Non-esterified polyunsaturated fatty acids distinctly modulate the mitochondrial and cellular ROS production in normoxia and hypoxia

There is an intense discussion about the subcellular origin of the generation of reactive oxygen species (ROS) under hypoxia. Since this fundamental question can be addressed only in a cellular system, the O-2-sensing rat pheochromocytoma (PC12) cells were used. Severe hypoxia is known to elevate non-esterified fatty acids. Therefore, the site(s) of ROS generation were studied in cells which we simultaneously exposed to hypoxia (1% oxygen) and free fatty acids (FFA). We obtained the following results: (i) at hypoxia, ROS generation increases in PC12 cells but not in mitochondria isolated therefrom. (ii) Non-esterified polyunsaturated fatty acids (PUFA) enhance the ROS release from PC12 cells as well as from mitochondria, both in normoxia and in hypoxia. (iii) PUFA-induced ROS generation by PC12 cells is not decreased either by inhibitors of the cell membrane NAD(P) H oxidase or inhibitors impairing the PUFA metabolism. (iv) PUFA-induced ROS generation of mitochondria is paralleled by a decline of the NADH-cytochrome c reductase activity (reflecting combined enzymatic activity of complex I plus III). (v) Mitochondrial superoxide indicator (MitoSOXred)-loaded cells exposed to PUFA exhibit increased fluorescence indicating mitochondrial ROS generation. In conclusion, elevated PUFA levels enhance cellular ROS level in hypoxia, most likely by impairing the electron flux within the respiratory chain. Thus, we propose that PUFAs are likely to act as important extrinsic factor to enhance the mitochondria-associated intracellular ROS signaling in hypoxia.