Flow cytometric 96‐well microplate‐based in vitro micronucleus assay with human TK6 cells: Protocol optimization and transferability assessment

An automated approach for scoring in vitro micronuclei (MN) has been described in which flow cytometric analysis is combined with compound exposure, processing, and sampling in a single 96‐well plate (Bryce SM et al. [2010]: Mutat Res 703:191‐199). The current report describes protocol optimization and an interlaboratory assessment of the assay's transferability and reproducibility. In a training phase, the methodology was refined and collaborating laboratories were qualified by repeatedly testing three compounds. Second, a set of 32 chemicals comprised of reference genotoxicants and presumed non‐genotoxicants was tested at each of four sites. TK6 cells were exposed to 10 closely spaced compound concentrations for 1.5‐ to 2‐cell population doublings, and were then stained and lysed for flow cytometric analysis. MN frequencies were determined by evaluating ≥5,000 cells per replicate well, and several indices of cytotoxicity were acquired. The prevalence of positive results varied according to the MN‐fold increase used to signify a genotoxic result, as well as the endpoint used to define a cytotoxicity limit. By varying these parameters, assay sensitivity and specificity values ranged from 82 to 98%, and 86 to 97%, respectively. In a third phase, one laboratory tested a further six genotoxicants and five non‐genotoxic apoptosis inducers. In these experiments assay specificity was markedly improved when top concentration selection was based on two cytotoxicity endpoints—relative survival and quantification of ethidium monoazide‐positive events. Collectively, the results indicate that the miniaturized assay is transferable across laboratories. The 96‐well format consumes considerably less compound than conventional in vitro MN test methods, and the high information content provided by flow cytometry helps guard against irrelevant positive results arising from overt toxicity. Environ. Mol. Mutagen. 54:180–194, 2013. © 2013 Wiley Periodicals, Inc.

[2]  Raffaella Corvi,et al.  ECVAM retrospective validation of in vitro micronucleus test (MNT) , 2008, Mutagenesis.

[3]  R. D. De Abreu,et al.  Bcl-2 prevents loss of mitochondria in CCCP-induced apoptosis. , 2004, Experimental cell research.

[4]  GUIDANCE DOCUMENT,et al.  Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use , 2008 .

[5]  S. McKiernan,et al.  In vitro micronucleus assay with Chinese hamster V79 cells - results of a collaborative study with in situ exposure to 26 chemical substances. , 2000, Mutation research.

[6]  Stephen D Dertinger,et al.  In vitro micronucleus scoring by flow cytometry: Differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability , 2006, Environmental and molecular mutagenesis.

[7]  M. González-Cid,et al.  Comparison of the aneugenic effect of vinorelbine and vincristine in cultured human lymphocytes. , 1999, Mutagenesis.

[8]  J. Tischfield,et al.  Replication Stress Induces Micronuclei Comprising of Aggregated DNA Double-Strand Breaks , 2011, PloS one.

[9]  M. Kirsch‐Volders,et al.  The in vitro micronucleus test: a multi-endpoint assay to detect simultaneously mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction. , 1997, Mutation research.

[10]  B. Epe,et al.  Reactive oxygen species derived from the mitochondrial respiratory chain are not responsible for the basal levels of oxidative base modifications observed in nuclear DNA of Mammalian cells. , 2004, Free radical biology & medicine.

[11]  J. Bemis,et al.  Miniaturized flow cytometry‐based CHO‐K1 micronucleus assay discriminates aneugenic and clastogenic modes of action , 2011, Environmental and molecular mutagenesis.

[12]  Stephen D Dertinger,et al.  Miniaturized flow cytometric in vitro micronucleus assay represents an efficient tool for comprehensively characterizing genotoxicity dose-response relationships. , 2010, Mutation research.

[13]  Raffaella Corvi,et al.  Recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests: a follow-up to an ECVAM workshop. , 2008, Mutation research.

[14]  R. Walmsley,et al.  Nongenotoxic apoptosis inducers do not produce misleading positive results in the TK6 cell-based GADD45a-GFP genotoxicity assay. , 2012, Toxicological sciences : an official journal of the Society of Toxicology.

[15]  J. Nath,et al.  Inhibition of methotrexate-induced chromosomal damage by folinic acid in V79 cells. , 1998, Mutation research.

[16]  S Albertini,et al.  Comparison of different methods for an accurate assessment of cytotoxicity in the in vitro micronucleus test. I. Theoretical aspects. , 2008, Mutation research.

[17]  T. Sofuni,et al.  Validation study of the in vitro micronucleus test in a Chinese hamster lung cell line (CHL/IU). , 1999, Mutagenesis.

[18]  S. Attia,et al.  Molecular cytogenetic evaluation of the mechanism of micronuclei formation induced by camptothecin, topotecan, and irinotecan , 2009, Environmental and molecular mutagenesis.

[19]  John Nicolette,et al.  Flow cytometric analysis of micronuclei in mammalian cell cultures: past, present and future. , 2011, Mutagenesis.

[20]  Stephen D Dertinger,et al.  In vitro micronucleus assay scored by flow cytometry provides a comprehensive evaluation of cytogenetic damage and cytotoxicity. , 2007, Mutation research.

[21]  P. Carmichael,et al.  Evaluation of an automated in vitro micronucleus assay in CHO-K1 cells. , 2007, Mutation research.

[22]  M. Pallardy,et al.  Apoptosis can be a confusing factor in in vitro clastogenic assays. , 2001, Mutagenesis.

[23]  David M. Reif,et al.  In Vitro Screening of Environmental Chemicals for Targeted Testing Prioritization: The ToxCast Project , 2009, Environmental health perspectives.

[24]  M. Fellows,et al.  Cytotoxicity in cultured mammalian cells is a function of the method used to estimate it. , 2007, Mutagenesis.

[25]  M. Nüsse,et al.  Flow cytometric analysis of micronuclei found in cells after irradiation. , 1984, Cytometry.

[26]  M. Nüsse,et al.  Flow cytometric detection of micronuclei by combined staining of DNA and membranes. , 1995, Cytometry.

[27]  M. Fenech,et al.  Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells. , 2011, Mutagenesis.

[28]  M. Kirsch‐Volders,et al.  In vitro primary human lymphocyte flow cytometry based micronucleus assay: simultaneous assessment of cell proliferation, apoptosis and MN frequency. , 2011, Mutagenesis.

[29]  Stephen D Dertinger,et al.  Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay. , 2008, Mutation research.

[30]  Ronald D Snyder,et al.  SFTG international collaborative study on in vitro micronucleus test III. Using CHO cells. , 2006, Mutation research.

[31]  Jing Shi,et al.  Further evaluation of a flow cytometric in vitro micronucleus assay in CHO-K1 cells: a reliable platform that detects micronuclei and discriminates apoptotic bodies. , 2010, Mutagenesis.