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Kafe 바로가기주관연구기관 | 한국화학연구원 Korea Research Institute of Chemical Technology |
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발행국가 | 대한민국 |
언어 | 한국어 |
발행년월 | 2002-01 |
주관부처 | 환경부 Ministry of Environment |
등록번호 | TRKO200200052606 |
DB 구축일자 | 2013-04-18 |
I. 제 목 환경오염물질 독성평가의 신기술 개발 (국내 화학물질의 유해성 시험 및 평 가기법 개발) II. 연구개발의 목적 및 필요성 국내 화학물질 관리체계를 선진화하고, 국제적인 화학물질 프로그램에 능동적으로 대처하기 위하여, ① 국내 화학물질의 유해성 평가에 선진적 개념을 도입하고, ② 국내 시험연구기관의 독성시험능력을 제고 하고, ③ 이를 통하여 유해화학물질로부터 우리의 환경과 국민 건강을 보호하고, OECD 회원국으로서 능동적인 입장을 확보하고자 한다. III. 연구개발의 내용 및 범위 1. 난용성
I. 제 목 환경오염물질 독성평가의 신기술 개발 (국내 화학물질의 유해성 시험 및 평 가기법 개발) II. 연구개발의 목적 및 필요성 국내 화학물질 관리체계를 선진화하고, 국제적인 화학물질 프로그램에 능동적으로 대처하기 위하여, ① 국내 화학물질의 유해성 평가에 선진적 개념을 도입하고, ② 국내 시험연구기관의 독성시험능력을 제고 하고, ③ 이를 통하여 유해화학물질로부터 우리의 환경과 국민 건강을 보호하고, OECD 회원국으로서 능동적인 입장을 확보하고자 한다. III. 연구개발의 내용 및 범위 1. 난용성 및 휘발성 물질의 유해성 시험방법 난용성물질 3종류(phenyl salicylate, 2,4-di-tert-butylphenol, dibenzoyl peroxide)에 대해서, 유수식으로 독성시험을 수행하고, 지수식으로 할 때와 결과를 비교하였다. 휘발성 물질 3종류(methyl acrylate, n-heptane, allyl methacrylate)에 대해서는 밀폐식과 노출식 장치에서 독성시험을 수행하고 결과를 비교하였다. 2. 수서생물을 이용한 독성시험법 연구 1) 어류 embryo yolk sac-fry 시험법 OECD에 등재된 시험법 212을 참고로, 본 연구는 송사리(Oryzias latipes)와 왜몰개(Aphyocypris chinensis)에 있어 수정란(embryo) 단계뿐만 아니라 자어(sac-fry) 단계에 대한 화학물질(zinc) 독성을 조사하였다. 따라서 본 연구는 어류의 각기 다른 두 생활사 단계에 대한 화학물질의 독성 영향을 알아보고, 각 단계별 사이에서 독성 영향의 민감성을 비교 분석하였다. 2) Lemna 독성시험법 OECD Test Guideline에 따라 Lemna독성시험을 할 수 있도록, Lemna gibba를 입수, 사육기술을 확립하고, 이 종을 이용하여 화학물질의 독성시험을 실시하였다. 이 결과를 이용하여, 확립된 기술이 재현성 있게 운용이 되는지를 평가하였고, 생물사육 및 독성시험에 대한 지침서를 제작하였다. 3) 화학물질의 퇴적물 독성시험법 국제적으로 많이 사용하는 Chironomus riparius에 대한 사육기술을 확립하고, 이종을 이용하여, sediment-water system using spiked sediment에서 독성시험하는 기술을 확립한다. 그리고 생물사육 및 독성시험에 대한 시험지침서를 작성하였다. 3 육상식물을 이용한 독성시험법 OECD Test Guideline 208에 따라 육상식물을 이용한 독성시험을 할 수 있도록, 3종의 식물종자와 두가지 시험물질(m-nitrobenzene, 3,4-dinitrotoluene)로 독성시험을 실시, 재현성과 기존자료와 비교하였다. 아울러 독성시험법에 대한 지침서를 작성하였다. 4. 수질· 토양에서의 화학물질 거동연구 1) 광분해 시험법 OECD guideline을 중심으로 비교한 결과, 인공광을 이용한 실제 광분해 시험시, system 적인 측면에서 광원, 필터, 용기 및 온도조절에 있어서 guideline의 기준에 맞추기가 어렵기 때문에 이러한 항목들에 대하여 연구하였다. 2) 토양대사 및 토양 미생물을 이용한 분해 시험법 최근까지의 문헌 검색과 US EPA, OECD guideline의 토양 대사 및 토양 미생물에 의한 분해 시험법을 비교 검토한 결과, OECD guideline을 중심으로 시험법을 확립하였다. 이때 방사능 표지 화합물을 이용하여 flow-through system내에서 토양 대사 시험과 토양 미생물에 의한 분해 시험을 병행하였다. 5. 환경오염물질의 진보된 독성평가 신기술 개발연구 1) 세포를 이용한 신속 정확한 in vitro 시험법 정립 연구 세포를 이용한 신속 정확한 in vitro 시험법으로서 single cell gel electrophoresis의 IWGTP의 논의에 따른 세포주 선정, 최적 lysis, unwinding time, 전기영동조건, 대사활성존재 및 부재하의 양성대조물질의 조건확립 및 cytokinesis blocking 소핵 시험법의 양성대조물질들의 조건 확립. 2) 유전자를 이용한 신속 정확한 진보된 독성 평가 기법 정립 유전자를 이용한 신속 정확한 독성평가기법으로 Thymidine kinase gene forward mutation (1차년도만 수행) 및 Transgenic mutagenesis system확립연구 - Single cell gel electrophoresis 기법 의 화학물질 독성평가에의 활용 및 응 용연구 - Cytokinesis blocking assay의 화학물질 독성평가에의 활용 및 응용연구 - Transgenic Cell Line을 이용한 lac I gene mutation 활용연구 6. 환경오염물질의 위해성 평가를 위한 조류독성 시험법 개발 본 연구는 1996년에 SETAC/OECD workshop에서 제안하고 있는 조류 독성 시험법 (OECD's Guidelines for the Testing of Chemicals, Monograph Series on Testing and Assessment, Report of the SETAC/OECD Workshop on Avian Toxicity Testing)을 근거로 하여 본 연구에서는 국내의 실정에 맞는 조류를 이용한 독성 물질의 평가 법을 확립하기 위해 메추리를 부화 사육하였고, 급성경구독성시험법과 식이성 독성시험법을 메추리, 꿩등의 조류에 수행하였다. 7. 화학물질의 최기형성 평가를 위한 FETAX 기법 연구 본 연구의 제 1 차년도에는 FETAX 기법의 확보 및 기술 전파를 위한 기반연구를 수행하였다. 주요 연구 내용으로는 시험생물의 사육, 수정란 채취, 그리고 정상적 배아의 선별 및 배양 조건의 확립 등이었다. 또한, 표준 시험 물질을 이용한 배아 사멸능, 기형 발생능을 연구 또는 조사하였다. 제 2 차년도에는 제 1 차년도에서 확보된 기법의 신뢰성을 검증하는 기술 안정화 연구 즉, Validation study for FETAX Phase I and II를 수행하였다. 대표적 유해화학물질을 대상으로 최기형성 평가 시험을 하였으며, 얻어진 FETAX 결과와 문헌상의 자료를 상호 비교하였다. 제 3 차년도에는 확보된 FETAX 기법을 적용하여 환경 시료의 생태독성을 평가할 수 있는 새로운 기술 개발과 응용 연구를 수행하였다. IV. 연구개발결과 1. 난용성 및 휘발성 물질의 유해성 시험방법 난용성물질(phenyl salicylate, 2,4-di-tert-butylphenol, dibenzoyl peroxide)을 대상으로 지수식과 유수식으로 급성어독성시험을 하여, 독성값을 비교해 본 결과, 유수식으로 시험한 급성독성값이 2배에서 27배 낮음을 알 수 있었다. 휘발성 물질(methyl acrylate, n-heptane, allyl methaclylate)로 closed system과 open system에서 시험한 결과. closed system에서 시험한 급성독성값이 약 2.4배에서 11배 정도 낮았다. 이로써 유해화학물질관리를 위해서 독성자료를 생산할 때, 난용성 및 휘발성이 높은 물질에 대해서는, 시험용액내 농도를 유지할 수 있는 노출시스템에서 시험을 하도록 하여야 할 것이다. 미생물분해시험은 난용성물질인 diazinon과 fenitrothion을 이용하여, 계면활성제 Tween80와 초음파 처리가 분해울에 미치는 영향을 평가하였다. Diazinon과 Fenitrothion은 초음파처리에 의하여 오히려 분해율이 감소하였으며, Tween 80처리에 의해서도 분해율은 상승하지 않았다. 이러한 이유는 이러한 처리가 오히려 미생물의 활성에 영향을 주어 분해율이 감소하는 것으로 판단된다. 그리고 난용성 및 휘발성 물질과 같이 시험하기 어려운 물질을 가지고, 환경독성시험을 할 때 필요한 지침서를 작성하였다. 2. 수서생물을 이용한 독성시험법 연구 1) 어류 embryo yolk sac-fry 시험법 송사리(Oryzias latipes)와 왜몰개(Aphyocypris chinensis)에 대한 반복실험결과, 두 어류의 수정란(embryo)은 모두 zinc 최고 농도인 63.7 mg/L에서 높은 치사율과 낮은 부화율의 독성 영향을 나타냈다. 반면에 자어(sac-fry) 단계에서는 송사리의 경우 20 mg/L에서부터, 왜몰개의 경우 더 낮은 6.25 mg/L 농도에서부터 높은 치사율이 발견되었다. 더불어 왜몰개는 송사리보다 더 짧은 부화기간을 보였다. 