to CdCl2 168
• 4.2.2.4. Integrated genes and metabolites in CdCl2-specific responses 173
• 4.2.3. Species-specific and common molecular pathway 177
• 4.3. Arsenic trioxide 180
• 4.3.1. Toxicological mechanisms of As2O3 in Zacco platypus 180
• 4.3.1.1. Multi-omics profiling in responses to acute exposure 180
• 4.3.1.2. Antioxidant defense and apoptosis responses 184
• 4.3.1.3. Integrated transcriptomic and proteomic response to As2O3 186
• 4.3.1.4. Integrated genes and metabolomic response to As2O3 193
• 4.3.2. As2O3-targeted pathways in Neocaridina denticulata 197
• 4.3.2.1. Multi-omics responses to acute exposure 197
• 4.3.2.2. Antioxidant defense and apoptosis responses 201
• 4.3.2.3. Integrated transcriptomic and proteomic responses to As2O3 203
• 4.3.2.4. Integrated genes and metabolites in As2O3-specific responses 208
• 4.3.3. Species-specific and common molecular pathway 212
• Chapter 5. Discussion 215
• References 219
• 국문초록 232
• Acknowledgements 235