Development of bioassays for the multiplex biomarker analyses using UV-crosslinkable hydrogels

최웅선 Graduate School, Korea University 2022 국내박사

Today, as of 2021, we are experiencing various inconveniences and difficulties from a life that has changed so much from the past in the pandemic era by COVID-19. Meanwhile, as diagnostic methods such as PCR for COVID-19 testing have become widely known, interest in the field of molecular diagnosis has emerged socially. The most important thing in molecular diagnosis is proper biomarker selection for disease and condition and accurate detection of biomarkers through bioassays. Bioassay can be primarily divided into solution-phase and solid-phase according to the state of a probe. Solution-phase bioassays are relatively easy to detect biomarkers because they have the same phase as most biomarkers. However, there are limitations for solution-phase bioassays from the perspective of multiplexing capacity. On the other hand, solid-phase bioassays are accessible to multiplex but have a limitation of having low sensitivity due to low probe density. Therefore, in this study, the goal was to develop bioassays that can simultaneously have the strengths of the solution-phase and the solid-phase using a substance called hydrogel. Based on the solution-like nature of hydrogels, which are mostly composed of water, and the method of integrating probes capable of detecting biomarkers into the 3D structure of hydrogels, this study presents methods for detecting various biomarkers in a multiplexed manner as sensitive as solution-phase bioassays. To this end, we utilized polyethylene glycol diacrylate (PEGDA), which is bio-friendly and capable of photocrosslinking by UV, and wide use of hydrogels was also suggested through various photocrosslinking conditions. First, Chapter 1 introduces the introduction of biomarker and bioassay and the motivation for conducting this study. Chapter 2 discusses simultaneous multiple genotyping of single nucleotide polymorphism (SNP) using hydrogel microparticles. In Chapter 3, we will discuss the simultaneous multiplexed detection of miRNAs associated with Alzheimer's disease using hydrogel microposts. In Chapter 4, we will develop a 3D-based electrochemical sensor that is more advanced than a 2D-based electrochemical sensor using hydrogel scaffold and present multiple simultaneous detection of proteins related to Alzheimer's disease. Finally, Chapter 5 summarizes the developed hydrogel-based bioassays and describes the directions that bioassays should pursue.

Genetics and Bioassay of Rice Allelopathic Potential against Barnyardgrass (Echinochloa crus-galli (L.) Beauv.) : 벼 품종의 피(Echinochloa crus-galli (L.) Beauv.)에 대한 알레로파시 성능의 검정법과 유전연구