위의 결과를 볼 때, 본 연구는 OECD에 의해 요구되어지는 조건들이 만족되는 시험법을 확립하였고, 왜몰개도 송사리와 같은 시험종으로서 요구된 조건들을 가진다는 것을 발견하였으며, 더 나아가 왜몰개가 송사리보다 자어 단계에서 화학물질에 대한 좀더 민감한 독성영향을 가졌다 2) Lemna 독성시험법 AAP 배지의 최적의 배양조건으로는 모든 측정항목(잎의 면적, 습중량 및 건중량)과 입의 doubling time에서 가장 높은 성장조건으로는 0.5% sucrose를 포함한 9,300 Lux의 조도조건이었다 M-Hoagland's 배지의 경우는 0.5% sucrose를 첨가하지 않은 9,300 Lux의 조도조건에서 모든 측정항목에 대해 가장 빠른 성장을 기록하였다. 대조물질을 이용한 독성시험결과, potassium dichromate의 독성값(EC50값)은 30 ㎎/L 또는 58 ㎎/L 로 확인되었고, 이 독성값은 기존자료의 범위(8~30 ㎎/L) 내에 존재하였다. 2.4-dichlorophenol의 경우, 3회 반복시험한 독성값((EC50값)은 11∼17 ㎎/L이었다. 3) 화학물질의 퇴적물 독성시험법 연구결과, 실험실 사육조건에서 사육한 Chironomus riparius의 성장인자는, 기존의 자료와 비교해 볼 때, 매우 유사한 값을 보여, 실험실 조건에서 성공적으로 사육되고 있음을 확인할 수 있었다. 3회 독성시험한 결과를 보면, 회차간 독성값의 변이가 매우 낮아서, 독성시험에서 정밀도(precision)가 상당히 높았다. 유충의 발달단계별 감수성을 보면, 카드뮴의 경우는 1령충의 감수성이 3령충의 감수성보다 약 28배 높았고, pentachlorophenol은 약 2배 정도 높아, 1령충이 화학물질에 대한 감수성이 매우 높음을 알 수 있었다. 그리고 생존율, 성장률, 성체발현율과 같은 관찰점에서, 반수영향농도는 성장률이 가장 민감한 관찰점이었고, 무영향농도와 최저영향농도를 결정할 때는 성체발현율이 가장 예민한 관찰점이었다. 3 육상식물을 이용한 독성시험법 시험종에 대한 m-nitrobenzene의 독성시험 경우, 밀은 대조군의 평균 발아율이 93%였고 모든 측정항목에서 EC50값이 100 ㎎/kg 이상이었다. 양상치의 경우, 대조군의 평균 발아율은 83%이며, EC50값은 10~100 ㎎/kg의 범위 내에 존재하였다. 순무우는 88%의 평균 발아율을 기록하였고, EC50값도 100 ㎎/kg 이상이었다. 3.4-dinitrotoluene의 경우, 대조군의 평균 발아율이 밀 91%, 양상치 87% 및 순무우 91%로 모두 정상적이었다. 3종 식물에 대한 EC50값은 100 ㎎/kg 이상이었다. 4. 수질· 토양에서의 화학물질 거동연구 1) 광분해 시험법 인공광을 이용한 system 적인 측면에서 광원, 필터, 용기 및 온도조절 등의 연구에서 첫째로, 광원으로 두 guideline에서 xenon arc, mercury lamp 등에 borosilicate 재질의 필터를 장착하여 사용하는 것을 제안하지만, xenon arc lamp에 borosilicate filter의 사용이 spectrum의 분포가 자연광과 유사하기 때문에 인공광의 분해도를 자연광의 분해도로 환산하는 경우, 보다 근접할 수 있을 것으로 판단된다. 둘째로, 시험에 사용되는 시험용기는 빛의 투과율 때문에 EPA에서는 흡광하는 파장에 따라 재질을 제안하고 있으나, OECD guideline에서는 투과율과 관계없이 적절한 재질의 시험용기를 제안하고 있다. 이는 재질에 따라 분해율이 달라지지만, 광량을 측정하여 표준화 할 수 있기 때문에 OECD에서 제안한 것과 같이 파장에 관계없이 pyrex 또는 borosilicate 재질를 사용할 수 있을 것으로 사료된다. 셋째로, 시험온도를 OECD 25 5 , EPA 20∼30 ( 2 )로 유지할 것을 요구하지만, lamp에서 많은 열이 발생되기 때문에 유지가 어렵다. 따라서, 시험용기에 water jacket을 달아 시험에 이용하는 경우, 25 로 유지할 수 있었다. 넷째로, 두 type의 상업용 장치인 suntest와 weather-ometer는 분해율에 있어서 차이가 있었으나, 단지, lamp의 watt 차이기 때문에 두 장치 모두 사용이 가능할 것으로 사료된다. 2) 토양대사 및 토양 미생물을 이용한 분해 시험법 호기성 토양 조건에서 14C-flupyrazofos의 flow-through system내에서의 토양 대사 및 토양 미생물에 의한 분해시험을 실시하였다. 토양은 논토양의 대표적인 토성인 양토였으며, 20 2 의 온도에서 0, 4, 7, 14, 30, 60일 간격으로 시료를 채취 분석한 결과, 시험기간 동안의 미생물 균수는 97 106 (No./g soil)이었고, 14C-flupyrazofos는 처리 후 30일에 비살균 토양에서 처리 방사능의 52.5%로 감소하였으며 토양중 반감기는 31.5일로 계산되었다 (R2=0.91). 5. 환경오염물질의 진보된 독성평가 신기술 개발연구 1) 포유동물세포를 이용한 Mouse Lymphoma L5178Y tk+/- (thymidine kinase) Gene Forward Mutation Assay (MOLY) tk gene내의 point mutation 또는 tk gene을 포함하는 11번 chromosome내에서 발생하는 넓은 범위의 유전적 변화를 인식할 수 있다는 장점을 지니는 MOLY 방법을 사용하여 benzoyl chloride와 2-nitroaniline의 mouse lymphoma L5178Y tk+/- cell line에서의 돌연변이 유발 빈도를 알아보고자 하였다. 먼저, benzoyl chloride에 대한 MOLY 방법의 결과를 보면, 대사활성계의 부재와 존재하에서 benzoyl chloride의 각 농도에 대해서 용매 대조군과 비교하였을 때 대사활성계 부재시 최고 농도인 500 ㎍/㎖과 대사활성계 존재시 최고농도인 600 ㎍/㎖에서 통계적 유의성을 나타냈으며, 또한 mutation frequency의 농도의존성은 대사활성계 존재 및 부재시 모두에서 관찰 할 수 없었다. 따라서 본 연구를 통하여 benzoyl chloride는 mouse lymphoma tk(+/-) gene assay를 이용한 본시험 조건에서는 각각의 최고 적용 농도인 대사활성계 부재시 500 ㎍/㎖과 대사활성계 존재시 600 ㎍/㎖에서 돌연변이를 유발하는 변이원성 물질임을 알 수가 있었다. 다음으로 2-nitroaniline을 살펴보면, 대사활성계의 존재와 부재하에서 2-nitroaniline의 각 농도에 대해서 용매 대조군과 비교하였을 때 대사활성계 부재하에서는 통계학적 유의성이나 농도의존성을 보이지 않으나, 대사활성계 존재하에서는 최고 농도인 625 ㎍/㎖에서 통계학적 유의성을 나타냈으며. mutation frequency의 유의성 있는 linear trend 또한 대사활성계 존재하에서만 관찰되었으나, 농도 의존성은 관찰 할 수 없었다. 따라서 본 연구를 통하여 2-nitroaniline는 mouse lymphoma tk(+/-) gene assay를 이용한 본시험 조건에서는 대사활성계 존재하 625 ㎍/㎖에서 돌연변이를 유발하는 변이원성 물질임을 알 수가 있었다. 2) 포유동물 세포를 이용한 Single Cell Gel Electrophoresis (Comet) Assay Cell level에서 DNA damage를 측정할 수 있는 single cell gel electrophoresis assay 방법에 대한 international harmonization의 일환으로 시험 적용 최적 조건을 설정하는 연구를 수행하였다. 그 결과로서, 시험에 사용할 양성 대조물질과 최적 용량은 대사활성계 부재시에 150 M의 methyl methanesulfonate (MMS)를, 대사활성계 존재시에는 50 M의 benzo(a)pyrene (BaP)으로 결정하였다. 또한, 실험 결과 및 편차 등 여러면을 고려하여 lysis time은 1.5시간으로 unwinding time은 20분으로 결정하였다. 이와 같이 결정된 최적조건하에서, L5178Y cell line을 이용하여 7종의 phthalate analogue들의 DNA 손상 유발여부를 살펴보았다. 대사 활성계 부재시butyl benzyl phthalate와 di-n-octyl phthalate 두 종의 phthalate만이 통계적으로 유의한 tail moment의 증가를 나타내어 본 시험 조건에서 DNA 손상을 유발할 수 있는 물질임을 알 수 있었으며, 대사 활성계 존재시에는 diallyl phthalate, diisodecyl phthalate, dibutyl phthalate가 DNA에 손상을 줄 수 있는 물질임을 확인 할 수 있었으며, 앞으로 이들 물질의 대사 및 대사체의 규명을 통해 추가적인 연구가 수행되어야 하리라 사료된다. 이러한 연구 결과를 통하여 포유 동물 세포를 이용하는 single cell gel electrophoreis assay 방법은 다른 유전 독성연구에 사용되는 여러 방법들에 비하여 보다 신속하고 정확하게 DNA 손상 유발 여부를 측정할 수 있는 방법임을 확인 할 수 있었으며, 앞으로 여러 화학 물질의 환경 및 인간에 미치는 독성 평가에는 물론 직업병 진단 등 여러면에서 활용성이 크리라 사료된다. 3) in vitro cytokinesis-block micronucleus assay 연구 in vitro 독성평가시험법인 cytokinesis-blocking assay에 대해 1차년도에는 기본적인 연구방법의 설정 및 확립에 대한 연구를 수행하였다. 그 결과를 살펴보면, 양성 대조물질로써 대사 활성계 부재하에서는 0.3 ㎍/㎖의 mitomycin C를 대사 활성계 존재하에서는 3 ㎍/㎖의 cyclophosphamide를 설정하였다. 2, 3차 연도에는 이미 확립된 기초연구방법을 이용하여 대사활성계 부재하에서와 대사활성계 존재하에서 dibutyl phthalate (DBP)와 atrazine에 대하여 응용연구를 수행하였다. 