Chinese Hamster Lung Cell을 이용한 in vitro 소핵시험의 세포질 최적화 연구
Study on Optimization of Cytoplasm Conditions for In Vitro Micronucleus Test Using Chinese Hamster Lung Cells
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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 cytokinesisblock 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.
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 cytokinesisblock 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.
참고문헌 (12)
- Fenech, M, "Cytokinesis-block micronucleus assay evolves into a 'cytome' assay of chromosomal instability, mitotic dysfunction and cell death", Mutation Research, 2006, pp.600(1), 58~66
- Kirkland, D., "Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens: I. Sensitivity, specificity and relative predictivity", Mutation Research, 2005, pp.584(1), 1~256
- Kirsch-Volders, M., "in vitro genotoxicity testing using the micronucleus assay in cell lines, human lymphocytes and 3D human skin models", Mutagenesis, 26(1), pp.177~184 (2011)
- Kirsh-Volders, M., "Towards a validation of the micronucleus test", Mutation Research, 392, pp.1~4 (1997)
- Könen-Adıgüzel, S., "in vitro evaluation of the genotoxicity of CeO2 nanoparticles in human peripheral blood lymphocytes using cytokinesis-block micronucleus test, comet assay, and gamma H2AX", Toxicology and Industrial Health, 34(5), pp.293~300 (2018)
- Lee, H., "Validation study of the in vitro micronucleus test", Journal of Alternatives to Animal Experiments, 5(1), pp.11~50 (2011)
- Lee, Y. H., "Recent advances in micronuclei assay for the monitoring of genotoxic effects of environmental pollutants in human populations", The Korean Journal of Public Health, 47(1), pp.16~25 (2010)
- Matthews, E. J., "An analysis of gentic toxicology, reproductiveand developmental toxicity and carcinogenicity data : I. identification of carcinogens using surrogate end points", Regulatory Toxicology and Pharmacology, 44(2), pp.83~96 (2006)
- Miller, B. M., "Evaluation of the micronucleus test in vitro using Chinese hamster cells: results of four chemicals weakly positive in the in vivo micronucleus test", Environmental and Molecular Mutagenesis, 26(3), pp.240~247 (1995)
- Parry, J. M., "The detection and assessment of the aneugenic potential of environment chemicals: the European Community aneuploidy project", Mutation Research, 287, pp.3~15 (1993)
- Ruzin, S. E., "Plant microtechnique and microscopy", pp.190~203 (1999), Oxford University Press
- Yamamoto, M., "The optimized conditions for the in vitro micronucleus (MN) test procedures using chamber slides", Environmental Mutagen Research, 27(3), pp.145~151 (2005)