쥬나에디 건국대학교 대학원 2008 국내박사

A series of research has been conducted to study allelopathic potential in rice (Oryza sativa L.). These researches encompass three topics, i.e.: (1) Impro-vement bioassay for rice allelopathic potential against barnyardgrass (Echinochloa crus-galli (L.) Beauv.) assessing in laboratory and field; (2) Genetic study by diallel analysis of rice allelopathic potential against barnyardgrass; and (3) Differentially expressed genes related with allelopathic potential in rice as response against barnyardgrass. The objective which relates with the topic as follows: (1) To develop and evaluate more appropriate bioassay method in the laboratory and field, especially for breeding purposes; (2) To understand genetic information of rice allelopathic character that would be important in the breeding program; (3) To explore information in the molecular aspect of genes expression in the rice allelopathic potential varieties. From these studies, the results may contribute for more understanding of allelopathy in rice and to make it closer for implementing allelopathic potential rice cultivar in practical cultivation. 1. Bioassay Improvement of Rice Allelopathic Potential against Barnyardgrass. Screening methods that can evaluate more exactly the allelopathic potential are necessary to develop rice cultivars as one option for sustainable weed management. This study was performed to evaluate new laboratory bioassay, the Double Pots Allelopathy Bioassay (DPAB) and rice-mixture exudates bioassay, and a new field bioassay, the Rice Ratoon Interplanting Barnyardgrass Seedling (RRIBS) bioassay. In the DPAB barnyardgrass (Echinochloa crus-galli (L.) Beauv. var. frumentaceae) was used as a plant indicator and they were grown for 2 wk in soil media co-planting with rice after ratooning at 4 wk after seeding. In RRIBS bioassay, 2 wk barnyardgrass seedling was transplanted nearby a hill of rice that was ratooned at 5 wk after transplanting. Rice-mixture exudates were tested on the growth of radish (Raphanus sativus L.) and barnyardgrass. The DPAB and RRIBS bioassay technically performed some advantages and both bioassays could determine allelopathic potential among tested rice accessions. The DPAB and RRIBS bioassay showed significant correlation in assessment of eight rice accessions, but it was not significant in assessment 180 Recombinant Inbred Lines (RILs). The RRIBS bioassay could minimize the competition factors between rice and barnyardgrass. The rice-mixture exudates bioassay using radish as test-plant could determine rice allelopathic potential significantly, but using barnyardgrass as test plant was not significant. The RRIBS bioassay may prove useful for assessment rice allelopathic potential in field condition especially for the breeding program, in which numerous lines and limited seed numbers have to be assessed. Some prospective allelopathic rice lines have been selected from the RILs of Nongan/Sathi cross. 2. Diallel Analysis of Rice Allelopathic Potential against Barnyardgrass. A complete diallel cross was performed with six parents, viz.: Dular and Nongan that were considered as low allelopathic potential, Sathi, IR50363, Yamayutaka and Cica4 that were considered as high allelopathic potential against barnyardgrass. Parents and F1 of 30 crosses were assessed for allelophatic potential using Rice Ratoon Interplanting Barnyardgrass Seedling bioassay in field condition. Barnyardgrass was used for rice allelopathic potential indicator which was observed in their height and shoot dry weight, and suppression percentages were calculated by comparing to barnyardgrass grown in control plot (no rice). Results found that general combining ability showed no significant effect, whereas specific combining ability and reciprocal effect showed significant. Variance analysis confirmed that genetic control of allelopathic potential in rice against barnyardgrass was mostly directed by dominance gene effect. The direction of dominance effect was dominant gene associated with high allelopathic potential expression and recessive gene associated with low allelopathic potential. Heritability in broad sense for barnyardgrass dry weight suppression was around 41%; whereas for barnyardgrass height suppression was 51%. However, heritability in narrow sense was very low. The finding suggests that in reference with the specific combining ability of cross combinations, accumulation of genes associated with allelopathic potential would be a strategy to develop promising varietes. 3. Differentially Expressed Genes of Potentially Allelopathic Rice in Response against Barnyardgrass. Differentially expressed gene(s) (DEG) was identified in a rice variety, Sathi, an indica rice showed high allelopathic potential against barnyardgrass. Rice plants were grown with and without barnyardgrass and total RNA was extracted from rice leaves at 45 days after seeding. DEG full-screening was performed by GeneFishingTM method. The differentially expressed bands were re-amplified and sequenced. The nucleotides sequence was analyzed by BLAST searching for homology sequence identification. Gel electrophoresis of GeneFishing showed 9 possibility genes associated with allelopathic potential in Sathi. Among these, six genes namely DEG-1, 4, 5, 7, 8 and 9 showed more expressed, and three genes namely DEG-2, 3 and 6 showed less expressed. cDNA sequences showed that DEG-7 and 9 has same sequence. BLAST searching resulted that DEG-1, 2, 5, 6, 7 and 8 has homology with nr blast-n, DEG-3 has high significant similarity with nr blast-n, and DEG-4 has similarity both in nr and EST blast-n. It is suggested from blast-x and blast-n results that DEG-1 is gene for S-adenosylmethionine synthetase (SAMS), DEG-2 is a chloroplast gene for ribulose 1,5-bisphosphate carboxylase large subunit, DEG-6 is the nicotineamine aminotransferase, DEG-3 is similar to oxygen evolving complex protein in photosystem II, DEG-5 may relates with histone 2B protein, DEG-7 may relate with putative serin/threonine protein kinase and putative transposable element, DEG-8 may relate with oxysterol-binding protein, and DEG-4 is unknown protein. In another study, rice-60k oligomicroarray was performed to Sathi and Nongan varieties treated with and without barnyardgrass. Expression profiling summary showed that number of significant spots was higher in Nongan than Sathi, 51237 compare with 20959 spots; number of significant spots induced with barnyardgrass that expressed more than two fold was higher in Sathi than Nongan, 98 compare with 34 spots; and number of significantly repressed spots that expressed less than two fold was lower in Sathi than Nongan, 13 compare with 170 spots. Genes for information storage and processing, cellular processing and signaling, and for metabolism were mostly higher expressed in Sathi, i.e. 24 genes up regulated and 4 genes down regulated; whereas in Nongan for the same cluster were mostly suppressed, i.e. 10 genes up regulated and 63 genes down regulated. Eight genes, one suggested as membrane protein and others unknown function, identified both in Sathi and Nongan. Among these, two genes showed up regulated and six genes showed down regulated both in Sathi and Nongan treated with barnyardgrass. 벼 (Oryza sativa L.)의 알레로파시 (타감작용) 성능을 연구하기 위한 일련의 과제를 수행하였다. 본 연구는 (1) 실험실과 포장에서 벼의 알레로파시 성능 분석을 위한 검정법 개선, (2)이면교배 분석을 통한 피에 대한 벼 알레로파시 성능의 유전적 연구 및 (3) 피에 대한 반응으로서 벼 알레로파시 성능과 연관되어 있는 유전자의 발현 차이의 세가지 주제를 가지고 수행되었다. 이와 같은 주제와 관련한 본 연구의 목적은 (1) 육종을 목표로 실험실 및 포장에서 활용할 수 있는 정확한 검정법의 평가 및 개선, (2) 육종 설계에서 중요한 벼의 알레로파시 특성에 대한 유전적 정보 이해 및 (3) 알레로파시 잠재성을 보유한 벼 품종에 있어서 유전자 발현의 분자수준에서의 정보 탐색으로 요약할 수 있다. 일련의 연구를 통해 벼의 알레로파시 특성을 더욱 이해하여 알레로파시 잠재성을 보유한 벼 품종을 실질적인 경작에 적용하기 위한 좀더 구체적인 접근을 할 수 있을 것으로 기대한다. 1. 벼 품종의 피에 대안 알레로파시 성능에 검정법 개선. 정확한 알레로파시 잠재성을 평가할 수 있는 선발방법은 지속가능한 잡초 관리를 위한 방안의 하나로 벼 품종을 육성하기 위해 요구되고 있다. 본 연구는의 새로운 검정법 (이중 포트 알레로파시 검정법 (DPAB) 및 벼 혼합 삼출액 검정법)과 포장 수준에서의 새로운 검정법 (벼 라툰-피 혼합 육묘 (RRIBS) 검정법)에 대한 평가를 하고자 수행되었다. DPAB법에서 피 (Echinochloa crus-galli (L.) Beauv. var. frumentaceae)는 단일 식물 지표로 사용되었는 바, 파종 후 4주째에 라투닝 (ratooning)을 실시한 벼와 함께 토양 배지에 심어 2주간 생육시켰다. RRIBS법에서는 2주간 생육시킨 피 묘를 이식 후 5주째에 라투닝한 벼가 자라고 있는 근처에 이식하였다. 벼 혼합 삼출액은 무 (Raphanus sativus L.)의 생장에 대해 시험되었다. DPAB법과 RRIBS법 기술적인 편리성을 보였으며, 두 분석법 모두 공시한 벼 계통간의 알레로파시 잠재성을 측정할 수 있었다. DPAB법과 RRIBS법간의 상관은 8개 벼 계통에 있어서 유의한 것으로 나타난 반면, 180개 유전자 재조합 계통 (RILs)에서는 유의하지 않았다. RRIBS법은 벼와 피간의 경합요인들을 최소화할 수 있었다. 실험대상으로 무를 이용한 벼 혼합 삼출액 분석법은 벼 알레로파시 잠재성을 유의적으로 측정할 수 있었으나 피를 대상으로 실험한 결과에서는 유의하지 않았다. RRIBS법은 많은 계통과 한정된 종자수를 갖고 분석해야 하는 포장 수준에서의 육종 프로그램에서 벼 알레로파시 잠재성을 분석하는데 유용한 것으로 판명되었다. 농안 (Nongan) /사티 (Sathi) 교배 계통으로부터 몇몇 유용한 알레로파시 계통을 얻었다. 2. 피에 대한 벼의 알레로파시 잠재성에 대한 이면분석. 알레로파시 잠재성이 낮은 것으로 간주되는 Dular, 농안과 피에 대해 알레로파시 잠재성이 높은 것으로 알려진 사티, IR50363, 야마유타카, Cica4 등 6종의 교배친을 이용하여 완전 이면교배를 실시하였다. 교배친과 30개의 1대 교배계통에 대하여 포장에서 벼 라툰-피 혼합 육묘법을 이용하여 알레로파시 잠재성을 분석하였다.

