Korean Journal of Environmental Agriculture (한국환경농학회지)

The Korean Society of Environmental Agriculture (한국환경농학회)
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Study on Optimization of Cytoplasm Conditions for In Vitro Micronucleus Test Using Chinese Hamster Lung Cells

Chinese Hamster Lung Cell을 이용한 in vitro 소핵시험의 세포질 최적화 연구

  • Paik, Min Kyoung (Chemical Safety Division, Department of Agro-food Safety and Crop Protection, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Kim, Areumnuri (Chemical Safety Division, Department of Agro-food Safety and Crop Protection, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Shin, Hye Rim (Chemical Safety Division, Department of Agro-food Safety and Crop Protection, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Chon, Kyongmi (Chemical Safety Division, Department of Agro-food Safety and Crop Protection, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Park, Kyung-Hun (Chemical Safety Division, Department of Agro-food Safety and Crop Protection, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Ryu, Ji-Hyeok (Chemical Safety Division, Department of Agro-food Safety and Crop Protection, National Institute of Agricultural Sciences, Rural Development Administration) ;
  • Moon, Byeong Chul (Chemical Safety Division, Department of Agro-food Safety and Crop Protection, National Institute of Agricultural Sciences, Rural Development Administration)
  • 백민경 (농촌진흥청 국립농업과학원 농산물안전성부 화학물질안전과) ;
  • 김아름누리 (농촌진흥청 국립농업과학원 농산물안전성부 화학물질안전과) ;
  • 신혜림 (농촌진흥청 국립농업과학원 농산물안전성부 화학물질안전과) ;
  • 전경미 (농촌진흥청 국립농업과학원 농산물안전성부 화학물질안전과) ;
  • 박경훈 (농촌진흥청 국립농업과학원 농산물안전성부 화학물질안전과) ;
  • 류지혁 (농촌진흥청 국립농업과학원 농산물안전성부 화학물질안전과) ;
  • 문병철 (농촌진흥청 국립농업과학원 농산물안전성부 화학물질안전과)
  • Received : 2018.08.14
  • Accepted : 2018.09.21
  • Published : 2018.09.30
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Abstract

BACKGROUND: in vitro micronucleus test (vitMNT) is one of the promising alternative testing methods in genotoxicity test and was adopted as OECD test guideline for chemical registration. This study was conducted to optimize the cytoplasm conditions in vitMNT using Chinese hamster lung (CHL) cell. METHODS AND RESULTS: In this study cytokinesis-block micronucleus test was conducted. Mitomycin C and colchicine were used as positive control chemicals and were treated for three hours (short time) or twenty-four hours (long time). Giemsa solution was used for cell staining. For optimization of vitMNT, the final fixative was prepared as five concentrations (0%, 1%, 3%, 5%, and 25%) of acetic acid in methanol, and treatment times of the final fixative were varied under four conditions (immediately, one hour, four hours, and one day). CONCLUSION: Acetic acid at 1% in methanol as the final fixative was most adequate to preserve the cytoplasm around the nucleus in the interphase cells. Also, fixative treatment time of cell suspension for one to four hours may minimize the cell rupture. These results can be helpful for getting an accurate result promptly due to clear visual distinction to score micronucleus in vitMNT using giemsa solution.

In vitro 소핵시험(vitMNT)은 유전독성의 유망한 대체시험법 중 하나로, OECD에서 TG로 채택되어 화학물질의 등록에 사용되고 있다. 본 시험에서는 CHL cell을 사용한 vitMN test에서 소핵을 판별하기 위한 최적화된 세포질 조건을 찾고자 하였으며, 양성대조물질로 MMC와 Col을 사용하고 세포 염색을 위해 giemza 용액을 사용하였다. 시험결과, 세포현탁을 위해 사용되는 고정액의 acetic acid의 농도는 1%로 하는 것이 band의 두께와 세포질의 퍼짐성 측면에서 적당하였다. 또한 세포의 깨짐을 최소화하는 최종 고정액의 적하시간은 현탁 후 1~4시간이었다. 이러한 결과는 vitMNT에서 소핵관찰의 신속성, 용이성 및 정확성을 확보하는데 도움이 될 것이다.

Keywords

References

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