Development and validation of an in vitro micronucleus assay platform in TK6 cells

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• 作者：Zhanna Sobolp> ; p>p>Zhanna.Sobol@pfizer.comp> ; Michael L. Homiski ; Donna A. Dickinson ; Richard A. Spellman ; Dingzhou Li ; Andrew Scott ; Jennifer R. Cheung ; Stephanie L. Coffing ; Jennifer B. Munzner ; Kelley E. Sanok ; William C. Gunther ; Krista L. Dobo ; Maik Schuler
• 关键词：In vitro micronucleus assay ; Aneugen ; Clastogen ; Metabolic activation ; Lymphoblastoid cell line ; TK6
• 刊名：Mutation Research/Genetic Toxicology and Environmental Mutagenesis
• 出版年：2012
• 期刊代码：31_13835718
• 类别：pha
• 出版时间：4 July, 2012
• 卷：746
• 期：1
• 页码：29-34
• 文件大小：212 K

摘要

The Organization for Economic Co-operation and Development (OECD) has recently adopted Test Guideline 487 (TG487) for conducting the in vitro micronucleus (MNvit) assay. The purpose of this study is to evaluate and validate treatment conditions for the use of p53 competent TK6 human lymphoblastoid cells in a TG487 compliant MNvit assay. The ten reference compounds suggested in TG487 (mitomycin C, cytosine arabinoside, cyclophosphamide, benzo-a-pyrene, vinblastine sulphate, colchicine, sodium chloride, nalidixic acid and di(2-ethylhexyl)phthalate and pyrene) and noscapine hydrochloride were chosen for this study. In order to optimize the micronucleus response after treatment with some positive substances, we extended the recovery time after pulse treatment from 2 cell cycles recommended in TG487 to 3 cell cycles for untreated cells (40 h). Each compound was tested in at least one of four exposure conditions: a 4 h exposure followed by a 40 h recovery, a 4 h exposure followed by a 24 h recovery, a 4 h exposure in the presence of an exogenous metabolic activation system followed by a 40 h recovery period, and a 27 h continuous direct treatment. Results show that the direct acting clastogens, clastogens requiring metabolic activation and aneugens caused a robust increase in micronuclei in at least one test condition whereas the negative compounds did not induce micronuclei. The negative control cultures exhibited reproducibly low and consistent micronucleus frequencies ranging from 0.4 to 1.8%(0.8 卤 0.3%average and standard deviation). Furthermore, extending the recovery period from 24 h to 40 h produced a 2-fold higher micronucleus frequency after a 4 h pulse treatment with mitomycin C. In summary, the protocol described in this study in TK6 cells produced the expected result with model compounds and should be suitable for performing the MNvit assay in accordance with guideline TG487.