Quorum sensing 저해물질 검색법 개발

김영희 부산대학교 대학원 2002 국내석사

Biofilms are found in many places in nature including the human body and are of great concern because they can result in virulence by the infected microorganisms or cause environmental hazards. They are known to be formed through a unique mechanism called "quorum sensing" which involves membrane-permeant signaling molecules, AHLs (acylated homoserine lactones). The cells in biofilms, are more resistant to antibiotics than their free-living counterpart. de Nys et al. have recently isolated halogenated furanone analogues having antifouling activity from the red algae, Delisea pulchra, that were chemically similar to the AHLs. The isolation of these analogues have prompted research in the discovery of mechanism based quorum sensing inhibitors from natural as well as synthetic products. However, the absence of a sensitive bioassay has hindered the screening of active compounds, since bioactive secondary metabolites are usually present at very low concentrations. The aim of this research was therefore to establish simple, effective and reproducible bioassay systems to detect AHL analogues. A plate bioassay system employing the cross-feeding assay by Fuqua et al using an AHL-responsive A. tumefaciens strain has been developed to identify new AHL-like compounds. By varying the X-gal concentration, incubation time and the sample loading procedure, it was possible to detect low level AHLs up to 10 nM concentrations. The length of the acyl chain of the AHLs was found to affect the sensitivity of this bioassay. The activity was observed to increase when the carbon number in AHLs increased up to 8~12 but decreased with further increase in carbon length. This implies that the binding affinity of AHLs is affected by their lipophilicity A luminometry assay system employing the quorum sensing bacteria V. harveyi was explored for detecting AHL analogues. This system could detect AHLs up to 102nM. The sensitivity of this system seemed to be less than that of the plate bioassay but it was interesting to note that inhibition of luminescence activity was observed by adding AHLs to V. harveyi. This suggests that the AHLs could be quorum sensing inhibitors by competing with the AI-1 in a cell density-dependent regulatory system-1. Moreover, unlike the plate bioassay which could serve as a universal assay system for detection of AHL analogues (whether they are agonists or antagonists), the luminometry assay with V. harveyi seems to be a more specific system. However, similar to the plate bioassay, lipophilicity seemed to affect the binding affinity of AHLs to the receptor. The universal plate bioassay system with A. tumefaciens was also modified into a liquid culture assay system. This system would give more quantitative results by measuring the absorbance intensity caused by the blue color response which is generated from the lacZ gene expression of reporter strain activated by the AHL analogues. AHLs could be detected up to 10-1nM, which makes this the most sensitive system developed so far. Several natural products were fractionated and tested in the above systems. The marine sponge, Petrosia, which has been known to possess antifouling activities did not show activity as a quorum sensing inhibitor which suggests that its activity may have been due to its cytotoxic activity. The significance of this study was in developing sensitive bioassay systems for quorum sensing inhibitors from natural and synthetic products.

환경시료의 생태 독성평가를 위한 Microtox-물벼룩 Tandem Bioassay

류성민 仁濟大學校 大學院 2003 국내석사

Daphnia magna를 이용한 생물학적 독성 시험법은 오염된 수층을 평가하는 대표적인 시험법으로 어독성 시험법보다 간단하고 신속하기 때문에 환경 독성 평가시 많이 이용되고 있는 방법이며, 독성물질에 민감할 뿐 아니라 혼합물의 독성상호작용을 인지하는데 효과가 뛰어나다. 또한 Microtox 발광성 미생물은 중금속과 polychlorinated byphenyls(PCBs) 등의 유기 화합 물질류에 민감하게 반응하기 때문에 유해 화학 물질로 오염된 퇴적물의 생물학적 독성 평가에 많이 이용되고 있다. 본 연구에서는 이같은 각 bioassays의 특성을 이용하여 환경독성 검색시 상호 보완적이 될 수 있는 두가지 기법을 병행하는 Tandem bioassay를 확립하였다. 아울러, 수계의 오염원인 물질 중 중금속, IGRs 그리고 호소 퇴적물에 대한 생태학적 독성을 평가하기 위해 D. magna-Microtox Tandem Bioassay를 적용하였다. 중금속에 대한 D. magma의 급성 독성 수준은 Cu>Cd)Se>Zn>As 순으로, Microtox는 Zn>Cu>Cd>Se>As의 순서로 독성이 높았다. IGR농약의 독성 수준은 .D. magna와 Microtox 시험에서 비슷한 경향성을 보였다. 또한, IGRS의 D. magna의 급성 독성 수준은 IGRS가 함유된 살충제에 비해 낮았다. 이같은 현상은 원제의 독성뿐만 아니라 살충제에 함유되는 부재료의 독성이 부가된 결과라 사료된다. IGRS는 급성 독성 시험시 무영향 농도 범위에서도 .D. magna의 번식에 영향을 주는 것으로 나타났다. 특히 살충제인 인쎄가의 경우, 번식독성과 함께 어린 개체의 기형 유발 효과까지 관측되었다. 이밖에 퇴적물의 공극수의 일반수질평가와 호소퇴적물의 독성과는 상관성이 없었다. 소양호 및 한강수계 정점의 퇴적물의 급성 독성 수준은 D. magna와 Microtox에 따라 다르며, 이것은 각 정점마다 미지의 물질이 다르게 있으며 이는 곧, 생물체에 미치는 독성이 다르다는 것을 의미한다. 일련의 연구들을 통하여 Daphnia magna 와 Microtox 기법에 의한 생물학적 독성 평가는 적은 비용으로 신속하게 오염도를 파악하는데 좋은 방법이며, 더욱이 D. magna의 만성독성 시험기법은 잠재적인 독성수준을 예측할 수 있는데 좋은 자료가 될 수 있다. 또한 D. magna-Microtox Tandem Bioassay의 활용은 환경시료의 건강성을 평가하기 위한 기초 자료로 유용하게 사용할 수 있을 것이라 사료된다. The purpose of this study was to develop and evaluate the usefulness of the Daphnia-Microtox Tandem Bioassay as a screening tool for toxicity testing of environmental pollutants. This tandem bioassay was composed with the Microtox test which uses the marine bacteria V. fischeri and Daphnia magna acute/chronic toxicity test. Daphnia magna has been found to be very sensitive species to the presence of a large number of chemicals in the environment and more conveniently used for toxicity testing compared to fish. Microtox^(�) is now one of the most widely used microbial tests and has been shown to be sensitive to a wide range of chemicals as well as environmental matrix. Daphnia-Microtox Tandem Bioassay was conducted to evaluate the environmental toxicity of heavy metals, IGRs and freshwater sediment. The acute toxicity (48-h immobilization) of heavy metals to the D. mgna, in decreasing order, was as follow : Cu>Cd>Se>Zn>As and Microtox was : Zn>Cu>Cd>Se>As. The IGRs toxicity to the D. mgna and K fisheri seemed to be quite correlated with each other. For the comparison of results obtained with active ingredients of IGRs and commercial products (INSSEGA^(�), CASCAEID^(�) and ATABRON^(�)) using D. magna, commercial products was significantly more toxic than the respective active ingredients. The results obtained until now suggest that insecticide adjuvants may affect partly to the toxicity of commercial products. Even though the EC_(50) (immobilization) of INSSEGA^(�) was quite low compared to order insecticides, it showed higher effects to the reproduction of D. magna and the malformation of juveniles. The sediment toxicity by D. mgna and V. fischeri was different at all sites and there is no relevancy between the chemical analyses(pH, TDS and Conductivity) of the porewater and sediment toxicity. It means that each site has different specific compounds responsible for different toxicity. According to these results, we suggest that Daphnia-Microtox Tandem Bioassay can serve as rapid screening and comprehensive test for detecting environmental toxicity of pollutant and a freshwater sediment. Also this tandem bioassay may be a useful tool to evaluate toxicity with relative low cost, rapidity and simplicity compared with the traditional ecotoxicity tests.