먼저 V79 cell line에서 dibutyl phthalate의 binucleated micronucleus 유발 유무를 살펴본 결과, 0.1, 1, 10 ㎍/㎖ 농도의 dibutyl phthalate에 대해서 대사 활성계 부재시 농도에 따라 소핵 유발 빈도가 증가지만 최고 농도인 10 ㎍/㎖의 처리군은 오히려 감소하는 것을 관찰 할 수 있었다. 이는 이 농도에서의 세포독성에 기인하는 것으로 사료된다. 또한 통계적 유의성 여부를 살펴보기 위하여 대조군과 비교하여 ANOVA를 적용하였다. 그 결과, 1 ㎍/㎖의 DBP 처리군이 통계적으로 유의한 소핵 유발 빈도를 나타내는 것을 알 수 있었다 (P<0.05). 동일 농도에서 대사 활성계 존재시에는 농도에 따라 소핵 유발 빈도가 증가하는 양상을 볼 수 있었고, 특히 최고농도인 10 ㎍/㎖의 DBP 처리군은 대조군과 비교시 통계적으로 유의한 차이를 나타내는 것을 알 수 있었다 (P<0.05). 또한 Transgenic mutagenesis assay에서 사용하였던 cell line인 Big Blue Rat2 fibroblast를 이용하여 atrazine의 binucleated micronucleus 유발여부를 알아보고자 하였다. 양성대조물질로서 mitomycin C (MMC) 0.3 ㎍/㎖을 사용하였으며, 농도에 따른 소핵유발 양상을 볼 수는 있었으나 대조군과 비교하여 통계 처리를 한 결과, 유의성있는 증가 여부를 관찰 할 수 없었다. 4) Transgenic Big Blue 배양세포를 이용한 환경유해물질의 돌연변이 발 현기전 및 변이spectrum 연구(Mutation Mechanism and Spectrum Study in Transgenic Big Blue Mutagenesis System) 4.1. 1,2-dibromo-3-chloropropane (DBCP) 4.1.1. DBCP의 처리 농도 결정 세포에 0.1, 0.2, 0.4, 0.8, 그리고 1.6 mM의 DBCP를 처리하였을 때 medium control과 비교시 각각 82, 81, 68, 44, 그리고 5%의 생존률을 나타냄을 알 수 있었다. 위의 결과로부터 얻어진 DBCP의 50% 생존률을 나타내는 농도(IC50)는 0.75 mM로 결정되었으며, 생존률에 대한 DBCP의 농도 의존성 (y=-55.3686x + 91.4365) 역시 correlation efficiency (r2= 0.9875)를 지니는 것을 관찰할 수 있었다. 이러한 결과를 바탕으로 본실험에 적용한 세 농도는 IC50을 최고농도로 하고 공비 2로 희석하여 얻어진 두 농도로 결정하였다. 4.1.2 DBCP의 mutant frequency (MF) lacI transgenic Big Blue Rat2 fibroblast 세포에서 DBCP의 mutagenicity를 결정하기 위해 lacI gene에서의 mutant frequency를 구하였다. Medium과 용매인 1% DMSO를 처리한 세포에서의 평균 MF값 ( 10-5, SEM)은 각각 6.43 0.616과 5.28 1.086이었다. 양성대조군으로는 현재까지 Big Blue cell line에서의 mutant frequency가 보고된 유일한 물질인 MNU(metyl nitrosourea)를 사용하여 실험의 재현성 여부를 확인하였다. 그리고 0.21, 0.39 와 0.75 mM의 DBCP를 처리한 세포에서의 MF값은 각각 8.09 1.02, 10.86 2.17 과 12.26 0.79 로 결정되었으며 농도 의존적으로 증가함을 알 수 있었다. (ANOVA, P=0.007). 특히 0.39 과 0.75 mM DBCP 처리군은 medium 대조군에 비해 통계적으로 유의한 차이를 나타내었다 (ANOVA, P<0.05). 이와 같은 결과로부터 DBCP는 lacI transgenic Big Blue Rat2 세포주에서 mutagenic 함을 알 수 있었다. 기존의 보고들에 따르면 DBCP는 multi-organ animal carcinogen이며 possible human carcinogen으로 알려져 있으며, 또한 몇몇 in vitro 및 in vivo 박테리아와 포유동물 세포를 이용한 실험을 통해 genotoxic한 것으로 알려져 있다. 따라서 DBCP는 강력한 돌연변이원이며, lac I gene에서 돌연변이를 일으켰듯이 발암과 관련있는 tumor supressor gene이나 proto-oncogene상에 돌연변이를 일으켜서 각종 암을 유발함을 유추 할 수 있었다. 또한 transgenic mutagenesis assay가 돌연변이원을 민감하게 감지할 수 있는 뛰어난 방법이며 단순한 독성평가에 그치지 않고 구체적인 기작을 설명해 줄 수 있는 방법임을 본 연구를 통해 확인할 수 있어, 앞으로 환경유해물질의 독성 발현기전연구는 물론 환경유해물질의 신속한 검정과 발암성유발과의 상관성 연구 등에 다양하게 적용될 수 있는 진보된 연구기법으로 활용될 수 있으리라 사료된다. 4.1.3 DBCP의 Mutation spectrum 돌연변이원이면서 발암원인 DBCP가 유전자상에서 유도하는 돌연변이 형태나 위치등의 정보에 대한 연구 즉, mutation spectrum을 밝히는 연구는 돌연변이 발생과정에 대한 이해와 위에서 언급한 발암기작에 대한 이해에 도움이 되므로 본 연구에서는 감지된 mutant들의 염기서열 분석을 통해 Big Blue cell line에서 자연발생한 돌연변이와 DBCP에 의해서 유도된 돌연변이의 mutation spectrum을 밝히고자 하였다. Medium과 용매 대조군에서 얻어진 mutants (40개의 mutant중 33개에서 mutation 분석, 이중 28개가 independent함)와 0.75 mM DBCP에 의해 유도되어진 mutants (42개의 mutant에서 분석, 이중 40개가 independent함)에서 1,080 bp lacI gene의 sequence를 분석하였다. 대조군(21/28, 75%)과 DBCP 처리군(31/40, 77.5%) 모두에서 회수되어진 대다수의 mutation은 single base pair substitution이었다. DBCP에 의해 유도되어진 40개의 independent mutation중 31개가 single base pair substitution이었고 9개 (22.5%)는 deletion 혹은 insertion이었다. Single base pair substitution중 25개의 mutation(62.5%)이 G:C base pair에서 일어난 반면 6개(15%)는 A:T base pair에서 일어났다. Base substitution은 pyrimidine계 염기간(T가 C, C가 T로), purine계 염기간 (A가 G로, G가 A로)으로 염기가 전이되는 transition과, pyrimidine계 염기가 purine계로 또는 purine계 염기가 pyrimidine계로 전환되는 transversion의 두가지 유형으로 크게 나뉘어 구분된다. 회수한 mutation의 predominant class는 G:C A:T transition (40%, 16/40) 과 G:C T:A transversion (22.5%, 9/40)이었다. 또한 6개의 single base frameshift와 2 bp, 3 bp 그리고 4 bp deletion도 유도되었다 (22.5%). DBCP 처리군은 대조군과 비교시 mutation spectrum에서 유의한 차이 (P=0.05)를 보였다. G:C A:T transition은 대조군에 비해 2배 이상을 차지하였으며, G:C A:T transition의 경우 CpG site에서 67% (4/6)를 차지한 대조군과 달리 CpG site와 non-CpG site에서 동일한 비율(8/16, 50%)을 보였다. 반면 대조군에서 G:C C:G transversion이 많은 부분(21.4%)을 차지하지만 DBCP 처리군에서는 거의 유도되지 않았다. 그리고 위에서 설명한 base substitution에 관한 모든 결과를 아미노산의 변화, 즉 단백질로 표현되어 변성되는 측면에서 정리하면 다음과 같다. Spontaneous mutation의 경우에는 실질적인 아미노산서열의 변화를 초래하지 않는 silence mutation이 5.3%, 유전자내 신장사슬(extension chain)이 종결되는 nonsense mutation은 10.5%로 나타났으며 나머지 84.2%의 mutation는 다른 아미노산을 지정하는 코돈으로 바뀌는 missense mutation이였다. DBCP induced mutation의 경우에서는 silence mutation이 3.2%, nonsense mutation이 19.4%, missense mutation이 77.4%인 것으로 나타났다. 그리고 phenotypic change를 일으켰음에도 불구하고 sequence상에는 mutation site가 발견되지 않은 비율이 spontaneous의 경우 전체의 15%, DBCP induced mutaion의 경우 4.8%인 것으로 나타났는데 이는 lac I coding region이 아닌 operator, promoter region에서 mutation이 일어났기 때문인 것으로 생각되어진다. 이상의 결과를 종합, 요약해보면, DBCP의 mutant frequency결과가 mutagenesis를 연구하는데 널리 이용되는 기존의 방법들의 결과들과 일치함을 통해 본 실험에서 사용한 transgenic Big Blue cell line을 이용한 Transgenic Mutagenesis(TM) assay system의 유효함을 알 수 있었다. 