The effect of the lipophilic cation lucigenin on mitochondria depends on the site of its reduction.

The role of NAD(P)H-dependent oxidoreductases of the outer mitochondrial membrane (OMM) in the activation of lipophilic cationic dyes is poorly understood. In the present study we compared the rates of production of reactive oxygen species (ROS) and mitochondriotoxic effects of the redox-cycling lipophilic cationic dye lucigenin upon its activation by the respiratory chain and NAD(P)H-dependent oxidoreductases of the OMM. We found that, only in the presence of external NADH and NADPH, which are unable to penetrate the inner membrane, lucigenin stimulated a massive superoxide production and a fast permeabilization of mitochondrial membranes. The permeabilization was biphasic. The first, cyclosporin A-insensitive and Ca(2+)-independent phase was characterized by increased permeability of the inner mitochondrial membrane to solutes with molecular masses of <or=200 Da. The second phase was sensitive to the antagonists of the permeability transition pore (mPTP) and was characterized by permeability similar to that of mPTP (<or=1500 Da). A massive cytochrome c release was observed even at the first phase of permeability when the second phase was inhibited by mPTP antagonists. Whatever the site of lucigenin activation, antioxidants and scavengers of ROS that strongly decrease the ROS level were unable to delay or prevent the permeabilization of membranes, which casts doubt on the involvement of ROS in the regulation of permeability by redox-cycling lipophilic cations. Our results strongly support the idea that the NAD(P)H-dependent reductases of xenobiotics of the OMM can mediate the toxicity of cationic dyes.