CALUX bioassay as a highthroughput screening method for dioxins

정영희 이화여자대학교 대학원 2004 국내박사

다이옥신류 화합물은 환경 중에서 잘 분해되지 않고 잔류성이 강하며 먹이사슬을 통해 축적되어 최종적으로는 인간을 비롯한 생명체에 악영향을 미치는 환경오염물질이다. 또한, 이들 물질은 발암성을 지니고 있고, 급성, 만성, 면역, 유전 등 폭넓은 분야에 강한 독성을 나타내고 있어 관심이 매우 높은 물질 중의 하나이다. 현재 다이옥신류 화합물의 분석은 고분해능 가스크로마토그래피/질량분석법(HRGC/MS)을 이용하여 이루어지고 있는 실정이다. 그러나 이 분석방법은 복잡한 시료의 전처리 과정과 고가의 기기, 숙련된 전문 인력을 필요로 하기 때문에 많은 시간, 노력, 비용을 요구한다. 또한 각각의 이성질체를 분석하여 여기에 다이옥신에 대한 상대적 독성치(TEF, Toxic Equivalent Factor)를 곱해 다이옥신 등가값(TEQ, Toxic Equivalency)으로 환산해야 하므로 이 과정에서 누락되는 다이옥신류 화합물이 있을 수 있어 실제와 차이가 날 수 있다. 따라서, 최근에는 이를 대체할 수 있는 대량검색(highthroughput screening) 방법으로써 다양한 바이오 분석법이 개발되고 있다. 본 연구에서는 CALUX 바이오분석법의 적용성을 연구하고자 한국의 토양 및 수질 환경시료를 대상으로 HRGC/MS를 이용한 화학분석법과 CALUX법을 이용한 바이오분석법을 활용하여 다이옥신류 화합물을 분석하였다. 더 나아가 현재 화학분석법에서의 분석 대상인 17종의 PCDD/PCDF 이성체에 대하여 CALUX법으로부터 도출된 상대효능치(REP value)와 WHO에서 지정한 TEF 수치를 비교하여 보았다. 또한 두 가지 분석법의 결과를 비교함으로써 다이옥신류 화합물 오염의 초기검색 방법으로써의 CALUX법의 활용 가능성을 평가하였다. 먼저 HRGC/MS분석법으로 환경시료중의 다이옥신류 화합물의 농도 및 이성질체 분포특성을 조사한 결과를 살펴보면, 물 시료의 경우, HRGC/MS-TEQ는 전체적으로 0∼2.281 pg-TEQ/L의 범위로 나타났고 가장 높은 수치를 보인 시료를 제외한 나머지 물 시료의 TEQ 수치는 1 pg-TEQ/L 이하였다. 토양 시료는 0∼65.6 pg-TEQ/g (dry) 범위의 TEQ 수치를 나타내었으며, 과거에 근처에 제련소가 위치했던 마서 지역에서 가장 높은 TEQ 수치(65.6 pg-TEQ/g (dry))를 나타냈다. 다이옥신류 화합물의 이성질체 분포특성을 살펴보면, 물과 토양 시료 모두에서 OCDD가 주요 이성질체인 것으로 나타났다. 또한 가장 높은 수치를 보였던 물 시료의 경우, HpCDFs와 HxCDFs가 주된 이성질체로 포함되어 있었다. 두 번째로 CALUX법을 이용하여 환경시료의 다이옥신성 활성(dioxin-like activity)을 측정하였다. CALUX-TEQ 수치를 살펴보면 물 시료의 경우 0.7∼18 pg-TEQ/L, 토양 시료의 경우 0.8∼650 pg-TEQ/g (dry)의 범위로 나타났다. 대부분 물 시료의 다이옥신성 활성은 미미한 것으로 나타났으나, 토양 시료의 경우 현저한 다이옥신성 활성을 나타냈다. 이상의 두 가지 분석법으로부터 얻어진 TEQ 수치를 비교해 보았을 때, HRGC/MS-TEQ와 CALUX-TEQ는 매우 높은 상관성을 보였다(r^(2)=0.982). 그러나 그 절대적인 수치를 비교해보면 CALUX-TEQ 수치가 HRGC/MS-TEQ 수치에 비해 10배 이상 높은 것으로 나타났다. 그 이유를 살펴보기 위해, HRGC/MS 분석법의 대상인 17종의 다이옥신류 화합물에 대하여 CALUX법으로 REP값을 구해보았다. 그 결과, 다이옥신류 화합물의 구조에서 염소기의 수가 많을수록 REP 수치가 WHO-TEF 수치보다 높게 나타났다. EC_(50)에서의 REP값에 근거하여 TEQ 값을 계산한 결과(REP-TEQ), HRGC/MS-TEQ와 CALUX-TEQ 모두와 높은 상관성을 보였다. 절대적인 수치에 있어서 HRGC/MS-TEQ와 REP-TEQ는 비슷하게 나타난 데 반해, CALUX-TEQ값은 REP-TEQ값보다 10배 정도 높게 나타났다. 결론적으로, CALUX 바이오 분석법은 환경매체중의 다이옥신류 화합물을 측정하기 위한 초기검색 도구로써, 다량의 시료를 신속하고 민감하게 검색할 수 있는 "early warning system"으로 적극 활용될 수 있을 것으로 사료된다. CALUX 분석에서 양성으로 판정된 시료에 대해서는 화학분석법을 이용하여 오염원인 물질의 종류 및 농도를 규명하는 연구가 계속 수행되어지면 좀더 과학적이고 신뢰 할 수 있는 결과를 얻을 수 있을 것으로 생각된다. Dioxin-like compounds are ubiquitous environmental pollutants that could be accumulated in biological system through the food chain. These chemicals have been identified worldwide and can have a significant impact on the human health and well being of human and wildlife. Given these issues, the detection and quantification of these chemicals in biological, environmental, and food samples is of paramount importance. However, a typical strategy in testing is time-consuming and expensive, due to exhaustive clean up procedure to remove all the interference and the use of expensive instruments. Two different methods, HRGC/MS chemical analysis and CALUX bioassay were used for testing environmental water and soil samples if CALUX bioassay is applicable for the measurement of dioxin-like compounds. Furthermore, REPs for 17 kinds of dioxin-like compounds were determined and compared with WHO-TEFs of corresponding compounds. i) In the water samples, HRGC/MS-TEQs ranged from 0 to 2.281 pg-TEQ/L, and the highest TEQ value was 2.281 pg-TEQ/L. All samples except one sample showed total-TEQ values below the 1 pg-TEQ/L. HRGC/MS-TEQ values of soil samples was in a range of 0∼65.6 pg-TEQ/g (dry) and the highest TEQ value was found in Maseo, where the refinery used to be near. OCDD, which has relatively low TEF value, was predominant congener in both water and soil samples. Contribution of OCDD is much higher in soil samples (92%) than in water samples (50%). In the water samples, congener distribution was the order of OCDD, HpCDFs, HpCDDs, HxCDFs and OCDF. However, in the heavily contaminated sample, the major congeners were HpCDFs and HxCDFs. In soil samples, OCDD was the major congener observed in almost of soil samples and followed by HpCDDs, HpCDFs and OCDF. ii) All of the water samples showed minimal effect on the luciferase activity. However, most of the soil samples caused significant luciferase induction. CALUX-TEQs of water samples ranged from 0.7 to 18 pg-TEQ/L and CALUX-TEQs of soil samples ranged from 0.6 to 650 pg-TEQ/g (dry). iii) The correlation between CALUX-TEQs and HRGC/MS-TEQs was very high (r^(2)=0.982). However, the absolute values obtained by CALUX bioassay were always higher than that by HRGC/MS. To interpret this data, the REP values for 17 kinds of dioxin-like compounds were determined by CALUX bioassay. And REP values were compared with WHO-TEF values. As increase the numbers of chlorine in PCDD/PCDF chemical structures, REP values become higher than WHO-TEF values. iv) The correlation between estimated REP-TEQs and HRGC/MS-TEQs turned out to be very high (r^(2)=0.998) and the absolute values obtained by the analysis of HRGC/MS and estimated based on REP EC_(50) were appeared to be very similar. And also, there were good correlation (r^(2)=0.970) between estimated REP-TEQs and CALUX-TEQs. However, the absolute TEQ values obtained by CALUX bioassay were always about ten times higher than the estimated REP-TEQ values. Thus, the CALUX bioassay can be highly recommended as an "early warning system" for routine measurement of dioxin-like compounds in environmental matrices. CALUX bioassay could be used as a screening tool for routine measurement of dioxin-like compounds in environmental matrices because it has great deal of advantages; that is available with smaller sample volume, smaller finance cost, and shorter testing time.