또한 이 방법은 기존의 방법들의 단점이 보완되어 단순한 검색이나 정량평가 뿐만 아니라 기작연구의 기초가 될 수 있는 mutation spectrum의 연구가 가능하므로 매우 유용한 것으로 평가되며 다방면에 응용되어 사용되어 질 수 있으리라 사료된다. 또한, DBCP는 mutagen이며, 염기중 G에 주로 작용하는 base substitution mutagen으로서 특히, base substitution중에서도 transition을 주로 유도시킴을 mutation spectrum분석을 통해서 확인할 수 있었다. 이러한 사실은 mammalian cell 에서는 처음으로 보고되는 것이며 본 실험결과는 다른 시스템에서의 결과와 일치되는 양상을 나타내었다. lac I gene에서의 이와 같은 결과는 DBCP가 mutation을 일으키는 구체적 기작에 대한 정보를 제공하는 것으로서, DBCP가 carcinogen임을 고려할 때 다른 endogenous genes 특히 proto-oncogene, tumor suppressor gene, repair관련 gene들에게서 같은 효과를 나타내어 세포내 이상을 초래함으로써 암 발생의 개시를 유도하는 것이라 추측해 볼 수 있다. 따라서 chemical carcinogenesis연구에 있어서 mutation spectrum을 관찰하는 것이 매우 중요한 과정이라 생각되며, transgenic mutagenesis assay는 발암기작 또는 유전병을 비롯한 기타 질병에 관한 구체적 작용기작을 연구하는데 있어서 가치 있는 자료를 제공하는 수단이 되는 것으로 보여진다. 4.2. Atrazine 4.2.1 Atrazine의 처리 농도 결정 적당한 atrazine의 처리농도 결정을 위해 MTT assay를 시행하였고 본실험과 동일한 조건하에서 실시하였다. Figure 21에 제시된 결과를 보면, 20, 39, 78, 158, 313, 625 ㎍/ml의 atrazine을 처리하였을 때 solvent control과 비교시 각각 99, 103, 97, 86, 72 그리고 61%의 생존률을 나타냄을 알 수 있었다. 위의 결과로부터 처리농도는 selection pressure를 배제하고자 세포성장에 전혀 영향을 미치지 않는 농도로 선택하고자 하여, 본 실험에 사용한 세 농도는 각각 IC5, IC10, IC20 값인 65.42, 130.84, 261.68 ㎍/ml로 결정하였다. 4.2.2 Atrazine의 mutant frequency (MF) 65.42, 130.84, 261.68 ㎍/ml의 atrazine을 Big Blue cell에 3시간 처리하고confluent cell로부터 genomic DNA를 분리하였다. 분리한 DNA를 Transpack packaging extract와 섞어서 shuttle vector를 packaging시켰다. Transpack packaging extract는 shuttle vector에 있는 cos site를 인식하여 이 부위를 절단하여 lambda DNA를 package해서 infectious phage particle을 만든다. 각각의 package된 phage는 하나의 회수된 target gene을 포함하고 있으며, lac I에 발생한 돌연변이는 host E. coli에 infection시켜서 X-gal이 함유되어 있는 배지에 plating시켜 감지한다. Atrazine이 lac I gene에 돌연변이를 유발시킨 빈도는 plating후 24시간 이내에 생성된 blue plaque와 colorless plaque의 수를 세어 측정하였으며 음성대조군은 용매로 사용된 1% DMSO를 처리하였고, 양성대조군으로는 현재까지 Big Blue cell line에서의 mutant frequency가 보고된 유일한 물질인 MNU(metyl nitrosourea)를 사용하여 실험의 재현성 여부를 확인하였다. 그 결과, 음성대조군의 mutant frequency는 5.57 0.06 이었으며, 65.42, 130.84, 261.68 ㎍/ml의 atrazine을 처리한 군은 각각 5.27 3.79, 5.87 8.14, 6.13 1.25의 mutant frequency를 나타냄을 알 수 있었으며, 유의성 판정을 위해 ANOVA (P > 0.05)로 통계처리한 결과, atrazine 세 농도의 처리군이 음성대조군에??면 atrazine은 breast, ovary, uterus, testis cancer, leukemia, lymphoma등을 유발한다고 한다. 본 연구를 통해 atrazine에 의해 유발되는 발암의 원인이 유전자 상에서의 돌연변이 유발 기전에 의함이 아니라 어떤 다른 기작에 기인 할 것이라는 것을 유추할 수 있었다. 특히 endocrine disruptor로 작용하여 birth defect와 reproductive tumor를 일으킨 다는 보고는 이를 뒤받침 해준다고 볼 수 있다. 6. 환경오염물질의 위해성 평가를 위한 조류독성 시험법 개발 급성경구독성시험에서 각 농약의 메추리에 대한 LD50값은 암컷에서 diazinon 7.12 mg/kg, isazofos 16.26 mg/kg, pyraclofos 41.26 mg/kg, 및 methomyl 21.24 mg/kg이었고, 수컷에서는 diazinon 8.28, isazofos 21.44, pyraclofos 35.64 그리고 methomyl 28.28 mg/kg으로 diazinon이 다른 농약에 비해 암수 모두에서 좀더 높은 독성을 나타내었고, pyraclofos를 제외한 나머지 약물에서 암컷의 감수성이 더 높았다. 사망개체의 부검 결과 육안소견으로 십이지장과 공장, 회장에 미만성 점성 출혈이 발견되었고, pyraclofos와 isazofos 투여 군에서는 근위의 흑갈색 변화가 감지되었다. Brain acetylcholinesterase의 활성을 각 농약의 농도별, 투여 후 경과 시간별로 측정한 결과 사망 개체에서 88∼96%의 높은 활성 억제를 보였고, 투여 5시간 경과 후에도 유기인계 농약에 의한 억제는 회복되지 않았다. 카바메이트계 농약에 의한 활성 억제는 유기인계 농약에 비해 빨리 회복 되었다. 꿩에서는 성성숙이 일어난 수컷 꿩을 사용하여 isazofos와 pyraclofos에 대해 up and down test를 하였고 각 농약의 꿩에 대한 LD50값은 수컷에서 diazinon 11.22 mg/kg, isazofos 11.89 mg/kg, pyraclofos 28.28 mg/kg, 및 methomyl 67.27 mg/kg이었다. Diazinone이 역시 다른 농약에 비해 좀더 높은 독성을 나타내었다. 식이성 독성시험은 일본 메추리를 이용하여 유기인계 농약 2종(pyraclofos, isazofos)에 대해 실시하였고 실제로 동물이 약물에 노출되는 경로와 유사하며, 강제 투여시 나타날 수 있는 regurgitation을 막을 수 있고, handling stress가 없어 일반 외부 광범위한 지역의 오염 환경 정도를 조사할 수 있는데 그 이점이 있다. 또한 위의 농약에 대한 식이성 독성 시험으로 LC50를 구하였으며 메추리의 실험동물로서의 유의성도 확인하였다. 7. 화학물질의 최기형성 평가를 위한 FETAX 기법 연구 제 1 차년도에는 개구리 배아를 이용한 최기형성 평가 기법 확립 연구(보고서, 제 1 편)를 수행하였다. 연구 결과, 시험생물인 Xenopus laevis의 사육방법, 정상적 배아의 판별과 배양 방법 등을 확립하였으며, 표준 시험물질에 대한 최기형성 시험을 수행하여 FETAX 기법을 확보하였다. 제 2 차년도에는 FETAX 기법의 Phase I 및 II 검증 연구(보고서, 제 2, 3 편)를 통하여, 생산하고자 하는 실험 결과의 신뢰성을 제고시켰다. 제 3 차년도에는 확보된 FETAX 기법을 적용하여 환경 시료의 생태독성을 평가할 수 있는 새로운 기술 개발(보고서, 제 4 편)과 응용 연구(보고서, 제 5 편)를 수행하였다. 연구 결과, FETAX/Microtox tandem bioassay 기법이 매우 유용한 하천생태계 위해성 평가 수단인 것으로 사료되었다. 한편, 수입종인 Xenopus를 대체하기 위한 타당성 연구로서, 한국 산개구리 배아를 이용한 최기형성 평가 기법 연구(보고서, 제 6 편)를 시도하였다. 연구 결과, 산개구리 배아의 발생계를 활용한 화학물질 및 환경오염물질 독성 평가가 가능한 것으로 판단되었으며, 향후 독성 발현 기작을 밝히는 기법으로도 활용이 가능할 것으로 사료되었다. 한편, 본 연구 결과물인 확보 기술을 외부 기관에게 전파하기 위하여 FETAX 시험 지침서(Test Manual)를 작성하였으며, 수 차례의 워크샵을 개최하였다. V. 연구개발계획의 활용계획 이 연구결과들은 국내 화학물질관리의 선진화에 도움이되는 시험법들 이다. 따라서 이 과제들은 화학물질관리 정책에 반영하여야 할 것이다. 이 연구에서 확립된 진보된 독성평가 기법을 활용하여 환경 유해물질을 신속, 정확하게 평가할 수 있으므로, 국내 유해화학물질관리법의 '화학물질유 해성 시험방법'에 채택되어, 국내에서 시험하는 모든 유해성 시험에 적용할 수 있음. 아울러, 본 연구에서 나온 manual들은 국가기관, 기업체, 학교 등 관련 시험 연구를 하는 기관에 널리 배포하여 국가 유해성 시험의 표준화를 달성할 수 있음. OECD/SIDS와 같은 국제적인 화학물질 관리 프로그램에 능동적으로 참여할 수 있게 됨. OECD GLP수준에 적합한 국제적 독성 시험기관의 육성 국내 정밀화학 관련제품의 개발 및 해외 수출에 적극적으로 대응 환경오염 매체에 대한 환경관리사업에 활용 : 산업폐수, 하천수, 하천퇴적물, 지정폐기물 등에 대한 환경독성을 평가하는 수단으로 매우 적절한 기법이다. 따라서 개발된 기술들은 환경계에서 유해화학물질의 관리에도 응용할 수 있 어, 환경관리의 선진화에 기여할 수 있을 것이다.
