Miniaturized flow cytometric in vitro micronucleus assay represents an efficient tool for comprehensively...

This laboratory has developed a flow cytometric approach for scoring in vitro micronuclei (In Vitro MicroFlow(®)) whose characteristics are expected to benefit studies designed to comprehensively investigate genotoxicity dose-response relationships. In particular, new experimental designs become possible when automated scoring is combined with treatment, processing and sampling that all occur in microtiter plates. To test this premise, experiments described herein investigated micronucleus (MN) formation in TK6 cells treated with genotoxic agents applied at 22 closely spaced concentrations in quadruplicate, with 10,000 cells analyzed per replicate. The genotoxicants colchicine, vinblastine sulfate, ethyl methanesulfonate, methyl methanesulfonate, ethyl nitrosourea, methyl nitrosourea, and bleomycin were applied continuously for 24-30 h. Following treatment, all cell processing, sampling and data acquisition steps were accomplished in the same 96-well plate. Data acquisition occurred in a walk-away mode via the use of a high throughput sampling device. The resulting flow cytometric MN values were evaluated with a statistical model that indicated non-linear relationships describe the data better than linear fits. The one exception was bleomycin, where MN induction was consistently best described by a linear dose-response relationship. Collectively, these results suggest that flow cytometry represents a practical and efficient approach for thoroughly examining the dose-response relationship, and clearly benefits studies that seek to characterize no observable genotoxic effect levels, lowest observable genotoxic effect levels, and/or benchmark doses.