The genetic toxicity of time: Importance of DNA-unwinding time to the outcome of single-cell gel electrophoresis assays

Abstract

Single-cell gel electrophoresis assays (comet assays) are described in which DNA damage is assessed in mouse skin keratinocytes treated with N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) and β-propiolactone (BPL) either in vitro or in vivo. The positive results observed under both conditions of test encourage the further development of the mouse skin comet assay as a screen for direct-acting in vivo genotoxins. From the outset of the present experiments we were struck by the compacted nature of the DNA in mouse skin keratinocytes. Under similar conditions of assay, rodent hepatocytes presented a uniform `unwound' distribution of DNA over the whole nuclear region. In order to study this effect we varied what seemed to be the most obviously related assay parameter: the DNA-unwinding time. A series of experiments was conducted in which control and MNNG-treated cells were exposed to a range of alkaline DNA-unwinding times (0.3–18 h) followed by measurement of the three comet tail parameters (length, DNA content, and their product, tail moment). Each of these parameters increased with increasing time of unwinding such that the tails observed for MNNG-treated cells with 0.3 h of DNA unwinding were similar in length to the tails of control cells exposed to an 8 h DNA-unwinding time. It is concluded that DNA-unwinding time is a critical parameter of the comet assay and that it may require optimisation for each tissue/cell type studied. Further, the data alert to the prospect that agents that uniquely affect chromosomal protein superstructure may increase comet tail length/DNA content in the absence of chemically induced DNA damage. Thus, there may be two discrete classes of chemical interaction with chromosomal DNA that yield identical comet assay results, but which have different implications for the genetic toxicity of the test agent. Similar effects were observed for rat hepatocytes or mouse lymphoma cells exposed to an 18 h DNA-unwinding time, but no comet tails were produced by exposure of cells to the lysis conditions (pH 10.0) for 18 h.