Carcinogenesis, Teratogenesis & Mutagenesis

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Establishment of flow cytometric in in vitro micronucleus assay

OU Hongmei,ZHOU Changhui,TU Honggang,HUANG Pengcheng,CHANG Yan*   

  1. China State Institute of Pharmaceutical Industry, Shanghai Institute of Pharmaceutical Industry, National Shanghai Center for New Drug Safety Evaluation & Research, Shanghai 201203, China
  • Received:2014-08-13 Revised:2014-12-21 Online:2015-01-31 Published:2015-01-31

Abstract:

OBJECTIVE: Establish the flow cytometric 96-well microplate-based in vitro micronucleus assay in CHO-K1 cells,and explore the possibility of this method for early genetic toxicity screening during drug discovery. MEHTODS:The test included treatment with and without metabolic activation. For the treatment with metabolic activation,CHO-K1 cells were treated with three different concentrations of cyclophosphamide in the S9 mix medium for 4 h,then incubated with S9-free fresh medium for 20 h. For the treatment without metabolic activation,cells were incubated with three different concentrations of mitomycin C continuously for 24 h. In all cases,after a total of 24 h since initiation of the treatment,cells were processed for microscopic scoring or flow cytometric MN analysis. A flow cytometric method for scoring MN used EMA and SYTOX Green to label the cells in 96-well microplate,and then compared with cytokinesis-block micronucleus assay in cell culture disks based on microscopy. RESULTS:Mitomycin C and cyclophosphamide at different concerntrations caused statistically significant and dose-dependent increasess in micronucleus assay . Non-parametric Spearman's coefficients (rs) is 1.000. CONCLUSION:Similar to literature published, mitomycin C and cyclophosphamide induced positive results in flow cytometric based in vitro micronucleus assay. So the method of flow cytometric 96-well microplate-based in vitro micronucleus assay in CHO-K1 cells was established. The concordance between microscopic scoring and flow cytometric was good,therefore this method is promising for screening and evaluating genetic toxicity of chemicals.

Key words: CHO-K1 cells, in vitro micronucleus assay, flow cytometric, 96-well microplate