The resin monomer TEGDMA induces late apoptosis in murine macrophages

Methods: Murine RAW264.7 macrophages were exposed to 3mM TEGDMA in culture medium for 6 and 24h. Lipopolysaccharide (LPS; 0.1µg/ml) and 1µM camptothecin were used as control substances. After exposure cells were stained with annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry. Data acquisition and analyses were performed using CellQuest software. The percentages of viable cells, cells in early (annexin V+/PI-) and late (annexin V+/PI+) apoptosis, and necrotic cells (annexin V-/PI+) were determined. Differences between medians (n=4) of the percentages of cells in different stages and of cell death were statistically analyzed (Mann-Whitney-U-test; p<0.05).

Results: After a 6h incubation period, there was a statistically significant difference between untreated cultures (0.5%) and cultures treated with camptothecin (13%). No increase of apoptotic cells was detected in cultures treated with any other reagent. After 24h exposure, however, the percentage of cells in early apoptosis significantly increased in TEGDMA-treated cultures (6.5%) compared to untreated cultures (1%), and co-incubation with LPS/TEGDMA was similar effective. In TEGDMA-treated cultures even 32% of the cells were detected to be in late apoptosis compared to 7% in untreated cultures. Late apoptosis (24h exposure) was found for camptothecin (49%), LPS/TEGDMA (24%) and LPS (20%), and the amounts of cells in necrosis increased in parallel but to a lesser extent.

Conclusion: Cells in various stages of cell death were detected after a 24h exposure and indicated that TEGDMA-induced apoptosis is a late effect in RAW macrophages.

Supported by the Deutsche Forschungsgemeinschaft (Schw 431/11-1)