유전자수준에서 돌연변이 유발기전을 밝히는 Transgenic Mutagenesis Assay

류재천,윤지윤,조경혜,장일무,Ryu, Jae-Chun,Youn, Ji-Youn,Cho, Kyung-Hae,Chang, Il-Moo 한국환경성돌연변이발암원학회 1998 한국환경성돌연변이·발암원학회지 Vol.18 No.1

Transgenic animal and cell line models which are recently developed and used in toxicology fields combined with molecular biological technique, are powerful tools to study the mechanism of mutation in vivo and in vitro, respectively. Transgenic models, which have exogenous DNA incorporated into their genome, carry recoverable shuttle vector containing reporter genes to assess endogenous effects or alteration in specific genes related to disease processes. The lac I and lac Z gnee most widely used as a mutational target in transgenic systems. The assay is performed by treatment with putative mutagenic agents, isolation of genomic DNA from cells or tissues, exposure the isolated DNA to in vitro packaging extract, plating and sequencing. The results from these processes provide not only mutant frequency as quantitative evaluation but also mutational spectrum as qualitative evaluation of various agents. Therefore we introduce and review the principle, detailed procedure and application of transgenic mutagenesis assay system in toxicology fields especially in mutagenesis and carcinogenesis.

DK1002에 대한 급성독성시험 및 유전독성에 관한 연구

류재천,김경란,김현주,정상운,김명국,박희석,김용해,Ryu, Jae-Chun,Kim, Kyung-Ran,Kim, Hyun-Joo,Jung, Sang-Oun,Kim, Myung-Kuk,Park, Hee-Sock,Kim, Yong-Hae 한국독성학회 1998 Toxicological Research Vol.14 No.3

The acute and genetic toxicity of DK1002 was subjected in this study. DK1002 which is a morphine-like new drug candidate synthesized by Dong-Kook Pharmaceutical Co. Ltd. is now under developing as a analgesics that have better drug efficacy and least addictive property. In acute toxicity study, the 50% lethal doses ($LD_{50}$) of DK1002 were determined as>2000mg/kg (p.o.), 237.0mg/kg(i.p.), 57.5mg/kg(i.v.), and 1266.9mg/kg (s.c.). And also, to study the genotoxicity of DK1002, we performed bacterial reversion assay with Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and in vitro chromosomal aberration assay with Chinese hamster lung cells in the presence and absence of S-9 metabolic activation system. In vivo micronucleus assay using mouse bone marrow cells was also performed. From these results, DK1002 was revealed nonmutagenic potential in S. typhimurium TA98, TA100, TA1535, and TA537 both in the absence and presecne of metablic activation system. No clastogenicity of DK1002 was observed in chromosomal aberration assay in vitro as well as in micronucleus assay in vivo.

Chitosan-Phthalocyanine 결합체(Eco-Blue)의 중금속 흡착 및 제거 능력

류재천 ( Jae Chun Ryu ),전희경 ( Hee Kyung Jeon ),박경수 ( Kyung Su Park ) 한국키틴키토산학회 2004 한국키틴키토산학회지 Vol.9 No.2

진보된 세포, 분자 독성평가기법

류재천(Jae-Chun Ryu) 한국농약과학회 2002 농약과학회지 Vol.6 No.3

합성화학물질들의 유전독성평가(Ⅷ)-마우스의 골수세포를 이용한 8종 합성화학물질들의 생체내 소핵시험

류재천(Jae-Chun Ryu),김경란(Kyung-Ran Kim),김연정(Youn-Jung Kim) 환경독성보건학회 2003 환경독성보건학회지 Vol.18 No.2

합성화학물질들이 환경중에 유입되어 인체에는 물론 환경생태계에 많은 영향을 미치고 있어 이들의 유해성 검증은 매우 중요한 일이라 할 수 있다. 실제 산업체에서 사용되는 수많은 화학물질들의 유전적 손상 유발유무는 더욱이 중요한 일이라 할 수 있다. 이에 산업체 공정과정에서 널리 사용되는 것으로 알려진 8종의 합성화학물질에 대해 마우스의 골수세포를 이용한 in vivo 소핵시험을 수행하여, 소핵형성 유발유무를 관찰하였다. 양성내조군으로 사용된 mitomycin C는 음성대조군과 비교시 유의하게 소핵을 유발하는 반면, 비교적 마우스에서 높은 50% 치사량을 보이는 phenylisocyanate, m-aminochlorobenzene 및 2-chloro-4-nitroaniline등의 합성물질들을 비롯한 나머지 5종의 물질들은 본 실험결과 통계적으로 유의하게 소핵을 유발하지 않는 것을 관찰할 수 있었다. To validate and to estimate the chemical hazard play a very important role to environment and human health. The detection of many synthetic chemicals used in industry that may pose a genetic hazard in our environment is of great concern at present. Since these substances are not limited to the original products, and enter the environment, they have become widespread environmental pollutants, thus leading to a variety of chemicals that possibly threaten the public health.<br/> In this respect, the clastogenicily of 8 synthetic chemicals was evaluated with bone marrow micronucleus assay in mice. The positive control, mitomycin C (2mg/kg, i.p.) revealed significant induction ratio of percentage of micronucleated polychromatic erythrocytes/1,000 polychromatic erythrocytes compared to carboxymethylcellulose control. The chemicals with relatively high LD_(50) value such as phenylisocyanate(CAS No. 103-71-9), m-aminochlorobenzene(CAS No. 108-42-9) and 2-chloro-4-nitroaniline(CAS No. 121-87-9) revealed no significant inducion of micronucleated polychromatic erythrocytes in mice. From this results. 8 synthetic chemicals widely used in industry have revealed no significant micronucleus induction of clastogenicity in mice in this experiment.

