Mitochondrial Transmembrane Potential as an Indicator of HEMA-induced Apoptosis

Method: RAW246.7 mouse macrophages were exposed to HEMA (0-8mM) up to 24h. ROS production was detected by flow cytometry (DCFH-DA), and the expression of antioxidant enzymes (SOD1, GPx1/2, catalase) was analyzed by Western blotting. MTP was determined by flow cytometry after the staining of cells with the voltage-sensitive fluorescent dye JC-1. GSH synthesis was inhibited by buthionine sulfoximine(50µM BSO) or activated by 2-oxothiazolidine-4-carboxylate (5mM OTC) or N-acetylcysteine (10mM NAC). Differences between medians (plus 25%/75% percentiles; n=4) were statistically analyzed (Mann-Whitney-U test).

Result: ROS production significantly increased 2-fold in cultures exposed to 8mM HEMA for 30min to 6h. Expression of catalase, a H₂O₂-decomposing enzyme, was enhanced by HEMA, but expression of GPx1/2 and SOD1 which metabolize H₂O₂was reduced. Cell viability as indicated by a high MTP after JC-1 staining was significantly reduced from 93% in untreated cultures to 67% in cultures exposed to 8mM HEMA for 24h. While MTP further decreased to 16% in the presence of BSO and 8mM HEMA, OTC and NAC protected cells from a HEMA-induced reduction of the MTP. An effect of 8mM HEMA on MTP after exposure for 30min to 12h was not detected.

Conclusion: The decrease of MTP is causally related to the availability of GSH, and indicates that apoptosis is a late response in cells exposed to HEMA.

Supported by the Deutsche Forschungsgemeinschaft DFG (Schw431/13-1).