파래를 이용한 하천유입 오, 폐수내 독성물질의 생물진단에 관한 연구

장남 인천대학교 대학원 2009 국내석사

The present study is a preliminary investigation of application of Ulva bioassay to test various toxic compounds. In this study, several different water samples were studied including a) wastewaters obtained from a rubber industry (Seehwa Industrial Complex of Incheon), plating industry (Namdong Industrial Complex of Incheon), and YangJu wastewater (YangJu city of Gyeonggi-do); b) stream water from five streams in Incheon viz. Gongchon, Jangsu, Hagik and Gyesan stream; and c) seawater in the YeanAn. The aim of this study was to find a new effortless approach to detect and deal with water pollution. Pollutants have a dramatic ecological impact on aquatic environment and in particular the dwelling flora and fauna. Limited resources are available to predict the impact of pollutants based on their effect on living organisms. This study involves a bioassay using green marine macroalga, Ulva pertusa. The parameters tested here include growth, reproduction, chlorophyll a and chlorophyll fluorescence. The least significant difference (LSD) of the data obtained was also estimated in order to interpret the results. Chlorophyll a / fluorescence and growth rate / reproduction proved to be suitable end points for estimating the toxicity of rubber and plating effluents respectively using Ulva bioassay. The waters collected from the five streams, were tested using traditional chemical methods as well as the Ulva bioassay. The chemical analysis (BOD, COD, T-N, T-P and SS) carried out according to the Water Quality Standard-River and Water Quality Standard-Lake and the Ulva bioassay yielded similar results. Both the assays showed that Gulpo and Hagik Streams were most polluted. A comparative study of standard seawater and natural seawater was also conducted which revealed the presence of pollution in the natural seawater sample. Based on the results stated herein, it can be concluded that this bioassay method is more simple, inexpensive and sensitive as compared to the chemical counterpart. Thus it can be used as a replacement of the several laborious chemical experiments. It doesn't cause secondary pollution. The fact that Ulva pertusa is distributed throughout the world, indicates the potential use of this bioassay. 본 연구는 한국국내산 파래(Ulva pertusa)의 생리적 특성(면적 증가율, 생중량, 엽록소 형광, 색소)을 이용하여 인천 관내 하천유입 오, 폐수, 해수의 독성물질을 평가하고 가능성을 수행되었다. 연구표본은 폐수(고무수, 도금수, 양주폐수), 하천수, 바닷물로 나뉜다. 여기에서 a)고무수는 시화공단, 도금수는 남동공단, 양주폐수는 양주종말폐수처리장에서 채집하였으며 b)하천수는 공촌천, 장수천, 학익천, 계산천, 굴포천을 이용하였고 c)바닷물은 연안해수를 이용하였다. 본 연구의 목적은 오수(오염물질)에 대한 새로운 측정방법과 해결방법에 대해 분석하는 것으로서 이러한 오염물질은 이미 우리의 수자원환경에 대해 아주 큰 영향을 주고 있으며 특히 부근의 동식물에 대한 영향도 아주 크다. 본 연구에서는 생물체의 본질적인 특성을 이용하여 주변의 오염물질을 측정하였다. 즉 본 연구는 파래를 이용한 생물적 분석방법을 통하여 면적 증가율, 생 중량, 엽록소, 형광, 색소 등 데이터로 LSD 통계학적 방법을 이용하여 분석하였다. 이러한 실험은 아주 실용적이며 도금수, 고무수의 독성을 측정하는데도 아주 유효한 것이다. 논문에 언급되는 하천수 연구는 전통적인 화학적 분석방법과 선진적인 생물적 분석방법을 이용하였다. 화학적 분석방법은 BOD, COD, T-N, T-P, SS의 기초에서 진행되었고 하천수의 수질 기준에 근거하여 분석한 결과, 생물적 분석방법을 통하여 얻은 결과와 거의 비슷하였다. 즉 굴포천, 학익천은 분석결과 오염정도가 가장 높은 하천에 속하였다. 다음, 자연해수와 실험실의 인공해수를 비교분석한 결과 자연해수의 오염정도가 더욱 높았다. 결론적으로, 실험결과에서 알 수 있듯이 생물적 검정법과 화학적 검정법을 비교하여 보았을 때, 생물검정법이 보다 간편하고 저렴하며 결과에도 민감하다. 때문에 생물적 검정법으로 화학적 실험방법을 대체할 수 있으며 또한 화학적 실험방법으로 인한 2차 오염을 방지할 수 있다. 또 해조-파레의 세계적인 분포의 특성에 의해 이용 잠재력도 아주 크기 때문에 생물적 검정 법을 더욱 선호하는 바이다.

