연구논문 : 유전독성 대체시험법(in vitro소핵시험)의 국내검증시험연구

이현걸 ( Hyun Kul Lee ),김종극 ( Jong Geuk Kim ),이우주 ( Woo Joo Lee ),맹은호 ( Eun Ho Maeng ),이종윤 ( Jong Yoon Lee ),정영신 ( Young Shin Chung ),김창환 ( Chang Hwan Kim ),김윤순 ( Yun Soon Kim ),장미해 ( Mi Hae Jang ),이석종 ( 한국동물실험대체법학회 2011 동물실험대체법학회지 Vol.5 No.1

This validation study was performed to introduce and set up the in vitro micronucleus test, as an alternative to the existing chromosome aberration test, in the test facilities in Korea, and to establish the competence of each facility to perform the in vitro micronucleus test independently. The 10 coded substances, including 6 positive and 4 negative in genotoxicity assay, were tested. Six facilities were divided into two subgroups, A and B. Three coded positive compounds and two negative compounds were sent to each test facility. The compounds were tested according to the facility`s GLP system. In group A, positive results were obtained from testing of the three positive compounds, and negative results were obtained from testing of the two negative compounds in all three facilities. In group B, positive results were also obtained from testing of the three positive compounds, and negative results were obtained from testing of the two negative compounds in all three facilities. For the Urethane, an in vitro equivocal compound tested by group B, negative results were obtained. These results suggested that the compound may be effectively activated only in in vivo environment. The results show that the six test facilities are capable of performing the in vitro micronucleus test independently and producing consistent results.

유전독성 대체시험법(in vitro소핵시험)의 국내검증시험연구

이현걸 ( Hyun Kul Lee ),김종극 ( Jong Geuk Kim ),이우주 ( Woo Joo Lee ),맹은호 ( Eun Ho Maeng ),이종윤 ( Jong Yoon Lee ),정영신 ( Young Shin Chung ),김창환 ( Chang Hwan Kim ),김윤순 ( Yun Soon Kim ),장미해 ( Mi Hae Jang ),이석종 ( 한국동물실험대체법학회 2011 동물실험대체법학회지 Vol.5 No.1

This validation study was performed to introduce and set up the in vitro micronucleus test, as an alternative to the existing chromosome aberration test, in the test facilities in Korea, and to establish the competence of each facility to perform the in vitro micronucleus test independently. The 10 coded substances, including 6 positive and 4 negative in genotoxicity assay, were tested. Six facilities were divided into two subgroups, A and B. Three coded positive compounds and two negative compounds were sent to each test facility. The compounds were tested according to the facility`s GLP system. In group A, positive results were obtained from testing of the three positive compounds, and negative results were obtained from testing of the two negative compounds in all three facilities. In group B, positive results were also obtained from testing of the three positive compounds, and negative results were obtained from testing of the two negative compounds in all three facilities. For the Urethane, an in vitro equivocal compound tested by group B, negative results were obtained. These results suggested that the compound may be effectively activated only in in vivo environment. The results show that the six test facilities are capable of performing the in vitro micronucleus test independently and producing consistent results.

감잎차 추출액의 Sister Chromatid Exchange(SCE) 방법에 따른 항돌연변이 효과

송현순(Hyun-Soon Song),이현걸(Hyun-Kul Lee),장해동(Hae-Dong Jang),김종익(Jong-Ik Kim),박옥진(Ock-Jin Park),이미숙(Mee-Sook Lee),강명희(Myung-Hee Kang) 한국식품영양과학회 1996 한국식품영양과학회지 Vol.25 No.2

돌연변이 유발 물질인 mitomycin C(MMC)를 처리하여 배양한 Chinese hamster ovary(CHO) cell에 대한 감잎차 추출액의 항돌연변이 효과를 자매 염색 분체 교환(sister chromatid exchange, SCE) 시험법을 사용하여 측정하여 보았다. 감잎차 추출액 자체는 CHO 세포의 SCE 빈도수를 변화시키지 않았으며, 세포의 분열 주기중 S phase에 S9 mixture없이 감잎차 추출액이 처리되었을 경우 MMC로 유도된 SCE 빈도수를 감소시키지 않았다. 그러나 S9 mixture 존재하에 G₁ phase에서 MMC 처리 후 감잎차를 처리하는 후처리 방식으로 감잎차 추출액을 처리하였을 때, 저농도(≤40㎍/ml)에서 MMC로 인해 유발된 SCE 빈도수가 낮아지는 것을 볼 수 있었다. 이에 비해 고농도 (>40㎍/ml)에서는 SCE 빈도수의 감소 효과가 없었다.<br/> 본 연구 결과, MMC 처리된 CHO 세포에 대한 감잎차 추출액의 항돌연변이 효과를 볼 수 있었고, 이 효과는 S9 mixture 존재하에서 저농도의 감잎차 추출액이 G₁ phase에 처리되었을 때 나타났다. 감잎차 추출액의 이러한 항돌연변이 효과의 기전은 감잎차 추출액의 대사산물이 MMC 처리된 CHO 세포에 대한 DNA-excision repair activity를 촉진시키기 때문인 것으로 생각된다. The antimutagenic effects of persimmon leaf tea extracts(PLTE) on mutagen-induced sister chromatid exchanges(SCEs) were studied. These PLTE did not affect spontaneous SCEs in cultured Chinese hamster cells. The frequency of SCEs induced by mitomycin C(MMC) was not affected by the simultaneous treatment with PLTE without S9 mixture in the S phase of the cell cycle. However, when cells were posttreated with PLTE in the presence of metabolic enzymes of rat liver(S9 mix), the antimutagenic effects on the induction of SCEs by mutagen were observed. MMC-induced SCEs were suppressed by the posttreatment with PLTE at low concentrations(≤40㎍/ml) with S9 mix. In contrast, at a high concentration(>40㎍/ml) of PLTE with S9 mix, MMC-induced SCEs were not affected. The antimutagenic effects of PLTE were shown to occur in the G₁ phase of the cell cycle. The results suggested that PLTE could promote DNA-excision repair activity and resulted in an antimutagenic effect.

