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Alternatives to Laboratory Animals - ATLA

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Apoptosis Versus Cytotoxicity in HeLa Cells Exposed to Paracetamol

Katarína Ruppová, Miroslava Urban

Apoptosis is a programmed form of cell death which occurs in response to specific stimuli. It is distinguished from necrotic or accidental cell death by unique events, including the degradation of chromatin and a loss of cellular volume. In contrast to necrotic cell death, cell membrane integrity and mitochondrial function are thought to be maintained until the apoptotic process is well advanced. One of the novel assays for detecting apoptosis is flow cytometry. In our experiments, we used a flow cytometric assay to detect DNA changes in a human cell line (HeLa) exposed to paracetamol, by measuring propidium iodide binding. We were able to detect the apoptotic process in cells exposed to paracetamol. Apoptosis did not correlate with cytotoxicity, and was only found in samples exposed to 4–5mg/ml paracetamol for 8 hours in minimum essential medium and incubated in fresh medium without paracetamol for 14–19 hours. The greatest effect was noted 18 hours after paracetamol exposure. These results were confirmed by studying cell morphology and chromatin condensation by fluorescent microscopy with the fluorochromes acridine orange and ethidium bromide. Our results support the hypothesis that, in cultured cells, apoptosis is induced by a relatively narrow range of chemical concentrations which are known to inhibit the cell cycle, and that apoptosis and inhibition of cell proliferation coincide to some degree.