Eicosapentaenoic acid (EPA) was previously shown to induce
caspase-independent apoptosis in rat basophilic
leukemia cells (RBL2H3 cells) by translocation of
apoptosis-inducing factor (AIF) [Free Radic Res (2005) 39, 225-235]. Here, we attempted to investigate the mechanism of EPA-induced apoptosis. A rapid and sustained increase in
calcium was observed in mitochondria at 2 h after the addition of EPA prior to apoptosis. Coincidently,
hydroperoxide was generated in the mitochondria after exposure to EPA. Production of mitochondrial
hydroperoxide was significantly reduced by
ruthenium red, an inhibitor of
mitochondrial calcium uniporter, and
BAPTA-AM, a cytoplasmic
calcium chelator, indicating that generation of
hydroperoxide is triggered by an accumulation of
calcium in the mitochondria. The production of mitochondrial
hydroperoxide was markedly attenuated by overexpression of
phospholipid hydroperoxide glutathione peroxidase (PHGPx) in the mitochondria. Apoptosis was therefore, significantly prevented through inhibition of mitochondrial
hydroperoxide generation with mitochondrial PHGPx,
ruthenium red or
BAPTA-AM. However, accumulation of
calcium in the mitochondria was not prevented by mitochondrial PHGPx although apoptosis was blocked, indicating that elevated
calcium does not directly induce apoptosis. Taken together, our results show that
calcium-dependent
hydroperoxide accumulation in the mitochondria is critical in EPA-induced apoptosis.