Comet, TUNEL, and annexin V assays were used to identify DNA fragmentation and plasma membrane alterations occurring during staurosporine-induced apoptosis in Chinese hamster ovary cells. TUNEL assay detected apoptotic cells after 6 h treatment. The occurrence of annexin V immunofluorescence staining after 1 h treatment confirms that exposure of phosphatidylserine (PS) residues is an early biochemical feature of apoptosis. According to intensity, three annexin staining patterns were distinguished, related to different steps in the apoptotic process. The detection of highly damaged cells by the comet assay after 3 h treatment occurred earlier than the detection of DNA modifications by the TUNEL assay, but later than the exposure of PS residues. However, late apoptotic cells, otherwise characterized by plasma membrane disruption and high annexin V staining, were not detected by the comet assay. In this case, comet assay modified by omitting electrophoresis (halo assay) was more sensitive for an accurate quantification of the apoptotic fraction.

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The genotoxic potential of the fungicide malachite green (MG) and its reduced derivative leucomalachite green (LMG) was assessed in bacteria and mammalian cells using the standard Salmonella typhimurium/Ames and CHO/HGPRT tests. In vitro potential DNA damaging effects of MG and LMG were tested using the single‐cell gel electrophoresis (Comet) assay on CHO cells. Malachite green was found to be extremely cytotoxic to bacteria and mammalian cells. It did not have any mutagenic activity, in any bacterial strains, in the presence or absence of metabolic activation for doses up to 10 μg per plate. In the CHO/HGPRT test, the mutagenic potential of MG could be evaluated only for very low concentrations ranging from 0.001 to 0.05 μg ml−1 medium. When S9 fraction was added to the medium, the highest tested dose could be increased to 1 μg ml−1. In these experimental conditions, MG did not increase the number of thioguanine‐resistant mutants. Leucomalachite green was less toxic than MG to Salmonella typhimurium and did not have mutagenic activity in the Ames’ test for doses up to 2000 μg per plate. It was also less cytotoxic than MG to CHO cells and was tested at doses ranging from 5 to 100 μg ml−1. Overall results indicated that LMG was not mutagenic in the HGPRT test. In the Comet assay, MG induced DNA damage only at cytotoxic doses. Loss of cell viability was observed for doses of ⩾ 3 μg ml−1, with parallel increase in DNA alterations as measured by the tail moment. After metabolic activation, however, DNA damage was observed at doses (15–20 μg ml−1) inducing only low cytotoxicity. In this case, the direct genotoxicity of MG metabolites could not be excluded. In the absence or presence of metabolic activation, LMG did not have any effect on cell viability or DNA damage for doses up to 500 μg ml−1. This study indicates that LMG, which is the main residue found in fish tissues after treatment with MG, did not have any mutagenic or clastogenic effects in the experimental conditions used. Copyright © 1999 John Wiley & Sons, Ltd.

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