In-vitro bioassay를 이용한 소각잔사 혼입 매립지 침출수의 위해성 평가

이종삼 목포해양대학교 대학원 2007 국내석사

Leachate, generated from waste landfill during the process of waste decomposition, is very complex and various on its quality according to the types of buried waste, geological and hydraulic characteristics of the landfill, water content, landfill age, etc.. This study investigated a reverse mutation by Ames test and examined cytotoxicity by SRB assay (Sulforhodamine B) from leachates of 13 landfills which contain of municipal solid wastes (MSW) and incineration residues. And mutagenicity on the process water according to leachate treatment process was also investigated. For reverse mutation test, Salmonella typhimurium TA98 strain was inoculated into 25ml nutrient broth liquid, and cultivated in the slant medium for 10 hours at 37℃. Then test material and the cultivated liquid respectively were injected 0.1m1 into top agar, and completely mixed for 2-3 seconds with vortex shaker. Thereafter, the samples were coated in minimal glucose agar plates, were cultivated for 48 hours at 37℃, and then counted the number of revertants. The specimen which was over 2 times of the number of revertants cultivated DMSO (dimethylsulfoxide) solution used as standard material and increased with dependence on dose was turned out to be positive. TA98 used in the reverse mutation test has been received from Korea Research Institute of Chemical Technology, and genetic characteristics of TA98 has been confirmed using 2-aminoanthracene which is a standard material to check genetic characteristics of TA98 and existence of activation in S-9 fraction. Table 2 shows the number of revertants appearing in DMSO solution that was used as a negative comparison group and 2-aminoanthracene which is a positive comparison group. SRB assay has used Hep 3B which is a liver cancer cell of human being. In this assay, we made the number of cells 10^(4)cell/well, injected it into 96well plate, exchanged medium after 24 hours, and then injected test material in each well. And to fix the cells, we injects 100 ㎕/well of 10% TCA, and washed it with distilled water 5 times. After drying completely, we injected SRB (0.4%), dye, into each well, and then dyed it for 30 minutes at room temperature. After dyeing, the wells were washed with 1% acetic acid and dried completely. After drying, we injects 100 ㎕ of 10mM tris buffer into each well and then measures it with ELISA (565nm-690nm). As a result, in all samples of investigated concentration, reverse mutation effect increased depending on dosage and showed over 2.0 times which is a judgment criteria for positiveness by U.S. Environmental Protection Agency, so all investigated leachates were turned out to contain a reverse mutation- inducing substances. And cytotoxicity in all concentration dosed increased, meaning that all of leachates investigated were contained a cytotoxicity- inducing matter. From the correlation study between toxicity and general water quality items such as TOC, CODcr and BOD, the mutagenicity have a good correlation to CODcr. The mutagenicity using TA98 and TA 100 did not show a consistent tendency depending on landfill age, but showed a good inverse relationship to the ratio of incineration residues to total wastes, while the cytotoxicity seemed to decrease depending on landfill age. From the results of mutation tests using fractionation leachates according to solvent, the mutation effect was different depending on site as well as solvent. The mutation effect in a site (e.g. BS) appeared in the dichloromethane fraction containing hydrophobic substances with high molecular weight, while that in another site (e.g. CS) in methanol fraction containing a relatively hydrophilic substances with low molecular weight. From the results of mutation test about process water depending on leachate treatment process, mutagenicity-inducing substances were not removed as much as TOC, CODcr and BOD were removed. And the mutagenicity slightly increased after dosing a coagulant including metal ion such as aluminium (Al) or iron (Fe), which means the formation of mutagenicity- inducing substances after coagulation process. Finally, we could confirm that the leachates including process water investigated contain the substances inducing mutation and cytotoxicity and it seemed to be difficult to efficiently remove the substances in the current leachate treatment facility. Therefore, a specific regulation and management for those substances seem to be needed.

Establishment of bioassay- and molecular marker-based acaricide resistance monitoring protocols and elucidation of additional resistance factors in the Varroa mite, Varroa destructor

이준희 서울대학교 대학원 2023 국내석사

꿀벌응애는 꿀벌에 기생하는 외부 기생성 해충으로서 양봉 산업에 큰 피해를 입힌다. 꿀벌응애로 인한 피해를 줄이기 위해서 다양한 방제방법이 사용되는데, 그 중에서 편의성과 방제효율을 고려하여 합성 살비제 스트립 사용이 선호되고 있다. 하지만 이러한 합성 살비제의 과도한 사용은 저항성 문제를 야기하여 전세계적으로 꿀벌응애의 합성 살비제 저항성 발달에 따른 방제실패가 보고되고 있다. 저항성 발달을 관리하기 위해서는 지속적인 저항성 모니터링이 필요하나 국내에서는 지금까지 한차례의 저항성 모니터링만이 진행되었다. 이와 같이 꿀벌응애 저항성 모니터링이 빈번하게 진행되지 않는 이유로는 꿀벌응애의 채집과 생물검정 상의 어려움을 들 수 있다. 따라서 본 연구에서는 생물검정(잔류접촉바이알법)과 분자마커(정량적 염기서열 분석 방법)을 이용한 꿀벌응애 저항성 모니터링 방법을 개발하였고 이를 이용하여 전국에서 꿀벌응애 살비제 저항성 모니터링을 진행하였다. 잔류접촉바이알법을 이용한 저항성 모니터링 방법을 개발하기 앞서 감수성으로 추정되는 계통인 2021-SO 계통을 이용하여 LC 값을 구하였고 이를 진단농도로 사용하였다. 2021년에는 17지역에서 저항성 모니터링을 수행하였다. 17지역 중 2지역(2021-GR, 2021-UR)에서 쿠마포스와 플루발리네이트에 대한 사충률 감소가 관찰되었고, 3지역(2021-SJ2, 2021-DG, 그리고 2021-GJ)에서 플루발리네이트에 대한 사충률 감소가 관찰되었다. 2022년에는 42지역에서 저항성 모니터링을 수행하였는데, 이중 14지역에서 쿠마포스 저항성이 8지역에서 플루발리네이트에 대한 사충률 감소가 관찰되었다. 플루발리네이트 저항성의 경우 2021년보다 2022년에 관찰된 곳이 더 많았기 때문에 플루발리네이트 저항성이 한국에서 확산되고 있다고 생각할 수 있다. 정량적 염기서열 분석 방법을 이용하여 저항성 모니터링을 실시하기 전 실제 염기 비율과 염기서열 분석 결과를 보정할 예측식을 구하였다. 이렇게 구한 예측식을 이후에 수행한 저항성 모니터링 방법에 사용하였다. 2021년 17지역에서 분자마커 기반 저항성 모니터링을 실시하였고 4지역에서 플루발리네이트 저항성 돌연변이(L925I/M)가 검출되었다. 2022년에는 90지역에서 분자마커 기반 저항성 모니터링을 실시하였는데, 이중 83지역에서 플루발리네이트 저항성 돌연변이(L925I/M)가 검출되었다. 위 결과를 보면 한국에 플루발리네이트 저항성 돌연변이가 빠른 속도로 확산되고 있는 것을 알 수 있다. 또한 플루발리네이트에 의한 사충률과 저항성 돌연변이 빈도 사이의 연관성을 확인하였지만 연관이 없는 것으로 확인되었다. 플루발리네이트를 처리한 후 매시간마다 기절(knockdown)한 꿀벌응애에서 플루발리네이트 저항성 돌연변이 유전형 빈도를 비교하였다. 그 결과 7시간 이후에 기절한 꿀벌응애에서 저항성 동형접합 유전자형이 급격히 증가하였다. 이를 보아 RCV를 이용한 생물검정에서 감수성 개체와 저항성 개체를 구별하는데 초기 시점이 바람직하다는 것을 알 수 있었다. 또한 플루발리네이트 처리 후 7시간 뒤에 기절한 꿀벌응애에서도 감수성 동협접합 꿀벌응애가 검출되었는데, 이는 다른 저항성 인자의 존재 가능성을 암시한다. 따라서 플루발리네이트의 작용점인 전압개폐나트륨 채널에서 새로운 돌연변이를 확인한 결과, 총 10개의 새로운 돌연변이가 발견되었다. 또한 cytochrome P450(P450s)의 발현량을 확인하여 대사 인자로서 저항성과의 연관성을 확인하였다. 이를 위하여 qPCR을 위한 적합한 대조유전자(reference gene)를 먼저 조사한 결과, eEF1A1과 NADHD가 꿀벌응애에 있어서 살비제가 미치는 영향 비교 시 적합한 대조유전자로 선발되었다. P450의 발현량과 RCV를 통해 얻은 사충률 사이의 명백한 상관 관계가 없음에도 불구하고 2종의 P450s(Flv-2B4 및 Flv-3A19)는 발현량이 증가함에 따라 사충률이 감소하는 경향을 보여 플루발리네이트 저항성에 기여할 가능성을 암시하였다. The Varroa mite, Varroa destructor, is an ectoparasitic mite that is one of the major threats to apiculture. Acaricides, such as fluvalinate, coumaphos, and amitraz, have been used for the control of Varroa mites. However, the extensive use of acaricide has led to resistance development in field populations of Varroa mite. Resistance monitoring is essential in the management of pesticide resistance, but acaricide resistance monitoring for Varroa mites has rarely been conducted in Korea, perhaps due to the difficulties associated with mite collection and bioassay. In this study, bioassay (residual contact vial: RCV)- and molecular marker (mutations in voltage-gated sodium channel)-based acaricide resistance monitoring methods were developed, and acaricide resistance monitoring was conducted in Korea in two years (2021 and 2022). To establish the RCV bioassay protocol, the LC50 and LC90 values were determined for fluvalinate and coumaphos using the 2021-SO strain as a putative susceptible strain, and used as diagnostic concentrations. In 2021, acaricide resistance monitoring was conducted using LC90 as the diagnosis concentration for 14 regional mite populations. The 2021-GR and 2021-UR populations showed reduced mortalities to both coumaphos and fluvalinate, whereas the 2021-SJ2, 2021-DG, and 2021-GJ strains showed reduced mortalities to only fluvalinate, suggesting the possible development of resistance to fluvalinate and coumaphos. In 2022, acaricide resistance monitoring was conducted using LC50 as diagnosis concentration for 42 regional populations. Varroa mites from eight regions showed reduced mortalities to fluvalinate, suggesting that fluvalinate resistance began to spread in Korea. The molecular maker-based acaricide resistance monitoring method was developed using the quantitative sequencing protocol. In 2021, molecular marker-based acaricide resistance monitoring was conducted for mites collected from 17 regions. Fluvalinate resistance mutation (L925I/M) was detected in four out of 17 regions. The resistance monitoring conducted for 90 regional populations in 2022 revealed that fluvalinate resistance mutation was detected in 83 regions out of 90 regions, showing a rapid spread of resistance allele. In the correlation analysis between the 24-h mortality obtained from RCV and fluvalinate resistance mutation frequency, no apparent correlation was observed. The knockdown bioassay followed by genotyping revealed that only mites knocked down at an early time point (< 7 h post-treatment) possessed most of the susceptible homozygous and heterozygous genotypes at the L925I/M mutation site. This finding further implicated that the early time point is desirable for discriminating susceptible individuals from resistant ones in RCV bioassay. Intriguingly, the susceptible homozygous genotype was also detected in putatively resistant Varroa mites knocked down after 7 h post-fluvalinate treatment, implicating the possibility of other resistance factors. In order to identify other fluvalinate resistance factors, novel mutations were searched in the nearly entire domains of the voltage-gated sodium channel gene. As a result, ten novel mutations were identified, among which two appeared to have some potential function as a target site insensitivity factor. In addition, transcription levels of cytochrome P450 monooxygenase (P450s) were investigated to determine their possible association with resistance as metabolic factors. To this end, searching for reliable reference genes for quantitative PCR was attempted first. The eEF1A1 and NADHD were recommended as reference genes for the comparison of the effects of acaricide on the whole body. Despite the lack of apparent correlation between their expression level and the mortality obtained from RCV bioassay, two P450s (Flv-2B4 and Flv-3A19) showed a tendency of decrease in mortality as the P450 expression level increased, thus implying their possible role in fluvalinate detoxification.

