, 2011). In the presence of the Ca2+-uniporter blocker ruthenium red, nemorosone induced mitochondrial swelling in a way sensitive to the classic mitochondrial permeability transition (MPT) inhibitor cyclosporine A. Unlike nemorosone, GA uncoupled mitochondria through a non-protonophoric mechanism (result not shown). In addition, mitochondrial swelling elicited by GA was not inhibited by cyclosporine A or EGTA and, therefore, it does not correspond to the MPT process (Zoratti and Szabò, 1995). Rather, the evidence that GA increased mitochondrial membrane fluidity suggests GSK1120212 that a direct

interaction with mitochondrial membrane, whose major structural lipids are cardiolipins, accounts for its permeabilizing action on the organelle. The evidence that isocitrate partly prevented GA-induced NADPH oxidation/depletion and

mitochondrial swelling in isolated mitochondria, as well as cell viability decrease, ATP depletion and ROS levels increase in HepG2 cells, suggests that NADPH oxidation/depletion is at least partly involved in the GA permeabilizing action on mitochondria and its consequence on cells. Isocitrate is the substrate of NADP+-dependent isocitrate dehydrogenase, a major Androgen Receptor Antagonist library NADPH source in mitochondria with a key role in cellular defense against ROS (Jo et al., 2001). In citosol, NADPH is provided primarily by the pentose phosphate pathway, including glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. In this regard, the fact that HepG2 cells

incubated in medium with Plasmin low glucose levels were more sensitive to GA-induced death, mitochondrial membrane potential dissipation, ATP depletion and ROS levels increase reinforces the proposed GA toxicity mechanism. Low glucose impairs NADPH re-generation in citosol and may potentiate mitochondria-mediated cytotoxic actions. In conclusion, the present results suggest the following sequence of events for the GA action on mitochondria: 1) GA interaction with mitochondrial membrane increasing its fluidity and promoting its permeabilization; 2) mitochondrial membrane potential dissipation; 3) NAD(P)H oxidation/depletion due to inability of membrane potential-sensitive NADP+ transhydrogenase of sustaining its reduced state; 4) ROS accumulation inside mitochondria and cells; 5) additional mitochondrial membrane permeabilization due to ROS; and 6) ATP depletion. The evidence that Ca2+ efflux was only partially prevented by the Ca2+-uniporter blocker ruthenium red in isolated mitochondria and the inability of isocitrate to prevent mitochondrial membrane potential dissipation in HepG2 cells suggest that this latter is an early event associated to the GA action on mitochondria, which could ultimately, via energetic and oxidative stress implications, result in cell ATP depletion.