마우스 소핵(Micronuclei, MN) 시험방법을 이용한 감잎차 추출물의 돌연변이 억제효과

송현순(Hyun-Soon Song),이현걸(Hyun-Kul Lee),강명희(Myung-Hee Kang) 한국식품영양과학회 2000 한국식품영양과학회지 Vol.29 No.5

마우스에서 직접 혹은 간접 돌연변이 물질에 의하여 유발된 소핵에 미치는 감잎차 추출물의 억제 효과를 연구한 결과, 사람이 마시는 조건으로 추출한 감잎차 추출물을 하루 5잔 또는 10잔 섭취하는 양으로 투여했을 때 돌연변이 억제효과가 있음을 관찰하였다. 간접 돌연변이 유발물질인 MMC로 유발된 소핵 빈도수는 5잔 섭취량인 PLTE I군의 경우 MMC 투여 6시간 전과 3시간 후에, 10잔 섭취량인 PLTE II군의 경우 MMC 투여 24시간 전과 6시간 전에 감잎차 추출물을 투여하였을 때 마우스골수 소핵의 빈도수가 유의적으로 감소하였다. 이에 비해 직접 돌연변이 유발물질인 4-NQO로 유발된 소핵 빈도수는 PLTE I군의 경우 4-NQO 투여 6시간 전과 3시간후에, PLTE II군의 경우 4-NQO 투여 3시간 전과 3시간후에 감잎차 추출물을 투여했을 때 소핵의 유의한 감소를 나타내었다. 이와 같은 결과는 선행연구에서 녹차나 차의 주성분인 catechin이나 tannic acid를 돌연변이 물질 투여전에 투여했을 때에만 소핵 빈도수를 감소시켰던 것과 비교해 볼 때 감잎차에는 이들 물질 외에 아직 알려지지 않은 다른 돌연변이 억제 물질이 있는 것으로 생각되며 이에 관해서는 앞으로 더 자세한 연구가 요구된다. The antimutagenic effects of persimmon leaf tea extract (PLTE) at concentration levels consumed by human were examined in mice using micronucleus induction with MMC (mitomycin C) or 4-NQO (4-nitroquinoline-1-oxide). When mice received oral gavage of 10 equivalent to PLTE 24 hr and 6 hr before, and 5 equivalent to PLTE 6 hr before and 3 hr after intraperitoneal injection of MMC, a significant decrease in the frequency of micronuclei were observed. The induction of micronuclei by 4-NQO was suppressed by oral dosage of PLTE at 5 equivalent to PLTE 6 hr before and 3 hr after, 10 equivalent to PLTE 3 hr before and 3 hr after intraperitoneal injection of MMC. Though the components of PLTE have not been analyzed so far, our present results suggest the existence of several bio-antimutagens and/or desmutagens in PLTE, beside catechin, well-known antimutagen.

감잎차,녹차,우롱차 추출물이 돌연변이 물질로 유발된 Sister Chromatid Exchanges 빈도에 미치는 영향

송현순(Hyun-Soon Song),이현걸(Hyun-Kul Lee),최언호(Eon-Ho Choi),강명희(Myung-Hee Kang) 한국식품과학회 1999 한국식품과학회지 Vol.31 No.3

Salmonella typhimurium Strain TA98, 100에서 감잎차, 녹차, 우롱차 추출물의 돌연변이 억제효과

송현순(Hyun-Soon Song),이현걸(Hyun-Kul Lee),강명희(Myung-Hee Kang) 한국식품영양과학회 1999 한국식품영양과학회지 Vol.28 No.3