벤조페논-3의 항안드로겐 영향에 관한 연구

김병준 창원대학교 2020 국내석사

벤조페논-3(BP-3, benzophenone-3 2-hydroxy-4-methoxybenzophenone, oxybenzone)는 지난 40년 이상 가장 널리 사용되는 벤조페논 유형의 유기 자외선 차단제로, 벤조페논-3의 환경 노출 영향에 대한 보고의 증가와 그에 따른 대중의 인식이 높아짐에 따라 잠재적으로 유해한 독성물질로 주목을 받고 있다. 본 연구는 벤조페논-3의 항안드로겐 영향을 알아보기 위하여 거세한 수컷 Spraque-Dawley 랫드에서 Hershberger bioassay를 진행하였고, 생식세포와 비생식세포간의 독성을 비교하기 위하여 MTT assay를 진행하였다. Hershberger bioassay에서 랫드는 6개의 투여군으로(n=7) 나누고, 투여군은 vehicle control(VC, corn oil 10 ml/kg/day), negative control(NC, testosterone propionate; TP 0.2 mg/kg/day), positive control (PC, flutamide; FT 3 mg/kg/day + TP 0.2 mg/kg/day), BP-3 저용량군(40 mg/kg/day + TP 0.2 mg/kg/day), BP-3 중용량군(200 mg/kg/day + TP 0.2 mg/kg/day), BP-3 고용량군(1000 mg/kg/day + TP 0.2 mg/kg/day)로 두었다. 용매와 FT 및 BP-3는 경구투여로 진행하였고, TP는 피하주사를 통해 투여하였으며, 시험은 총 10일간 진행되었다. MTT assay는 TM3(mouse leydig) cell과 NIH-3T3 cell(mouse fibroblast)에서 control(DMSO 1%), BP-3 62.5~500 μM 농도를 처리한 뒤 세포생존율을 측정하였다. Hershberger assay에서 VC와 PC를 제외한 모든 BP-3 투여군에서 glands penis(GP), ventral prostate(VP), seminal vesicle(SV), levator ani-bulbocavernosus muscle(LABC), cowper's glands(CG)의 무게 감소가 관찰되었다. 특히 VP 무게는 BP-3 200, 1000 mg/kg/day에서 통계적으로 유의한 감소가 관찰되었으며, LABC 무게는 40, 200, 1000 mg/kg/day에서 통계적으로 유의적인 감소가 관찰되었다. MTT assay에서 NIH-3T3 cell은 125 μM, 250μM 처리한 농도에서 컨트롤에 비해 4.5%, 6.8% 통계적으로 유의한 감소를 보였으나 농도의존적인 감소는 관찰되지 않았으며, TM3 cell 에서는 62.5~500 μM에서 각각 19.7%, 27.2%, 42.7%, 51.2%씩 통계적으로 유의한 감소를 관찰되었으며 LC50는 444.55 μM로 산출되었다. 결과를 종합해 보면, Hershberger bioassay에서 벤조페논-3은 200, 1000 mg/kg/day 용량에서 양성 안드로겐 길항제로 판단하였고, MTT assay에서는 비생식세포보다 생식세포에서 독성이 더 나타나며, 이와 같은 결과를 바탕으로 인체에 적용하였을 때, 인체에서 항안드로겐 활성을 나타낼 가능성이 있는 의심 물질로 판단된다. Dept, of Bio health science Graduate school of Changwon National University Benzophenone-3 (BP-3, 2-hydroxy-4-methoxybenzophenone, oxybenzone) is the most widely used benzophenone type organic sunscreen for over 40 years, increasing reports of BP-3's environmental exposure impacts and increasing public awareness have resulted in the attention of potentially harmful toxicants. In this study, we conducted Hershberger bioassay in castrated male Spraque-Dawley rats to investigate the anti-androgenic activities of BP-3, and MTT assay was performed to compare the toxicity between germ and non-germ cells. In the Hershberger bioassay, rats were divided into six groups (n = 7) including vehicle control(VC, corn oil 10 ml/kg/day), negative control(NC, testosterone propionate; TP 0.2 mg/kg/day), positive control (PC, flutamide; FT 3 mg/kg/day + TP 0.2 mg/kg/day), BP-3 low dose group(BP-3 40 mg/kg/day + TP 0.2 mg/kg/day), BP-3 medium dose group(BP-3 200 mg/kg/day + TP 0.2 mg/kg/day), BP-3 high dose group(BP-3 1000 mg/kg/day + TP 0.2 mg/kg/day). Solvent, FT and BP-3 were orally administered, and TP was administered by subcutaneous injection. The administration was conducted in 10 consecutive days. In MTT assay, cell viability was measured after treatment with control (DMSO 1%) and BP-3 62.5 ~ 500 μM concentration in TM3(mouse leydig) cells and NIH-3T3 cells(mouse fibroblast). In the Hershberger bioassay, weight of glands penis (GP), ventral prostate (VP), seminal vesicle (SV), levator ani-bulbocavernosus muscle (LABC), and cowper's glands (CG) were decreased in all BP-3 groups. In particular, VP weights were significantly decreased at dose of 200, 1000 mg/kg/day BP-3. In addition LABC weights were significantly decreased at dose of 40, 200, 1000 mg/kg/day BP-3. In MTT assay, NIH-3T3 cell showed a significant decrease of 4.5% and 6.8% at 125 μM and 250μM compared to the control, but did not show a concentration-dependent decrease. TM3 cells showed significant decreases of 19.7%, 27.2%, 42.7%, and 51.2% at 62.5 ~ 500 μM, respectively, and LC50 was calculated as 444.55 μM. In conclusion, BP-3 was considered as a positive androgen antagonist at dose of 200, 1000 mg/kg/day in Hershberger bioassay, and MTT assay showed more toxicity in germ cells than non-germ cells. Based on these result, when applied to the human body, BP-3 is considered to be suspected substance that may exhibit anti-androgenic activity in the human body.

