Among the tests used to determine the mutagenic potential of chemicals, the chromosomal aberrations and micronucleus assays play an important role. These tests score either chromosomal structural aberrations at metaphase or micronuclei at interphase. One of the hallmarks of apoptosis is DNA fragmentation into 50-300 kpb leading to oligonucleosomal fragmentation that can interfere with the results of clastogenic assays. In this case, apoptosis may be a confusing factor in the evaluation of the mutagenic potential of molecules and lead to false positive results. For these reasons we have developed a cell line able to demonstrate the interference of apoptosis in two mutagenicity tests: the in vitro micronucleus test and metaphase analysis in vitro. We used a murine cytotoxic T cell line, CTLL-2 Bcl2, in which a stably transfected bcl2 gene is known to protect these cells from apoptosis induced by various stimuli. A comparison between results obtained in parental CTLL-2 cells and in CTLL-2 Bcl2 cells treated with non-genotoxic apoptosis inducers, such as dexamethasone or gliotoxin, leads us to conclude that apoptosis could give false positive results due to DNA fragmentation. Moreover, with etoposide, a clastogen that also induces apoptosis, we observed that the percentages of aberrant cells and numbers of micronuclei were significantly increased in CTLL-2 cells compared with CTLL-2 Bcl2 cells. This observation suggests that apoptosis leads to an overestimation of the genotoxic potential of chemicals. Finally, with nocodazole, an aneugen, we confirm that this model can also detect agents that have only genotoxic potential and thus allows a better estimation of the genotoxic threshold in studies with aneugens, thus avoiding overestimation of the mutagenic risk of such a compound.

The single cell gel electrophoresis assay, or Comet assay, is a powerful tool for measurement of DNA strands breaks, oxidative damage, and alkali labile sites, and the assay was recently modified to detect DNA cross-links. It has also been proposed as a measure of apoptosis since apoptotic cells are suspected to result in total migration of the DNA from the nucleus into the tail. Cells with this appearance are called ghost cells, clouds, hedgehogs, or NDCN (nondetectable cell nuclei). The aim of this study was to determine if ghost cells can be used to measure apoptosis in the standard alkaline comet assay. To answer this question, we made use of two cell lines: CTLL-2 cells that can enter apoptosis upon addition of apoptosis stimuli or IL-2 deprivation, and CTLL-2 bcl2 cells that are protected from apoptosis due to the overexpression of the apoptosis inhibitor gene bcl2. The two cell lines were treated with cytotoxins (nongenotoxic apoptosis inducers, nongenotoxic necrotic agents) or genotoxins. They were also subjected to growth factor withdrawal, which induced apoptosis in the CTLL-2 cell line. The level of apoptosis was measured by the Annexin V-FITC method in parallel with performing the Comet assay. The results obtained in the two cell lines suggest that apoptotic or necrotic death does not correlate well with the detection of ghost cells, presumably because these cells are lost upon electrophoresis. A variant of the alkaline Comet assay that was performed without electrophoresis (halo method) was able to efficiently detect cells undergoing apoptosis, but it was unable to clearly distinguish between apoptosis and genotoxic damage.

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In vitro assays for chromosome aberrations (i.e., in vitro micronucleus and in vitro metaphase analysis tests) frequently produce false-positive or exaggerated-positive results. Our previous work suggested that apoptosis interferes with these tests, producing misleading results. These previous studies were conducted by performing the in vitro micronucleus test in CTLL-2 cells and a CTLL-2 cell derivative stably transfected with the apoptosis inhibitor gene bcl2. In the present study, these previous observations were extended by examining micronucleus induction with a larger number of compounds in both CTLL-2 and CTLL-2 bcl2 cells and measuring apoptosis with annexin V-FITC. Both cell lines were treated with different classes of compounds that were anticipated to be exclusively apoptosis inducers, or compounds known to be clastogens or aneugens, some of which were anticipated to be both genotoxic and apoptotic. We were able to confirm that compounds that are only apoptogenic induced micronuclei in CTLL-2 but not CTLL-2 bcl2 cells, indicating that the positive responses are due to apoptosis in CTLL-2 cells. Some genotoxins (clastogens and aneugens) did not produce apoptosis by the annexin V assay and gave similar responses in CTLL-2 and CTLL-2 bcl2 cells. Finally, higher responses were induced in CTLL-2 cells compared to CTLL-2 bcl2 cells that were treated with aneugens or clastogens that were also apoptosis inducers, suggesting that the greater response in CTLL-2 cells is a consequence of both genotoxicity and apoptosis. Finally, it was demonstrated that just eliminating CTLL-2 cells having three or more micronuclei from scoring was not adequate for correctly evaluating agents that only produce apoptosis. The results indicate that coupling the in vitro micronucleus test in both CTLL-2 and CTLL-2 bcl2 cells with the measurement of apoptosis is able to distinguish the genotoxic effects of a test compound from its apoptotic potential and is able to avoid interference from apoptosis in the in vitro micronucleus test. These observations may provide the basis for a useful genotoxicity assay.

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