실제 음용 조건으로 추출한 한국산 감잎차, 녹차, 우롱차 추출물의 돌연변이 억제효과를 보기 위하여 간접 돌연변이 물질로 polycyclic aromatic hydrocarbon인 B[a]P, heterocyclic amine인 IQ, aromatic amine인 2-AA, 그리고 직접 돌연변이 물질인 4-NQO에 대하여 salmonella/microsome assay를 응용하여 실험하였다. 실험 결과, 음용하는 조건으로 추출한 감잎차, 녹차, 우롱차 추출물에는 간접 돌연변이 물질에 의한 colony 생성에 미치는 돌연변이 억제 효과가 탁월함을 볼 수 있었다. 간접 돌연변이 물질 중 B[a]P이나 IQ의 경우는 차 추출물의 농도 증가에 따라 돌연변이 물질에 대하여 농도 의존적으로 colony 생성을 억제시키었으나 2-AA의 경우는 저농도 첨가시 co-mutagenic하게 작용하여 colony 수를 증가시키다가 고농도 첨가시 colony 생성을 억제시킴을 보였다. 이와는 달리 직접 돌연변이 물질인 4-NQO의 경우는 저농도 첨가시에는 큰 변화가 없다가 고농도 첨가시 colony 생성을 더 증가시키는 결과를 보였다. 돌연변이 억제효과의 작용 양상을 보면, 각 차 추출물에 따른 차이는 크게 볼 수 없었으며 TA98과 TA100 등 균주에 따른 경향의 차이도 볼 수 없었으나 돌연변이 물질의 종류에 따라서는 많은 차이를 보였다. 본 실험 결과 실제 음용하는 조건으로 추출한 한국산 감잎차, 녹차, 우롱차 추출물이 간접 돌연변이 물질들에 대하여 억제효과를 나타내는 것을 확인할 수 있었으며, 직접 돌연변이 물질에 대해서는 억제효과를 볼 수 없었다. 이 결과로부터 본 연구에서 사용한 차 추출물의 돌연변이 억제효과 기전은 각 차 추출물들이 돌연변이 물질과 직접 결합하여 돌연변이를 억제한다기보다는 돌연변이 물질의 체내 활성화 대사과정을 억제하는 물질로 작용하는 것으로 생각되었다. Water extracts of persimmon leaf tea(PLTE), green tea(GTE) and oolong tea(OTE), at the concentration used for human consumption, were examined for inhibitory effects on the mutagenicity of major classes of dietary and environmental mutagens including indirect-acting mutagens, B[a]P(benzo[a]pyrene), IQ(2-amino-3-methylimidazo[4,5-f]quinoline), 2-AA(2-aminoanthracene) in the presence of S9 mix and direct-acting mutagen, 4-NQO(4-nitroquinoline-1-oxide) without S9 mix, using the modified Ames Salmonella/microsome assay. PLTE, GTE and OTE showed very potent and concentration-dependent antimutagenic effects against indirect-acting mutagens B[a]P and IQ. At the maximum concentration(16,200㎍/plate) of each tea extract, number of colonies decreased in a dose dependent manner up to 82~100%. Similar inhibition of PLTE, GTE and OTE were seen at higher concentration in the mutagenicity of the 2-AA following an initial increase in the activity at lower concentration. However, the mutagenicity of the direct-acting mutagen 4-NQO were not suppressed at lower concentration of the three tea extracts, and higher concentration of the tea extracts enhanced mutagenic activity of the mutagen. There were no differences in the mode of antimutagenesis between PLTE, GTE, and OTE, in both Salmonella typhimurium TA98 and TA100 strains against the same mutagen. In conclusion, the water extracts of persimmon leaf tea, green tea and oolong tea possess marked antimutagenic potential against a variety of important dietary and environmental indirect-acting mutagens, but the activity was not observed against the direct-acting mutagens. These results suggest that the mode of inhibitory action may not have resulted from direct interaction between tea extracts and the mutagens, but rather from indirect metabolic inactivation of mutagens by tea extracts.

랫드에서 CONP01에 대한 4주 반복투여 독성연구

흥동호(Dong Ho Hong),장호송(Hu-Song Zhang),김광호(Kwang-Ho Kim),길기현(Gi Hyun Gil),김재민(Jae Min Kim),한명규(Myong Kyu Han),배진숙(Jin-Sook Bae),김남두(Nam Du Kim),이현걸(Hyun Kul Lee),이정숙(Jung Suk Lee),이선경(Sun Kyung Lee),박찬구 한국독성학회 2004 Toxicological Research Vol.20 No.4

This study was performed to evaluate repeated-dose toxicities of CONP01 in Sprague-Dawley rats. CONP01, a new antiarthritic agent was administered orally to rats at dose levels of 0, 125, 500 and 2,000 mg/kg/day for 4 weeks. In present study, there were no dose response changes in mortality, clinical signs, body weight changes, food and water consumption, ophthalmoscopy, organ weights, urine analysis, hematological findings, and biochemical examination of all animals treated with CONP01. Gross and histopathological findings revealed no evidence of specific toxicity related to CONP01. These result suggest that no observed adverse effect level (NOAEL) of CONP01 may be over 2,000 mg/kg in rats.