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Mutagenesis Advance Access published online on October 20, 2009
Mutagenesis, doi:10.1093/mutage/gep040
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© The Author 2009. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

Further evaluation of a flow cytometric in vitro micronucleus assay in CHO-K1 cells: a reliable platform that detects micronuclei and discriminates apoptotic bodies

Jing Shi*, Rahel Bezabhie and Anna Szkudlinska

Department of Genetic Toxicology, BioReliance Corporation, 14920 Broschart Road, Rockville, MD 20850, USA

The in vitro micronucleus (MN) assay is widely used to assess genotoxic potential of the test substances by measuring frequency of MN in cultured mammalian cells. Traditionally, MN frequency has been determined by microscopy. In recent years, a flow cytometric method for enumeration of MN has been developed, which significantly shortens analysis time and enhances assay throughput. However, a major concern has been raised that the MN results obtained from flow cytometry can be impacted by chromatin bodies produced during apoptosis or necrosis. In this work, we further evaluated this flow cytometry-based in vitro MN assay with CHO-K1 cells in a 24-well platform. Our results showed that the MN frequency determined using the flow cytometric method was highly correlated with the microscopy results. Importantly, several non-genotoxic apoptosis inducers or cytotoxins that have been previously reported to produce ‘artificial positives’ in various in vitro genotoxicity tests were evaluated in this system. As a result, these non-genotoxic cytotoxins did not produce false-positive MN response in the flow cytometric system in CHO-K1 cells when cytotoxicity was <50 ± 10%. Moreover, a total of 21 compounds were evaluated in this work, including direct or indirect clastogens, aneugens and non-genotoxic chemicals. A sensitivity of 83.3% and a specificity of 100% were obtained from the compounds we tested. Finally, significant increase of incidents in the hypodiploid region, an aneugenic signature, was confirmed in our evaluation. In conclusion, the flow cytometric in vitro MN assay is a reliable method that can be used to detect clastogenic or aneugenic potential of the test substances in CHO-K1 cells.

* To whom correspondence should be addressed. Tel: +1 301 6102615; Fax: +1 301 7382362; Email: jing.shi{at}bioreliance.com

Received on August 14, 2009; revised on September 17, 2009; accepted on September 18, 2009.


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