High content flow cytometric micronucleus scoring method is applicable to attachment cell lines

A flow cytometric method for analyzing suspension cell cultures for micronucleus content has been previously reported [Environ. Molec. Mutagen. 47 (2006) 56–66]. The experiments described herein were undertaken to evaluate the compatibility of this method (In Vitro MicroFlow®) with attachment cells. Initially, CHO-K1 cells were studied in nine independent experiments using mitomycin C and cyclophosphamide. The results demonstrated the effectiveness of the cell processing procedure, and also provided historical control data that were useful for setting criteria for making positive calls. Subsequently, CHO-K1 cells were treated with methyl methanesulfonate, mitomycin C, etoposide, vinblastine sulfate, dexamethasone, and sodium chloride. Whereas the four genotoxicants were each observed to increase micronucleus frequencies, the non-genotoxicants induced no such response up to cytotoxic concentrations. Following this initial work, inter-laboratory transferability was evaluated across three sites using a common cell staining and analysis protocol for CHO-K1 or V79 cells that had been treated with the ten chemicals listed in Annex 3 of the OECD Draft Proposal for a New Guideline 487: In Vitro Mammalian Cell Micronucleus Test. With the exception of benzo[a]pyrene at one site, each laboratory observed increased micronucleus frequencies for the genotoxicants, whereas no significant induction occurred with the non-genotoxicants. Interestingly, the method appeared to distinguish between genotoxic modes of action, as only aneugens increased the average micronucleus fluorescence intensity and the frequency of hypodiploid nuclei. Collectively, these data suggest that flow cytometry is capable of providing reliable micronucleus counts, and that additional information is obtained that appears to discern genotoxic modes of action.