I. Title Establishment of Advanced Testing Methods for Hazardous Chemicals II. Objective and Justification To upgrade the domestic chemical management system and to participate actively in the international chemical program for; ① Introduction of advanced concept to risk assessment of domest
I. Title Establishment of Advanced Testing Methods for Hazardous Chemicals II. Objective and Justification To upgrade the domestic chemical management system and to participate actively in the international chemical program for; ① Introduction of advanced concept to risk assessment of domestic chemicals ② Improvement of capability of toxicity testing in domestic test research institutions ③ Protection of our environment and national health from hazardous chemicals, and ④ Security of active role as OECD member country. III. Area and Scope of the study 1. Toxicity testing of sparingly soluble and volitile chemicals 96 hr- LC50 values of three kind of sparingly soluble compounds, phenyl salicylate, 2,4-di-tert-butylphenol and dibenzoyl peroxide, were determined with ricefish to clarify the difference of toxicity values between the exposure system, static and continuous system. Also, Fish acute toxicity testing of volitile compounds, methyl acrylate, n-heptane and allyl methacrylat, were performed using the closed system and the difference of toxicity values depending on the exposure systems was assessed. 2. Toxicity testing using aquatic organism 1) Fish, embryo and sac-fry toxicity testing Based on the OECD test guideline 212, we examined chemical(zinc) toxicity not only on fertilized eggs but also on sac-fry of Japanese ricefish/Medaka (Oryzias latipes) and Chinese bleak (Aphyocypris chinensis). Therefore, we can determine effects of chemical toxicity on both stages of fish and compare sensitivity of toxicity effects between the stages. 2) Lemna toxicity test The study was to establish an rearing technique of Lemna for toxicity testing according to OECD Test guideline, and to perform toxicity testing of chemicals with the organism. With the result of the study, the established technique was evaluated in terms of precision of repeated tests, and a manual for rearing test organism and toxicity testing was prepared. 3) Sediment toxicity test The study was establish rearing technique of Chironomus riparius used well internationally, to establish an technique of toxicity testing in sediment-water system using spiked sediment with Chironomus riparius, and to make out a manual on organism rearing and toxicity testing 3. Toxicity test of terrestrial plants The study was to perform toxicity testing with terrestrial plants, according to OECD Test guideline 208. Toxicity testing was proceeded using three species of plant seeds and two test substances, m-nitrobenzene and 3,4-dinitrotoluene to compare with reference data and to confirm reproducibility between repeated tests. In addition, a manual for the toxicity testing was prepared. 4. Fate of chemicals in water and soil 1) Photolysis test As a result of comparison with OECD guideline as the central figure, it was systematically difficult to satisfy with conditions of the guideline such as light source, filter, test vessel, and temperature control in the photolysis test. Therefore we studied on these conditions. 2) Soil metabolism and biodegradation using soil microorganisms With review of soil metabolism and soil biodegradation of recent researches and OECD and US EPA guidelines, test method was established with the OECD guideline as center figure. At the same time, soil metabolism and soil biodegradation test was performed using radio labelled compounds in flow-through system. 5. Study on new techniques for toxicity evaluation of environmental hazardous chemicals 1) Single Cell Gel Electrophoresis (Comet) assay As one of the mechanisms of carcinogenicity, induction of DNA damage was ascertained by a comet assay, which is widely used for the detection and measurement of DNA strand breaks. Since Ostling and Johanson (1984) introduced microelectrophoretic technique, Singh et al. (1988) have modified and improved the microgel electrophoresis technique to evaluate DNA damage in single cells under alkaline conditions. The single cell gel electrophoresis (SCGE, comet or microgel electrophoresis) assay is rapid, sensitive, visual and simple technique to quantify DNA strand breaks in individual cells. It can be noticed that there are 3 major basic protocols: the first of Ostling and Johanson, the second of Singh et al. and the one of Olive et al.. Each protocol follows the principle of embedding cells in agarose, lysing the cytoplasmic material and exposing the remaining cell nuclei to a weak electric field. Large differences can be found in the physical and chemical conditions of each version of the method. If the agent causes the strand breakage, we can see the extent of tail from the head (nucleus) shaped like comet with staining of fluorescent dyes such as ethidiumbromide, acridine orange, propidium iodide and so on. However, there are some variations in procedure and conditions between laboratories a kind of cells used. So, to harmonize this variations in comet assay, International Workshop on Genotoxicity Test Procedure (IWGTP) was held at Washington D.C. on March, 1999 by Environmental Mutagen Society supported with OECD. Our laboratory also involved in this harmonization and published as preliminary form for OECD guideline with Tice et al.. Comet assay is useful in various fields such as radiation, human monitoring in work place and toxicity evaluation of some chemicals etc. General reviews on this technique have been published by Tice et al. and Fairbairn et al.. 1)-a : Cell culture and viability test The mouse lymphoma cell line L5178Y (tk+/- 3.7.2c) was used for the experiment. Cells were cultivated in 90% RPMI-1640 (Life Technologies, MD, USA) with 1 mM sodium pyruvate, 0.1% pluronic supplemented with 10% heat-inactivated horse serum and antibiotics in a humidified incubator at 37 with 5% CO2. For the determination of cell viability, about 106 cells were treated for 2 hr with test chemicals. BaP was used in combination with S9 mixture. After the staining of 0.4% trypan blue (Life Technologies, MD, U.S.A), the total number of cells and the number of unstained cells were counted in five of the major sections of a hemocytometer. The average number of cells per section was calculated. Cell viability of treated groups was compared to solvent controls in the presence and absence of S9 mixture. All experiments were repeated twice independently. 1)-b : Preparation of agarose coated glass slide For the comet assay, 8 x 105 cells were seeded into 12 wells plate (Falcon 3043) and then treated with test compounds. In the experiments with metabolic activation, parallel cultures were performed in the absence or presence of S9 mixture. After 2 min, cells were centrifuged for 5 min at 100 x g (about 1,200 rpm), and gently resuspended with PBS and 100 ㎕ of the cell suspension was immediately used for the test. Cells were mixed with 0.1 ml of 1% low melting point agarose (LMPA, Gibco BRL, Life Technologies, Inc., MD, USA) and added to fully frosted slide (Cat. No., 12-544-5, Fisher Scientific, PA, USA) which had been covered with a bottom layer of 100 ㎕ of 1% normal melting agarose (Amresco, OH, USA). The cell suspension was immediately covered with cover glass and the slides were then kept at 4 for 5 min to allow solidification. After removing the cover glass gently, the slides were covered with a third layer of 100 ㎕ of 0.5% LMPA by using a cover glass and then the slide were kept again at 4 for 5 min. 1)-c : Alkaline unwinding and alkaline electrophoresis The procedure follows the method described by Singh et al., (1988) with minor modification. The cells embedded in the agarose on slides were lysed for 1.5 hr in reaction mixture of 2.5 M NaCl, 0.1 M Na2-EDTA, 10 mM Tris-HCl (pH 10), 1% Triton X-100 at 4 . Slides were then placed in 0.3 M NaOH containing 1 mM Na2-EDTA (approximately pH 13) for 20 min to unwind DNA before electrophoresis. Electrophoresis was conducted at 25 V (about 1 V/cm across the gels) and approximately 300 mA for 20 min at 4 . All of the steps described above were conducted under yellow light or in the dark to prevent additional DNA damage. 1)-d : Evaluation and Statistics of DNA damage After the electrophoresis, the slides were washed gently to remove alkali and detergents which would interfere with ethidium bromide staining, by placing horizontally and flooding them three times slowly with 0.4 M Tris (pH 7.5) for 5 min. The slides were stained by 50 ㎕ of ethidium bromide in distilled water solution on each slide, and then covering the slide with a cover glass. Image of 100 randomly selected cells (50 cells from each of two replicate slides) was analysed from each sample. All experiments were repeated in an independent test. Measurement was made by image analysis with Komet 3.1 (Kinetic Imaging Limited, Liverpool, UK) system, determining the mean tail moment (percentage of DNA in the tail times tail length) of the 50 cells. Differences between the control and the other values were tested for significance using one way of analysis of variance (ANOVA). 2) Mouse lymphoma thymidine kinase (tk)+/- gene assay MOLY was performed with L5178Y tk+/- mouse lymphoma cells as described by Clements (1994) with minor modification. The cytotoxicity of chemical was determined by relative survival (RS) after 3 hr treatment at concentrations up to 5000 ㎍/ml with and without S-9 mixture. The highest concentration chosen was one with a 10-20% RS. This experiment consisted of one solvent control, one positive control and at least three test chemical concentrations in duplicate cultures. Cultures were exposed to the test chemical for 3 hr, then cultured for 2 days before plating in 96-well microtiter plates at 2000 cells/well with trifluorothymidine for mutant selection and at 1.6 cells/well for cell viability. The number of wells containing colonies was counted on day 12 after plating, and large and small colonies were scored. Mutation frequencies were analyzed by the statistical package, Mutant V2.31 program (Hazleton, England) in accordance with the UKEMS guidelines. The acceptable ranges of mean absolute plating efficiency (PE) for solvent control were 60-140% for survival (PE0) and 70-130% for viability (PE2), based on the consensus agreements. 