Contribution of apoptosis to responses in the comet assay

Apoptosis, a physiological process of selected cell deletion, leads to DNA fragmentation in typical segments of 180 base pairs. DNA strand breaks are also an effect induced by genotoxic compounds. The aim of this study was to compare these two types of damaging potentials by a known genotoxic substa nce and an apoptosis-inducing agent in HT-29 colon adenocarcinoma cells. Th e cells were incubated for 24 h with N-methyl-N'-nitro-N-nitrosoguanidin (M NNG), a potent DNA ge-inducing agent, staurosporine, an inhibitor of protei ne kinase C and apoptosis-inducing agent, and hydrogen peroxide, a source o f reactive oxygen species. Apoptosis was measured with the Annexin V affini ty assay which detects the translocation of phosphatidylserine (PS) from th e inner to the outer leaflet of the cytoplasmic membrane, an early event in the apoptotic process. DNA damage as an end point of genotoxicity was dete cted by single cell microgel electrophoresis, also called "comet assay". Th e results show that apoptosis does not necessarily need to correlate or coi ncide with DNA damage observed with genotoxic substances in the comet assay . The representative apoptosis-inducing agent (staurosporine) did not induc e strand breaks in the tested concentrations (0.5 and 1.0 muM); genotoxic d oses of the strand break inducing agent MNNG did not induce apoptosis. Ther efore, the comet assay can be used as a specific test for detecting genotox icity, and the results are not necessarily confounded by concomittant proce sses leading to apoptosis. (C) 2001 Elsevier Science B.V. All rights reserv ed.