Evaluation of the Genetic Toxicity of Systhetic Chemicals(Ⅸ) - a Synthetic Selective Herbicide, Pretilachor -

류재천(Jae-Chun Ryu),김연정(Youn-Jung Kim) 환경독성보건학회 2004 환경독성보건학회지 Vol.19 No.1

Pretilachlor[2-chloro-N-(2, 6- diethylpheny1)-N-(2-propoxyethy1) acetamide, C??H??CINO₂M.W.=311.9 CAS No. 51218-49-6]는 제초제의 일종으로 온 연구에서는 박테리아 복귀 돌연변이 시험과 포유동물 세포를 이용한 염색체 이상 시험 및 마우스를 이용한 in vivo 소핵 시험을 수행하여 pretilachlor의 유전독성을 평가하였다.<br/> 박테리아 복귀 돌연변이 시험에서 pretilachlor는 salmonella typhimurium TA98, TA 100, TA1535, TA1537 균주의 대사 활성계 존재 및 부재시 J313~5,000㎍/plate의 범위에서 농도의존적인 돌연변이율의 증가를 관찰할 수 없었다.<br/> 또한 포유동물 세포인 Chinese hamster lung(CHL) fibroblast를 이용한 염색체 이상 시험에서 preti-lachlor는 대사 활성계 존재 및 부재시 1.56~6.24㎍/mL의 농도에서 clastogenicity를 보이지 않았고, 137.5~550,1㎎/㎏의 pretilachlor를 복강 주사한 마우스의 골수세포를 이용한 in vivo 소핵 시험의 결과에서도 통계적으로 유의한 소핵 유발능을 관찰할 수 없었다.<br/> <br/>

Gas-Chromatography/Mass Selective Detector를 사용하여 쥐의 뇨시료 중 benzidine 대사체의 확인 및 in vitro 독성

류재천(Jae Chun Ryu),권오승(Oh Seung Kwon) 대한약학회 2000 약학회지 Vol.44 No.5

Metabolism study of the dye, benzidine, was performed by gas chromatography-mass selective detector (GC/MSD) in the urine of rats orally administered 100mg/kg benzidine. Urine samples were collected in metabolic cages for 0-24, 24-48, and 48-72 hrs. Ten ml of the urine was extracted with XAD-2 resin and the XAD-2 column was eluted with methanol. After evaporation, benzidine and its metabolites were extracted with diethyl ether (for non-conjugated fraction). For conjugated metabolites, beta-glucuronidase was added to the aqueous layer that was incubated for 1hr at 50oC and the aqueous layer was extracted as in non-conjugated fraction. Aliquot of trimethylsilylated derivatives was applied to the GC/MSD. The mutagenicity of benzidine and its acetylated metabolites was tested by histidine/reversion assay. Five metabolites observed and confirmed either by electron impact and chemical ionization modes of the GC/MSD, or authentic compounds were monoacetyl-, diacetyl-, hydroxyacetyl-, hydroxydiacetyl-, and hydroxy-benzidine. Monoacetyl-benzidine was more potent than benzidine in histidine/reversion assay. This data indicates that monoacetylation of benzidine may be one of the metabolites produced in metabolic activation process.