3) In vitro cytokinesis-block micronucleus assay The study of DNA damage at the chromosome level is an essential part of genetic toxicology because chromosomal mutation is an important event in carcinogenesis. The micronucleus assays have emerged as one of the preferred methods for assessing chromosome damage because they enable both chromosome loss and chromosome breakage to be measured reliably. Because micronuclei can only be expressed in cells that complete nuclear division a special method was developed that identifies such cells by their binucleate appearance when blocked from performing cytokinesis by cytochalasin-B, a microfilament-assembly inhibitor. The cytokinesis-block micronucleus (CBMN) assay allows better precision because the data obtained are not confounded by altered cell division kinetics caused by cytotoxicity of agents tested or sub-optimal cell culture conditions. The method is now applied to various cell types for population monitoring of genetic damage, screening of chemicals for genotoxic potential and for specific purposes such as the prediction of the radiosensitivity of tumors and the inter-individual variation in radiosensitivity. In its current basic form the CBMN assay can provide, using simple morphological criteria, the following measures of genotoxicity and cytotoxicity: chromosome breakage, chromosome loss, chromosome rearrangement (nucleoplasmic bridges), cell division inhibition, necrosis and apoptosis. The cytosine-arabinoside modification of the CBMN assay allows for measurement of excision repairable lesions. The use of molecular probes enables chromosome loss to be distinguished from chromosome breakage and importantly non-disjunction in non-micronucleated binucleated cells can be efficiently measured. The in vitro CBMN technique, therefore, provides multiple and complementary measures of genotoxicity and cytotoxicity which can be achieved with relative ease within one system. 4) Transgenic Mutagenesis assay system Because mutagens often produce characteristic patterns of DNA sequence alterations, called mutation spectrum, the elucidation of mutation spectrum of specific carcinogen provides a basis for understanding of cancer etiology and action mechanism of chemical carcinogenesis. Recently developed transgenic mutagenesis assay system including Big Blue mutagenesis system is a useful and powerful tool to evaluate the genotoxicity of chemicals, and it also provides a window of carcinogenesis and mutagenesis mechanisms of chemicals based on information such as mutation pattern, frequency, and location in sequence context of the lacI target gene. The lacI transgenic Big Blue Rat2 fibroblast cell line carries over 40 copies of lambda shuttle vector containing lacI gene as a target. The lacI gene, as a mutational target, is very useful for the study of the mutational characteristics of a carcinogen for several reasons. First, the relatively small size (1,080 bp of coding region) of lacI gene facilitates sequence analysis. Second, the expression of repressor protein permits a rapid colorimetric assay to screen for mutations. In addition, it is possible to make use of the large historical database in subsequent comparisons, allowing us to elucidate the underlying mechanisms leading to mutations. The mutations induced in the lacI gene can easily be quantified by mutant frequency (MF), and the precise mutation type and distribution can quickly be identified by direct sequencing. Moreover, considering that mutations in lacI gene induced by chemicals reflect the effects of mutagens on other endogenous genes such as proto-oncogenes and tumor suppressor genes, and that mutations occurred in these genes are the most common events in many types of human cancer, this assay may provide a powerful tool to predict the mutation spectrum induced in cancer-related genes more accurately. Our laboratory elucidated mutation spectrum of 4-nitroquinoline N-oxide and dibromo chloropropane. And also, we reported the mutant frequency of atrazine with cytogenetic analysis using Big Blue Rat2 cells. 4)-a: Cell culture The lacI transgenic Big Blue Rat2 fibroblast cell line was purchased from Stratagene (La Jolla, CA). This cell line is derived from a Rat2 embryonic fibroblast cell line (CRL 1764; ATCC, Rockville, MD) transfected with the Big Blue shuttle vector (lambda lacI / lacZ shuttle vector) and pSV2NEO plasmid, which provides an antibiotic selection marker. The shuttle vector, which has been integrated into the Rat2 genome at two sites (~60 copies/cell), is identical to that used for generating the Big Blue mouse. The lacI transgenic Big Blue Rat2 fibroblast cells were cultured as a monolayer in Dulbeccos modified Eagles medium (DMEM; Gibco BRL, NY) containing 50 units/ml penicillin, 50 mg/ml streptomycin, 200 mg/ml geneticin (G418), and 10% heat-inactivated defined fetal bovine serum (Gibco BRL, NY) at 37 in a humidified incubator containing 5% CO2 (Ryu et al, 1999a). Under these conditions, the average cell cycle time was ~19 h. Low passage (<5) cells were used in each experiment. Cells were treated at 30-40% confluence (~3.5x105 cells in a 25 cm2 flask). A solution of 0.25% trypsin-EDTA (Gibco BRL, NY) was used for subculture. 4)-b : Transgenic mutagenesis assay a) : Isolation of genomic DNA. Genomic DNA was isolated from lacI transgenic Big Blue Rat2 fibroblast cells using the RecoverEase protocol (Stratagene, La Jolla, CA). In essence, cell pellets were thawed on ice, resuspended in 8 ml of ice-cold lysis buffer (8.20 g NaCl, 0.22 g KCl, 120 g sucrose, 0.30 g EDTA, 10 ml Triton X-100, 1.58 g Tris-HCl (pH 8.3)) and homogenized with a tissue grinder. The homogenate was filtered through a 100 m nylon mesh filter into 50 ml tubes and centrifuged at 1,100 x g for 12 min at 4 . The supernatant was discarded and the tubes were swabbed with a cotton swab to remove residual supernatant. The pellet was incubated at 50 for 45 min in the presence of 70 ㎕ digestion buffer containing RNace-It ribonuclease cocktail (20 ㎕/ml digestion buffer; Stratagene, La Jolla, CA) and 70 ㎕ proteinase K solution. The contents of the tube were dialyzed for 48 h by placing the DNA extract on the wetted surface of a membrane floating on 10 mM Tris/1 mM EDTA buffer, pH 7.4. The viscous DNA was collected and stored at 4 until packaging. b) : Packaging and plating of DNA. After DNA concentrations were adjusted to 0.5 mg/ml, the genomic DNA was incubated with Transpack packaging extract (Stratagene, La Jolla, CA) to excise the lambda vector target and package it into a lambda head according to the Stratagene Big Blue instruction manual. The titer of the rescued phage was estimated by plating the packaged phage in serial dilutions. For plating, the volume of the phage equivalent to 15,000 plaque forming units was added to 2 ml of Escherichia coli SCS-8 cells (Stratagene, La Jolla, CA) in 10 mM MgSO4. Pre-warmed Big Blue top agarose containing 1.5 mg/ml of X-gal (dissolved in dimethylformamide) was added, and the contents were poured onto 25 x 25 cm2 assay trays containing 250 ml Big Blue bottom agar (agar, casein peptone, yeast extract, MgSO4 and NaCl). Trays were incubated for 18 h at 37 and scored for blue mutant plaques. The total number of plaques in each plate was estimated by counting four sectors of 5 x 5 cm2 areas, averaging these numbers and multiplying by a scaling factor. Blue mutant plaques were counted and picked into individual tubes containing 0.5 ml SM buffer (5.8 g NaCl, 2.0 g MgSO47H2O, 50 ml 1 M Tris-HCl (pH 7.5) and 5 ml of 2% gelatin (W/V)) for further characterization. To confirm the mutant phenotype and for future use in DNA sequence analysis, all recovered putative mutant phages were diluted to 1:200 and replated on 100 mm plates with 3.5 ml of top agarose containing 1.5 mg/ml X-gal. The sectored plaques that were observed were also verified for their phenotype as previously specified and confirmed sectored plaques were scored separately. The lacI mutant frequency (MF) value was calculated by dividing the number of verified mutant plaques by the total number of plaques analyzed. 4)-c : Mutation spectrum The lacI gene was sequenced from lacI mutants of solvent and medium control cells (collectively considered as spontaneous mutants) and of cells exposed to chemical. Each mutant was purified by replating on a 100 mm plate at low density. From each plate, a single mutant plaque was isolated, cored, placed into 25 ㎕ deionized water, and then boiled for 5 min. The lacI gene was amplified by PCR in 100 ㎕ PCR reaction mixture containing 15 ㎕ of phage DNA, 2 ㎕ of each of forward position 53 to 37, (5-CCCGACACCATCGAATG-3) and reverse position 1,201 to 1,185, (5-ACCATTCCACACAACATAC-3) Big Blue PCR primers, 25 mM dNTPs and 5 U Ex Taq polymerase (TaKaRa Shuzo Co. Ltd, Japan). After the initial denaturation step at 95 for 5 min and extension step at 72 for 5 min, 30 cycles of amplification were performed as follows: denaturation at 95 for 90 s, annealing at 55 for 90 s, elongation at 72 for 150 s with a final extension at 72 for 10 min using thermocycler (Robocycler 40 temperature cycler, Stratagene, La Jolla, CA). The resulting 1,300 bp fragment was purified with a PCR purification kit (Inje Biotech, Seoul, Korea) according to the manufacturers instructions, and visualized on a 1% low melting point (LMP) agarose gel containing 1 mg/ml ethidium bromide. The PCR products were sequenced with an ABI PRISM dye terminator cycle sequencing ready reaction kit (Perkin-Elmer, CA) on an Applied Biosystem 373 DNA sequencer (Perkin-Elmer, CA). The purified DNA sample corresponding to approximately 200 ng was added to 3.2 pmol/ml primer and terminator dyes. The sequencing reactions were carried out 25 cycles as follows: 96 for 30 s; 50 for 15 s; 60 for 4 min, according to the protocol established by Applied Biosystems. 4)-d : Statistical analysis Analysis of variance (ANOVA) test was performed to evaluate differences in lacI mutant frequency between treatment groups at the level of P = 0.05. A Dunnetts multiple comparison test was used to compare each treatment group with untreated controls. Chi-square ( 2) test was used for statistical treatment of mutational spectra. 6. The development of avian toxity test for the assessment of environmental contaminating materials This research was carried out by following the OECD's Guidelines for the Testing of Chemicals, Monograph Series on Testing and Assessment, Report of the SETAC/OECD Workshop on Avian Toxicity Testing at SETAC/OECD workshop in 1996. We would like to confirm the avian toxity test method applied to our national reality. Firstly, we hatched and raised the quails and secondly, performed the acute oral toxity and dietary toxity on quails and pheasants. 