Miniaturized flow cytometric in vitro micronucleus assay represents an efficient tool for comprehensively characterizing genotoxicity dose–response relationships

Abstract

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.

Introduction

The existence of a threshold dose, that is, a dose level below which no-observed-adverse effects occur, is a well-known concept in toxicology [1]. For certain classes of genotoxicants, primarily aneugens, there is general consensus that their dose–response relationships are thresholded [2], [3], [4]. The mode of action for these agents is considered a key variable. For instance in the case of spindle poisons, chromosome segregation is not affected unless multiple targets are disrupted, a situation that is predicted to result in a thresholded response [5]. Conversely, radiation and DNA-reactive gentoxicants have historically been assumed to exhibit non-thresholded dose–response relationships [6], [7]. This default assumption has important consequences, since by definition there is no dose without effect. Regulation of genotoxicants with this profile is therefore strict in order to prevent or at least minimize human exposure [8], [9]. However, new evidence is accumulating that suggests even DNA-reactive genotoxicants can exhibit thresholded dose–responses [10], [11], [12], [13], [14]. Cellular defenses mechanisms, especially DNA-repair capacity, are often cited as contributing to thresholds in these cases [15].

Given the important implications that dose–response relationships have in risk assessment, it is clear that assumptions about DNA-reactivity/non-reactivity and thresholded versus non-thresholded responses requires further study. This situation highlights the need for in vitro assays that can comprehensively evaluate the low end of the dose–response curve. Indeed, Johnson et al. [12] expressed the need for in vitro assays that allow for large numbers of individual cells to be analyzed, include many replicates, and utilize closely spaced dose levels. However, the desire for thorough dose–response data is problematic unless the assay is very efficient, with data acquisition occurring via an automated process.

This laboratory has previously reported on the development of a flow cytometric method for scoring in vitro micronuclei (commercially known as In Vitro MicroFlow^®) [16], [17], [18]. In this system, a non-ionic detergent is used to liberate nuclei and micronuclei into suspension. In conjunction with outer membrane lysis, a sequential staining procedure is used to differentially stain chromatin from dead and dying cells from that of “healthy cells”, thereby minimizing the impact that necrosis and apoptosis has on the micronucleus (MN) frequency determinations. The so-called miniaturized version of the assay, conducted in 96-well plates, exhibits several characteristics that address the requirements articulated by Johnson et al. For instance, all the cell processing steps occur in the same 96-well plate, thus enhancing assay efficiency. In this format, it becomes simple to examine a very large number of concentrations and to include many replicate wells. Furthermore, flow cytometric data acquisition allows one to interrogate many cells per replicate for MN formation. We hypothesized that these characteristics would provide a useful platform for studying genotoxicity dose–response relationships. To test this premise, we used this system to study several prototypical genotoxicants, both DNA-reactive and non-reactive, using many finely spaced dose levels. The resulting data were evaluated in a statistical model designed to estimate a threshold dose and its confidence limits [19]. The results are discussed in regards to the ongoing need for data that thoroughly evaluates dose–response relationships.