Mitomycin C 유도 소핵 생성 유발에 대한 배추김치 및 부추김치 추출물의 마우스 말초혈에서의 억제 효과

류재천(Jae-Chun Ryu),박건영(Kun-Young Park) 한국환경성돌연변이발암원학회 2001 한국환경성돌연변이·발암원학회지 Vol.21 No.1

Kimchi is a major Korean traditional fermented food, as a supplying source of vitamins and minerals which is prepared with various vegetables and condiments such as red pepper, garlic and salted fish etc. There are many types of Kimchi depending on the ingredients and preparation methods used. To investigate the<br/> clastogenicity and anticlastogenicity of Baechu (Chinese cabbage) Kimchi and Buchu (leek, Allium odorum) Kimchi in mouse, it was performed acridine orange supravital staining of micronucleus (AOSS-MN) assay using mouse peripheral reticulocytes. Baechu Kimchi and Buchu Kimchi were cultivated by organic agricultural technique,<br/> and Kimchi samples were prepared by methanol extraction and lyophilization. First of all, it was studied the clastogenicity of two Kimchi samples themselves (250-1,000 mg/kg) after oral adminstration in mouse. And also to study the anticlastogenic effect of oral administration of Kimchi samples, mitomycin C (MMC, 1 mg/kg, i.p.) was used as micronucleus inducing agent in this study. Dosing scheme was performed as simultaneous (co-treatment),<br/> 3 hr before (pre-treatment) and 3 hr after (post-treatment) with MMC treatment. Two Kimchi samples in the range of 250-1,000 mg/kg did not reveal any clastogenic effect in AOSS-MN assay in mouse. They also revealed anticlastogenic effects in post-treatment of Baechu Kimchi (1,000 mg/kg), and in pre-treatment of Buchu Kimchi (500 and 1,000 mg/kg) with statistical significance. The anticlastogenic effect revealed 1 and 6 hr after treatment of Baechu Kimchi, and Buchu Kimchi with 3 and 6 hr pretreatment. Consequently, it is suggested that antimutagenic and anticlastogenic mechanisms of Baechu and Buchu Kimchi in vivo attributed to sipindle formation and kinetic behavior of mutagens such as absorption and metabolism etc.

합성화학물질들의 유전독성평가(Ⅵ)-Chinese hamster lung 세포를 이용한 17종 합성화학물질들의 염색체 이상 시험-

류재천(Jae-Chun Ryu),김경란(Kyung-Ran Kim),김연정(Youn-Jung Kim),전희경(Hee-KyungJeon) 환경독성보건학회 2003 환경독성보건학회지 Vol.18 No.2

합성화학물질들이 환경중에 유입되어 인체에는 물론 환경생태계에 많은 영향을 미치고 있어 이들의 유해성 검증은 매우 중요한 일이라 할 수 있다. 실제 산업체에서 사용되는 수많은 화학물질들의 유전적 손상 유발유무는 더욱이 중요한 일이라 할 수 있다. 이에 따라 산업체 공정과정에서 널리 사용되는 17종의 화학물질에 대해 Chinese hamster lung(CHL) 세포를 이용한 염색체 이상 시험을 수행하여 이들이 염색체상에 구조적 이상을 유발하는지 관찰하였다. 그 결과, 본 연구에서 사용한 17종의 합성화학물질 중 2-nitroaniline(CAS No 88-74-4)만이 대사활성화계 부재시 86.3μg/ml의 농도에서 통계적으로 유의한 염색체 이상 유발능을 보였다. 반면 가장 높은 세포독성을 보인 1-chloroanthraquinone(CAS No 82-44-0)은 0.8~3.0μg/ml의 시험농도범위에서 대사활성 존재 유무와 무관하게 염색체 이상을 유발하지 않았으며, 다른 15종의 물질들 역시 본시험 적용 농도 범위에서 염색체 이상 유발능을 관찰할 수 없었다. The validation of many synthetic chemicals that may pose a genetic hazard in our environment is of great concern at present. Since these substances are not limited to the original products, and enter the environment, they have become widespread environmental pollutants, thus leading to a variety of chemicals that possibly threaten the public health. In this respect, the regulation and evaluation of the chemical hazard play a very importaut role to eovironment and human health.<br/> The c1astogenicity of 17 synthetic chemicals was evaluated in Chinese hamster lung fibroblast cells in vitro. 2-Nitroaniline (CAS No. 88-74-4) induced chromosomal aberrations with statistical significance at the concentration of 86.3μg/ml in the absence of metabolic activation system. 1-Chloroanthraquinone (CAS No. 82-44-0) which is one of the most cytotoxic chemical among 17 chemicals tested revealed no c1astogenicity in the range of 0.8~3.0μg/ml both in the presence and absence of S-9 metabolic activation system.<br/> From the results of chromosomal aberration assay with 17 synthetic chemicals in Chinese hamster lung cells in vitro, 2-Nitroaniline (CAS No. 88-74-4) revealed weak positive c1astogenic results in this study.

내분비계 장애물질의 개요와 검색법의 고찰

류재천(Jae-Chun Ryu) 한국농약과학회 2002 농약과학회지 Vol.6 No.3