7. A Study for FETAX as a Biassay for Teratogenecity of Chemicals During the first year of the research period, the preliminary research was carried out to establish FETAX. Main research topics were the handling and caring animals, collection of fertilized eggs, selection of normal embryos, and incubation condition for embryos during test. And the mortality and the terra were counted from embryos exposed to a standard teratogen. The validation study for FETAX Phase I and II was done during the following year. From this study, FETAX was stabilized for the generation of test data by our research group. In the third year, FETAX was applied to assess the quality and toxicity of environmental media, such as a sediment. Additionally, the possibility of substituting Xenopus to a domestic frog was studied. IV. Results 1. Toxicity testing of sparingly soluble and volitile chemicals The acute toxicity testing of sparingly soluble chemicals, phenyl salicylate, 2,4-di-tert-butylphenol and dibenzoyl peroxide, were performed with ricefish in terms of static and continuous flow systems. The 96 hr LC50 values generated from continuous flow systems were showed to be 2∼ 27times lower than those from static systems. Also, The acute toxicity testing of volitile chemicals, methyl acrylate, n-heptane and allyl methaclylate, were performed with ricefish in terms of colsed and open systems. The 96 hr LC50 values generated from closed systems were showed to be 2.4∼ 11times lower than those from open systems. From these results it was suggested that the aquatic toxicity testing of sparingly soluble and volitile chemicals should be performed in a proper exposure systems to maintain the concentration in test solution during testing period. In addition, the manual for environmental toxicity testing of pooly soluble and volitile chemical was prepared. 2. Toxicity testing using aquatic organism 1) Fish, embryo and sac-fry toxicity testing Based on the repeated tests on Japanese ricefish/Medaka (Oryzias latipes) and Chinese bleak (Aphyocypris chinensis), embryos of both fishes were affected by toxicity of 63.7 mg/L of zinc concentration with high mortality and low hatchability. On the other hand, severe mortality of sac-fry was occurred from 20 mg/L and 6.25 mg/L of zinc concentration in Japanese ricefish/Medaka and Chinese bleak, respectively. In addition, Chinese bleak showed shorter hatching days than Japanese ricefish/Medaka. Based on the results, we established test method satisfied with required conditions by OECD, found out that Chinese bleak had required conditions for being test fish as Japanese ricefish/Medaka, and had more sensitivity of chemical toxicity under sac-fry condition than Japanese ricefish/Medaka. 2) Lemna toxicity test When Lemna was reared in AAP culture medium, addition of 0.5% sucrose with 9,300 Lux condition showed the best results in terms of growth endpoints such as frond area, wet weight, dry weight, and doubling time. In the case of M-Hoagland's culture medium, only 9,300 Lux condition without 0.5% sucrose addition showed the best results. With the result of toxicity testing using a control substance, EC50 of potassium dichromate was 30 mg/L or 58 mg/L. In the case of 2,4-dichlorophenol, EC50 obtained from three repeated tests ranged 11 to 17 mg/L. 3) Sediment toxicity test Development factor of Chironomus riparius under the laboratory rearing condition was very similar with other's(EPA or Canada Environment). Therefore, it was confirmed that Chironomus riparius was reared successfully under the laboratory condition. With the results of repeated tests, variation of toxicity values among three tests was very low so that the precision in toxicity test was pretty high. With the sensitivity of development stage in larvae, sensitivity of first larvae on cadmium and pentachlorophenol was 28 and 2 times higher than third larvae's, respectively. According to the result, it was revealed that first larvae were more sensitive on the chemicals. Among the LC 50s in endpoints as survival, growth, and adult emergence rates, the growth rate was the most sensitive endpoint. On the other hand, the adult emergence rate was the most sensitive endpoint with determination of NOEC(no observed effect concentration) and LOEC(lowest observed effect concentration) 3. Toxicity test of terrestrial plants In the toxicity testing of m-nitrobenzene on test species, mean germinations of the control in wheat, lettuce and turnip were 93, 83, and 88%, respectively. In addition, EC50s of endpoints such as wet weight, dry weight, and number of germination were greater than 100 mg/kg in wheat and turnip, ranged 10 to 100 mg/kg in lettuce. In the case of 3,4-dinitrotoluene, mean germinations of the control were 91, 87, and 91% in wheat, lettuce, and turnip, respectively, and EC50s of the endpoints were greater than 100 mg/kg in all species. 4. Fate of chemicals in water and soil 1) Photolysis test We studied on light source, filter, test vessel, and temperature control in system with artificial light. In light sources, xenon arc and mercury lamps with borosilicate filter were applicable in two guidelines. Distribution of spectrum using xenon arc lamp with borosilicate filter was very similar with natural light so that it was judged to apply conversion of photolysis rate in natural light more effectively. Test vessel was recommended vessel materials according to wavelength of absorption by US EPA due to transmission rate of light, however, it was recommended proper materials of test vessel by OECD guideline regardless of transmission rate. Photolysis rate was variable depending on vessel materials. However, because photolysis rate could be standardized with measuring radiation intensity, it was considered that vessel of pyrex or borosilicate could be used regardless of transmission rate by OECD. Test temperature was required to maintain 25 5 by OECD and 20∼30 ( 2 ) by US EPA, but it was difficult to maintain the range due to radiated heat by lamp. Therefore, temperature could be maintained to 25 using test vessel with water jacket. There was difference of photolysis rate between two commercial equipments, suntest and weather-ometer, however, it was due to difference of lamp watt. Therefore, both equipments were applicable for the photolysis. 2) Soil metabolism and biodegradation using soil microorganisms The soil metabolism and soil biodegradation of 14C-flupyrazofos was performed in flow-through system under aerobic condition. Test soil was loam as representitive soil property of paddy soil in Korea. The study was performed with sampling intervals(0, 4, 7, 14, 30, 60 days) in 20 2 , dark condition. With the result, number of total microorganism was 97 106(No./g of soil) during the test duration, applied radiocarbon of 14C-flupyrazofos was decreased to 52% at 30 days after treatment, and half-life was 31.5 days(R2=0.91). 5. Study on new techniques for toxicity evaluation of environmental hazardous chemicals 1) Mouse lymphoma thymidine kinase (tk)+/- gene assay First of all, we performed mouse lymphoma thymidine kinase +/- gene forward mutation assay (MOLY). in vitro. The environmental hazardous materials, benzoyl chloride and 2-nitroaniline. were subjected to MOLY with mouse lymphoma L5187Y tk+/- 3.7.2c cells using the microtiter cloning techniques, and the results were analyzed with Mutant V2.34 program. In the range finding cytotoxicity test with benzoyl chloride, the concentration range(>20% relative survival) was determined as 32-500 g/ml in the absence of S9 mixture, and 38-600 g/ml in the presence of S9 mixture. The statistical significance(3hr treatment) of the mutation frequency(MF) of benzoyl chloride was revealed in the absence of S9 mixture with 500 g/ml and in the presence of S9 mixture with 600 g/ml. In the range finding cytotoxicity test with 2-nitroaniline, the concentration range(>20% relative survival) was determined as 40-625 g/ml in the absence and presence of S9 mixture. The statistical significance(3hr treatment) of the mutation frequency(MF) of 2-nitroaniline was revealed only in the presence of S9 mixture with 625 g/ml. No dose-dependent increase of MF was not observed both benzoyl chloride and 2-nitroaniline. 2) Single Cell Gel Electrophoresis (Comet) assay To determine the optimal conditions as recommended by IWGTP, we used L5178Y mouse lymphoma cells in the comet assay. The dose-dependent typical DNA migration patterns was induced by MMS, which was well adopted as positive control in comet assay. The optimal concentration of MMS in the absence of S-9 metabolic activation system was determined as 150 M. No remarkable differences were observed in lysis time and unwinding time of the comet assay in mouse lymphoma cells. From these results, we decided the optimal lysis time as 1.5 hr and unwinding time as 20 min. The optimal concentration of benzo(a)pyrene as positive control in the presence of S9 mixture was also determined as 4-5 x 10-6M. Based on these optimal conditions in comet assay, seven phthalate analogs were subjected. Statistical significant increase of tail moment of butyl benzyl phthalate and di-n-octyl phthalate was observed in the absence of S9 mixture. However, the tail moment of diallyl phthalate, diisodecyl phthalate and dibutyl phthalate was increased in the presence of S9 mixture with significance. From these results, it is suggested that phthalate analogs and its metabolite(s) will be also subjected in the comet assay 3) In vitro cytokinesis-block micronucleus assay In vitro cytokinesis block micronucleus assay also adopted to assess the environmental chemicals such as dibutyl phthalate and atrazine. First of all, the optimal concentration of positive controls, mitomycin C and cyclophosphamide was determined as 0.3 g/ml and 3.0 g/ml in the absence and presence of S9 mixture, rspectively. Using the V79 cell line, the frequency of binucleated micronucleus was increased with statistic significance in the absence and presence of S9 mixture as 1 g/ml and 10 g/ml of dibutyl phthalate, respectively. No statistical significant increase of the frequency of binucleated micronucleus was observed in the range of 65.42 - 261.68 g/ml of atrazine in the absence S9 mixture using Big Blue Rat2 cell line. 4) Transgenic Mutagenesis assay system Also, transgenic mutagenesis system, which the most advanced technique in toxicology fields was established. To investigate the mutational spectrum of 1,2-dibromo-3-chloropropane and atrazine which is well known mutagen and carcinogen in mammalian system, we used transgenic Big Blue cell line derived from Rat2 embryonic fibroblast with very useful lambda shuttle vector containing lac I gene as the mutational target. 4.1. 1,2-dibromo-3-chloropropane (DBCP) 4.1.1. Cytotoxicity of DBCP Relative survival of lacI transgenic Big Blue Rat2 fibroblast cells following exposure to a range of concentrations of DBCP was determined by recording their cumulative growth over a period of 180 h. The survival rate relative to medium control was determined as a percentage of the number of cells surviving 120 h after plating. Exposure of lacI transgenic Big Blue Rat2 fibroblast cells to 0.1, 0.2, 0.4, 0.8, and 1.6 mM DBCP resulted in 82, 81, 68, 44, and 5% survival compared to the medium control, respectively. The 50% inhibition concentration (IC50) of DBCP was determined as 0.75 mM in lacI transgenic Big Blue Rat2 fibroblast cells. In addition, dose-dependency (y = -55.3686x + 91.4365) of DBCP for survival rate was observed with correlation efficiency (r2 = 0.9875). 