꿀벌과 제브라피쉬에서 대사체학과 단백질체학을 이용한 살충제 abamectin의 독성학적 비교

학위논문 (박사) -- 서울대학교 대학원 : 농업생명과학대학 농생명공학부, 2021. 2. 김정한.Abamectin is an abamectin insecticide known to be isolated from fermentation of Streptomyces avermitilis, a naturally occurring soil Actinomycete. Due to its high toxicity in bees and fish, this study investigated the toxic mechanisms of abamectin in honeybee (Apis mellifera) and zebrafish (Danio rerio) using targeted metabolomics approach by gas chromatography-tandem mass spectrometry (GC-MS/MS) and liquid chromatography-high resolution mass spectrometry (LC-orbitrap-HRMS). All homogenized samples were extracted with 80% methanol (honeybee) or 50% methanol (zebrafish) and derivatized with TMS for GC-MS/MS analysis. In the targeted metabolomics approach, multiple reaction monitoring (MRM) mode of a GC-MS/MS for 396 metabolites was used to detect 239 metabolites in honeybee and 243 metabolites in zebrafish. With the results of metabolites detected in each sample, statistical analysis such as partial least squares‐discriminant analysis (PLS-DA), variable importance in the projection (VIP) and analysis of variance (ANOVA) were performed to identify important biomarkers. Metabolic pathways associated with those biomarkers were constructed using MetaboAnalyst 5.0. In the exposure experiment of honeybee to abamectin as a targeted metabolomics using GC-MS/MS and non-targeted metaboloimcs using LC-orbitrap-HRMS, metabolic pathways such as tyrosine metabolism, phenylalanine/tyrosine/tryptophan biosynthesis, citrate cycle, ascorbate/aldarate metabolism, and alanine/aspartate/glutamate metabolism were identified as the significantly perturbed pathways. While, zebrafish showed several metabolic pathways such as aminoacyl tRNA biosynthesis, glyoxylate/dicarboxylate metabolism, citrate cycle, and tryptophan metabolism were identified by exposure of abamectin. Such significant disturbance of important metabolites within key biochemical pathways by abamectin could result in biologically hazardous effects in honeybee and zebrafish. In toxicoproteomics study, the toxicological effects of abamectin in honeybee and adult zebrafisha were investigated using a label-free quantitative proteomic approach on LC-HRMS. The proteins of honeybee and zebrafish samples were extracted with 0.1M phosphate buffer (pH 7.4) and 200 μg of proteins were digested with trypsin using the in-solution filter-aided sample preparation (FASP) method. After LC-HRMS analysis, a total of 670 proteins were identified and 32 proteins were selected as biomarkers through volcano analysis in honeybee. In zebrafish, 2189 proteins were identified and 1050 proteins were selected as biomarkers by statistically analysis.Abamectin은 자연적으로 발생하는 토양 방선균인 Streptomyces avermitilis의 발효에서 분리 된 것으로 알려진 살충제다. 꿀벌과 어류의 독성이 높기 때문에 본 연구는 가스크로마토그래피-탠덤 질량 분석법 (GC-MS/MS) 및 액체크로마토그래피-고분해능 질량 분석법(LC-orbitrap-HRMS)에 의한 비표적 및 표적 대사 체학 접근법을 사용하여 꿀벌(Apis mellifera) 및 제브라피시 (Danio rerio)에서 abamectin의 독성 메커니즘을 조사했다. 모든 균질화 된 샘플을 80 % 메탄올(꿀벌) 또는 50 % 메탄올(제브라피쉬)로 추출하고 GC-MS/MS 분석을 위해 추출액을 TMS로 유도체화반응을 적용하였다. 표적 대사체학 접근법에서는 396 개의 대사산물에 대한 GC-MS/MS의 다중 반응 모니터링(MRM) 모드를 사용하여 꿀벌에서 239개의 대사산물과 제브라피시에서 243개의 대사산물을 검출했다. 각 샘플에서 검출 된 대사 산물의 결과를 바탕으로 PLS-DA 패턴분석, VIP score 및 ANOVA 분산 분석과 같은 통계 분석을 수행하여 중요한 바이오마커를 식별했다. 이러한 바이오 마커와 관련된 대사경로는 MetaboAnalyst 5.0을 사용하여 확인 할 수 있었다. GC-MS/MS를 사용하는 표적 대사체와 LC-orbitrap-HRMS를 사용하는 비표적 대사체학을 통하여 꿀벌의 노출 실험에서 tyrosine metabolism, phenylalanine/tyrosine/tryptophan biosynthesis, citrate cycle, ascorbate/aldarate metabolism, and alanine/aspartate/glutamate metabolism가 상당히 교란 된 것으로 확인되었다. 반면, 제브라피쉬는 aminoacyl tRNA biosynthesis, glyoxylate/dicarboxylate metabolism, citrate cycle, and tryptophan metabolism이 상당히 변화함을 확인하였다. Abamectin에 의한 중요한 대사산물의 심각한 교란은 꿀벌과 제브라피쉬에 생물학적으로 위험한 영향을 미칠 수 있음을 확인 할 수 있었다. 한편, 독성 단백질체학 연구에서는 꿀벌과 제브라피쉬에서 LC-HRMS를 통하여 비표지 정량을 활용한 단백질체학적 접근방식을 사용하였다. 꿀벌과 제브라피시 샘플의 단백질은 0.1M phosphate buffer (pH 7.4)로 추출하고 200μg의 단백질은 in-solution filter-aided sample preparation (FASP) 방법을 사용하여 트립신으로 분해하였다. LC-HRMS 분석 후 총 670개의 단백질이 확인되었고, 화산 분석을 통해 32개의 단백질이 바이오 마커로 선정되었다. 