Grain counting in the in vitro hepatocyte DNA-repair assay

The in vitro hepatocyte DNA-repair assay is a widely used useful method in assessing the genotoxic activity of both directly and indirectly acting chemical agents. This article discusses the criteria presently employed in the autoradiographic evaluation of unscheduled DNA synthesis, and suggests that the subtraction of either the average or the highest cytoplasmic grain count, usually carried out to obtain the net nuclear grain count, may represent a potential source of errors when the test compound is a weakly genotoxic or a non-genotoxic agent. As a matter of fact, a response can be classified as positive or negative depending on the procedure used to quantitate the cytoplasmic background, and the subtraction of this background from the nuclear count is not founded on a sound theoretical basis because of the following reasons: the different nature of the processes responsible for the generation of nuclear and cytoplasmic grains; and the quantitatively different effect that the test compounds may have on the nuclear and the cytosolic labelling.