4.1.2. Mutant frequencies (MFs) The mutagenicity of DBCP to the lacI transgene in lacI transgenic Big Blue Rat2 fibroblast cells was determined. The MFs obtained from each independent experiment showed dose-dependency. (ANOVA, P = 0.007). Cells exposed to medium only and to medium containing 1% DMSO (solvent control) showed mean MFs ( 10-5, SEM) of 6.43 0.616 and 5.28 1.086, respectively. The MFs ( 10-5, SEM) of cells exposed to 0.21, 0.39, and 0.75 mM of DBCP were 8.09 1.02, 10.86 2.17, and 12.26 0.79, respectively. Moreover, 0.75 and 0.39 mM DBCP-treated groups showed a statistically significant difference (ANOVA, P < 0.05) compared to the medium control. 4.1.3. Analysis of Mutational spectrum The sequences of lacI genes were determined in mutants recovered from medium and solvent controls (mutation identified in 33 out of 40 mutants, with 28 independent mutations) and 0.75 mM DBCP-induced mutants (mutation identified in 42 mutants, with 40 independent mutations). Mutants were considered to be siblings if the same mutation occurred more than once in independently exposed cell culture samples and considered to represent a single in vitro mutational event by clonal expansion. One mutant was found to have two mutations in DBCP-induced and spontaneous groups. For each mutant, the entire lacI gene (1,080 bp) was sequenced Single base pair substitution mutations are prevalent in both control and DBCP-induced groups. Percentages of single base pair substitutions from recovered mutations were 21 out of 28 (75%) in the control and 31 out of 40 (77.5%) in the DBCP induced group. Of 40 independent DBCP-induced mutations, 31 were single base pair substitutions and 9 (22.5%) were deletions or insertions . Among single base pair substitutions of the DBCP-induced group, it was observed that 25 mutations (62.5%) occurred at G:C base pairs, while 6 (15%) were at A:T base pairs. The predominant type of mutations after DBCP treatment were G:C A:T transition (40%, 16/40), followed by G:C T:A transversion (22.5%, 9/40). In addition, there were six single base frameshift mutations in DBCP-induced group. A double bp, triple bp, and quadruple bp deletions, leading to frameshift mutation were observed. In consequence, 9 frameshift mutations out of 40 total mutations (22.5%) after DBCP-treatment were recorded. The mutation spectrum in the DBCP-induced cells was significantly different (P = 0.05) when compared with controls. G:C A:T transition (47.5%) occurred more than 2-fold when compared with spontaneous groups (21.4%). Also, unlike the spontaneous group (4/6, 67% at CpG site), G:C A:T transition at CpG and nonCpG sites occurred in the same proportion (8/16, 50%). While G:C C:G transversion in control group occupied an enormous proportion (21.4%), few mutation occurred in DBCP treatment group. The background lacI mutational spectrum generated in these experiments was very similar to the background mutational spectra previously reported in lacI transgenic Big Blue Rat2 fibroblast cells, and to those of untreated lacI transgenic rats and mice. Among 19 spontaneous base substitution mutations, two mutations occurred in a stop codon (nonsense mutation) and one silent mutation with no change in amino acid sequence. The remaining mutations were all missense mutations. Specifically, G:C A:T transitions were the predominant mutation in the background spectrum. Many of these (4/6) occurred at CpG dinucleotides. These transition mutations at CpG sites are thought to be a result of hypermethylation of the lacI transgene by mammalian DNA methylase and subsequent deamination of these 5-methylcytosine residues at CpG sites. The DBCP-induced lacI mutation spectrum was significantly different from the spontaneous mutation spectrum. In contrast to the lower rate of G:C A:T transitions that occurred at non-CpG sites in control cells, the proportion of these mutations was increased in DBCP-exposed cells. The most common DBCP-induced mutations were G:C A:T transitions (40%), followed by G:C T:A transversions (22.5%). Among 31 single base pair substitutions, 25 mutations (62.5%) occurred at G:C base pairs while 6 (15%) occurred at A:T base pairs. Consequently, we obtain that DBCP is a potent base substitution mutagen, especially affecting guanine bases. Furthermore this study is the first report of the mechanism involved in DBCP-induced mutagenesis in lacI gene using the transgenic Big Blue mutagenesis system. The possibility of DBCP causing mutations in endogenous genes (proto-oncogene, tumor suppressor gene, repair related gene, etc) strongly suggests that the mechanisms, which allow DBCP to exert its mutagenic effect, are involved in the initiation stage of carcinogenesis. Furthermore, to provide a better understanding of the mechanism of DBCP mutagenesis in vivo, further investigations are required of the mutagenic properties of DBCP and its metabolites in different tissues and in different species. 4.2. Atrazine 4.2.1. Cytotoxicity of atrazine To determine the exposure concentration of atrazine, we performed MTT assay. Relative survival of lacI transgenic Big Blue Rat2 fibroblast cells following exposure to a range of concentrations of atrazine was determined by absorbance at 540 nm. The 50% inhibition concentration (IC50) of atrazine was determined as 654.20 g/ml in Big Blue Rat2 cells. In addition, dose-dependency of atrazine for survival rate was observed with correlation efficiency (r2=0.9279). From this result, we decided the treatment concentrations as 65.42, 130.84, and 261.68 g/ml which is relevant to 5, 10, and 20% cell growth inhibition, respectively. 4.2.2. Mutant frequencies (MFs) The mutagenicity of atrazine to the lacI transgene in lacI transgenic Big Blue Rat2 fibroblast cells was determined. Cells exposed to medium containing 1% DMSO (solvent control) and 100 g/ml MNU (positive control) showed mean MFs ( 10-5, SEM) of 5.57 0.06 and 41 18.0, respectively. The MFs ( 10-5, SEM) of cells exposed to 65.42, 130.84, and 261.68 g/ml atrazine were 5.27 3.79, 5.87 8.14, and 6.13 1.25, respectively. The MFs obtained from each independent experiment showed dose-dependency. (ANOVA, P=0.007). No statistically significant increase in lacI mutant frequency was observed at any of the atrazine exposure concentrations following 24h exposure in Rat2 cells, compared to the medium control (ANOVA, P > 0.05). 6. The development of avian toxity test for the assessment of environmental contaminating materials The acute oral LD50 toxicity values of isazofos, pyraclofos, diazinon and methomyl were determined for Japanese quail and pheasants (Phasianus colchicus) based on OECD guideline. The LD50 of isazofos, pyraclofos and diazinon was 16.26 mg/kg, 41.26 mg/kg and 7.11 mg/kg body weight in female quails respectively. And the LD50 Of each chemicals in male quails was 21.44, 35.64, 8.28 mg/kg body weight respectively. Diazinon was the most susceptible compounds to Japanese quail in both sexes. The LD50 of methomyl was 21.24 mg/kg body weights in female quails, and 28.28 mg/kg body weight in male quails respectively. Diazinon, isazofos and methomyl were more toxic in the female quails than male. The symptoms of poisoning were similar in quails administrated with each chemicals. The clinical signs in Japanese quail were ataxia, salivation, diarrhea, ruffled feather and convulsion at dead point. There were severe hemorrhage and catarrhal inflammation from duodenum to ileum in all compounds. In Japanese quail treated with organophosphorus and carbamate compounds, brain acetylcholinesterase was inhibited by 88-96% at death. The recovery was not observed after 5h in sublethal dose. There was no relationship between toxicity and acetylcholinesterase activity among organophosphate compounds. The LD50 of diazinon, isazofos, pyraclofos and methomyl on male pheasants (Phasianus colchicus) was 11.22 mg/kg, 11.89 mg/kg, 28.28 mg/kg and 67.27 mg/kg body weight. The dietary LC50 toxicity value of isazofos, pyraclofos was 47.88 mg/kg, 91.81 mg/kg body weight in female quails. And the LC50 in male was 47.88 mg/kg, 103.86 mg/kg body weight. There were severe hemorrhage and necrotic villi in lamina propria in all compounds at death. The recovery was after 1 week in sublethal dose. 7. A Study for FETAX as a Biassay for Teratogenecity of Chemicals The preliminary study for the establishment of FETAX(the first part of the result section in the final report) was carried out during the first research year. As results, methodologies for handling and breeding of frogs, selection of normal embryos, etc., were set up in our research facility. During the second year, FETAX was stabilized by the validation study of FETAX Phase I and II (the second and third part of the result section). In this study, the malformation of embryos was characterized and scored for calculating the teratogenic index. The tandem bioassay of FETAX/Microtox was developed to use for the purpose of the ecological risk assessment of contaminated environmental matrix in the final research year(the fourth and fifth part of the result section). In a parallel study, the possibility of using a domestic mountain frog, Rana dybowskii as a substituted test animal to Xenopus was evaluated(the sixth part of the result section). In addition, the Test Manual was distributed during workshops to transfer the skill and technique to other possible FETAX user group. V. Plan for Application of Results The outputs from this projects were to set-up the new test methods in each laboratory and to disseminate to the outputs obtained from this projects the pertinent parties. The application of outputs was summarized as following. o The test methods estsblished through this projects should be applied to the laws and regulations on chemicals in Korea. o The manuals published in this projects were distributed to a pertinent parties, e.g. academia, government, and private sectors, which are interested in these study areas and thus could contribute to level-up the their testing capacity. o The methods established in participating laboratory were newly introduced or draft methods in OECD and/or ICH Test Guidelines and the expansion of testing ability of laboratories of this country could participate in an internationa chemical programs, e.g. SIDS and ring-tests in OECD member countries, on an equal footing. o From the viewpoint of environmental management, these methods could be applied to control of a toxic chemicals from wastewater, waste dumping site, and contaminated sediments via the screening the toxicity of environmental samples.
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