제브라 피쉬에서는 2189개의 단백질이 확인되었고 통계 분석을 통해 1050개의 단백질이 바이오 마커로 선택되었다.Abstract.............................................................................................................i Table of Contents...................................................................iii List of Tables...........................................................................vi List of Figures.................................................................................................viii Preface.......................................................................................................................1 Chapter I. Toxicometabolomics of abamectin in honeybee (Apis mellifera) and zebrafish (Danio rerio).............................................................................................3 Introduction.........................................................................................................5 Methodology of metabolomics............................................................................8 Application of metabolomics.............................................................................21 Purose of study………………………………………………………………….23 Materials and Methods......................................................................................24 Chemicals and reagents........................................................................................24 Experimental animals…………………………………………………………24 Chemical exposure...............................................................................................24 Sample preparation..............................................................................................25 Targeted profiling and identification of metabolites by GC-MS/MS...................26 Non-targeted profiling and identification of metabolites by LC-Orbitrap-HRMS…………………………………………………………..………………26 Statistical analysis and biomarker selection.........................................................30 Metabolic pathway analysis.................................................................................34 Measurement of MDA contents………………………………………………...34 Results and Discussion.......................................................................................36 Chemical exposure and sample preparation…….................................................36 Targeted profiling and identification of metabolites by GC-MS/MS...................36 Non-targeted profiling and identification of metabolites by LC-Orbitrap-HRMS…………………………………………………………………………..52 Statistical analysis and biomarker selection…………………………………….58 Metabolic pathway analysis…………….............................................................74 Abamectin effects on MDA in zebrafish………………………………………..84 Conclusions........................................................................................................88 Chapter II. Toxicoproteomics of abamectin in honeybee (Apis mellifera) and zebrafish (Danio rerio)...........................................................................................91 Introduction.......................................................................................................93 Proteomics...........................................................................................................93 Methodology of proteomics.................................................................................94 Application of proteomics..................................................................................105 Purpose of study.................................................................................................180 Materials and Methods....................................................................................109 Chemicals and reagents......................................................................................109 Experimental animals and chemical exposure……………………………….109 Sample preparation............................................................................................109 Profiling and identification of proteomes by LC-Orbitrap-HRMS.....................111 Statistical analysis……………………….......................................................114 Enrichment analysis...........................................................................................114 Results and Discussion.....................................................................................115 Protein profiling using LC-Orbitrap-HRMS......................................................115 Proteomic alteration induced by abamectin exposure........................................120 Enrichment analysis...........................................................................................186 Conclusions......................................................................................................199 References.............................................................................................................200 초록...............................................................................................................212Docto

A Study on Relation among Perceived Value, Attitude, and Behavior Intention According to Yes or No Recognition of Native Local Foods" Storytelling

openAbstract Ⅰ. 서론 Ⅱ. 이론적 배경 Ⅲ. 연구방법 Ⅳ. 실증분석결과 Ⅴ. 결론 및 시사점 참고문