THE GENETIC TOXICITY OF TIME - IMPORTANCE OF DNA-UNWINDING TIME TO THE OUTCOME OF SINGLE-CELL GEL-ELECTROPHORESIS ASSAYS

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 P-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 th e outset of the present experiments we were struck by the compacted na ture of the DNA in mouse skin keratinocytes. Under similar conditions of assay, rodent hepatocytes presented a uniform 'unwound' distributio n 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 whi ch 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 co met tail parameters (length, DNA content, and their product, tail mome nt). Each of these parameters increased with increasing time of unwind ing 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 exp osed 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 requir e optimisation for each tissue/cell type studied. Further, the data al ert to the prospect that agents that uniquely affect chromosomal prote in superstructure may increase comet tail length/DNA content in the ab sence of chemically induced DNA damage. Thus, there may be two discret e classes of chemical interaction with chromosomal DNA that yield iden tical comet assay results, but which have different implications for t he genetic toxicity of the test agent. Similar effects were observed f or rat hepatocytes or mouse lymphoma cells exposed to an 18 h DNA-unwi nding time, but no comet tails were produced by exposure of cells to t he lysis conditions (pH 10.0) for 18 h.