Methods: Murine macrophages (RAW264.7) were exposed to HEMA (0-8mM) and LPS (0.1µg/ml) in the absence or presence of N-acetylcysteine (NAC) and pharmacological inhibitors of MAPK pathways, such as PD98059 (ERK1/2), SB203580 (p38) or SP600125 (JNK). Cell survival was evaluated using the crystal violet assay and apoptosis was determined by flow cytometry after a 24h exposure period. Differences between medians (25% and 75% percentiles) from individual values of at least four independent experiments were statistically analyzed using the Mann-Whitney-U test.
Results: HEMA dose-dependently reduced cell survival in RAW264.7 macrophages. Although the presence of LPS further decreased cell survival in HEMA-exposed cell cultures, LPS significantly protected cells from cytotoxic effects caused by high HEMA concentrations. Furthermore, the amount of apoptotic cells was increased up to 40% in macrophages exposed to 8 mM HEMA, and the presence of LPS also reduced monomer-induced apoptosis here. The inhibition of the ERK1/2 pathway (PD98059) and JNK pathway (SP600125) increased apoptosis in LPS-exposed cultures, but no clear effect of the inhibitors on HEMA-induced apoptosis in RAW264.7 was observed. The antioxidant NAC counteracted HEMA- as well as LPS-induced apoptosis.
Conclusions: HEMA-induced cell death via apoptosis as a result of monomer-induced oxidative stress was not directly mediated through activated MAPKs.