Allelopathic potential evaluation and allelochemicals identification from Rumex acetosella : 애기수영(Rumex acetosella)의 allelopathic potential 평가 및 allelochemical 동정

강요셉 경북대학교 대학원 2023 국내석사

애기수영(Rumex acetosella)은 국내 목초지, 밭, 과수원과 같은 농경지에서 자생하는 외래 잡초이며, 생태계 교란종으로 지정되어 있다. 타 선행연구에서 애기수영으로부터 Catechol, Chrysophanic acid와 같은 살초활성물질을 분리 및 동정했지만, 이러한 연구들은 애기수영 전식물체를 이용한 실험이었기 때문에 애기수영 지상부, 지하부 각각을 분리해 식물에 미치는 영향을 확인 및 평가한 연구가 미비하다. 이에 본 연구는 애기수영 지상부, 지하부 분리 후 각각의 추출물이 토끼풀의 생육에 미치는 영향을 확인하고, 생육을 더 억제하는 추출물에 대해 분리 및 정제하여 조성을 확인하고자 한다. 실험이 사용된 애기수영 추출물은 지상부와 지하부로 분리 및 균질화 과정을 거쳐 methanol으로 추출 후 감압 농축하여 사용하였다. 애기수영 추출물을 이용해 토끼풀 종자에 대한 seed bioassay를 진행하였고, 토끼풀 유묘의 생체중에 대한 IC50을 측정하였다. Seed bioassay 결과, IC50값이 더 낮았던 애기수영 지상부 추출물을 이용해, 토끼풀에 경엽처리를 진행하였다. 경엽처리 한 토끼풀 식물체의 생육 및 엽록소 함량을 조사하였고, 샘플링 후 ROS, 항산화효소 활성, 식물호르몬 함량을 분석하였다. 생육 및 엽록소 함량 조사 결과, 추출물의 농도가 높아짐에 따라 토끼풀의 초장 및 생체중이 감소하였으며, 엽록소 함량 역시 감소하는 경향을 나타냈다. 또한, 처리 농도가 높아짐에 따라 O2- , H2O2 같은 ROS 함량 및 항산화효소인 SOD, CAT, POD 활성 역시 증가하였다. 식물 호르몬 분석 결과, 처리농도가 높아짐에 따라 ABA, SA 함량이 증가하였으며, JA 함량은 감소하였다. 애기수영 지상부 추출물에 대한 액-액 분배, TLC, Open column chromatography를 통해 분리 및 정제과정을 진행하였고, 분리된 층에 대한 seed bioassay 진행하였으며, 토끼풀 종자의 IC50이 가장 낮은 층에 대해 GC/MS 분석하였다. GC/MS 분석 결과, .gamma. -Sitosterol 이 가장 높은 함량이 보였다. 본 연구를 통해 애기수영 연구의 기초자료로 활용될 수 있을 것으로 기대된다. Red sorrel (Rumex acetosella) is an exotic weed that is found in agricultural lands such as domestic pastures, fields, and orchards and is designated as an invasive alien species. In other previous studies, herbicidal active substances such as Catechol and Chrysophanic acid were isolated and identified from R. acetosella. However, since the previous experiments were experiments using the whole plant of R. acetosella, there is a lack of research that has confirmed the effect on plants by separating each of the shoot and root parts of the R. acetosella. Therefore, this study is to investigate the effect of each extract on the growth of white clover (Trifolium repens) after separating the shoot and root parts of R. acetosella, and to isolate, purify, and identify the composition of the extract from the part that is more inhibitory to growth. The extract used in the experiment was separated into shoot and root parts, homogenized, extracted with methanol, and then concentrated under reduced pressure. In vitro seed bioassay was performed on T. repens seeds using the extract of R. acetosella, and the IC50 was measured for the fresh weight of T. repens seedlings. The foliar application was performed on T. repens using extracts of the shoot parts of R. acetosella, which had a lower IC50 value in the seed bioassay. The growth and chlorophyll content of foliar-treated clover plants were investigated, and after sampling, ROS content, antioxidant enzyme activity, and plant hormone content were analyzed. As a result of the growth and chlorophyll content investigation, as the concentration of the extract increased, the plant height and fresh weight of T. repens decreased, and the chlorophyll content also showed a tendency to decrease. In addition, the content of ROS such as O2- and H2O2 and the activity of antioxidant enzymes SOD, CAT, and POD also increased as the treatment concentration increased. Phytohormone analysis showed that ABA and SA content increased with increasing treatment concentration, while JA content decreased. Liquid-liquid extraction, Thin-layer chromatography, and open column chromatography were performed on the extracts of the shoot part of R. acetosella, and seed bioassay was performed on the separated layer. GC/MS analysis was performed on the layer with the lowest IC50 of T. repens seeds. GC/MS analysis results. gamma. -Sitosterol showed the highest content among all compositions. It is expected that this study can be used as basic data for R. acetosella research.